Opportunities and challenges for the development of covalent chemical immunomodulators

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Bioorganic & Medicinal Chemistry journal homepage: www.elsevier.com/locate/bmc

Opportunities and challenges for the development of covalent chemical immunomodulators Keriann M. Backus , Jian Cao, Sean M. Maddox ⁎

Departments of Biological Chemistry and Chemistry and Biochemistry, University of California Los Angeles, USA

ARTICLE INFO

ABSTRACT

Keywords: Covalent inhibitors Cysteines Kinase inhibitors Phosphatase inhibitors Protease inhibitors Electrophiles Chemoproteomics Chemical immunology Covalent immunomodulators

Compounds that react irreversibly with cysteines have reemerged as potent and selective tools for altering protein function, serving as chemical probes and even clinically approved drugs. The exquisite sensitivity of human immune cell signaling pathways to oxidative stress indicates the likely, yet still underexploited, general utility of covalent probes for selective chemical immunomodulation. Here, we provide an overview of immunomodulatory cysteines, including identification of electrophilic compounds available to label these residues. We focus our discussion on three protein classes essential for cell signaling, which span the ‘druggability’ spectrum from amenable to chemical probes (kinases), somewhat druggable (proteases), to inaccessible (phosphatases). Using existing inhibitors as a guide, we identify general strategies to guide the development of covalent probes for selected undruggable classes of proteins and propose the application of such compounds to alter immune cell functions.

1. Introduction Immune dysregulation contributes to the etiology and progression of many diseases, including diabetes, obesity, cancer, inflammation, and neurodegeneration. Small molecule and biologic modulators of the immune system, including both immunosuppressive and immunostimulatory agents have proven efficacious in the treatment of cancer and autoimmune disorders.1–3 Increasing T cell proliferation through administration of IL2 or inhibition of immunosuppressive pathways (e.g. antibodies targeting PD-1, PD-L1, and CTLA-4) has led to tumor regression and even cures, notably even in refractory cancers.4–11 These successes underscore the need for other immunomodulatory agents, in particular cell-penetrating small molecules that can target proteins inaccessible to monoclonal antibodies and other biologics, which comprise most immunotherapeutic agents. Recently, cysteine-reactive compounds have reemerged as powerful tools for altering protein function, especially for tough-to-drug classes of proteins.12–15 These compounds, which are frequently referred to as covalent compounds, feature electrophilic moieties that react either

irreversibly or reversibly with the thiol side chain of certain cysteine residues. The preferential labeling of specific cysteines is driven by a combination of factors, including (1) the intrinsic reactivity of the thiol, (2) the nature and relative reactivity of the electrophile, and (3) the molecular recognition of the binding portion of the molecule.16 The identification of inherently reactive cysteines and the choice of electrophile will be discussed in more detail below. Cysteine is an exciting amino acid to chemically target for several reasons. Due to the unique chemistry of the cysteine thiol, cysteine residues play key roles in the structure and function of most human proteins. Cysteines frequently serve as catalytic nucleophiles in enzymes such as proteases. Cysteines also coordinate metals, form structural and redox active disulfides, are frequently post-translationally modified, and can serve as sensors of oxidative stress. Cysteine-reactive compounds can be designed to access small and poorly defined binding sites and can efficiently block high-affinity interactions (e.g. proteinprotein interactions) or compete with high concentrations of endogenous biomolecules (e.g. ATP).14,17–19 There are numerous examples of cysteine-reactive clinical candidates and drugs, including

Abbreviations: ABPP, activity-based protein profiling; isoTOP-ABPP, isotopic tandem orthogonal proteolysis-activity-based protein profiling; LC-MS/MS, liquid chromatography tandem mass spectrometry; CuAAC, copper-catalyzed azide–alkyne cycloaddition or “click” chemistry; IAA, iodoacetamide alkyne; SILAC, stable isotope labeling by amino acids in cell culture; ROS, reactive oxygen species; RNS, reactive nitrogen species; TEV, tobacco etch virus; cmk, chloromethyl ketone; fmk, fluoromethyl ketone; PD-1, programmed death protein 1; PD-L1, programmed death ligand 1; CTLA-4, cytotoxic T-lymphocyte-associated protein 4, which is also known as cluster of differentiation 152; Cys-Ox PTMs, cysteine oxidative modifications; CKI, covalent kinase inhibitor; TF, transcription factor; TAMs, tumor associated macrophages; LPS, lipopolysaccharides; IFNγ, interferon gamma; IL-2, interleukin 2; IL-4, interleukin 4 ⁎ Corresponding author at: 615 Charles E Young Dr. South, BSRB 350A, Los Angeles, CA 90095, USA. E-mail address: [email protected] (K.M. Backus). https://doi.org/10.1016/j.bmc.2019.05.050 Received 12 February 2019; Received in revised form 24 May 2019; Accepted 31 May 2019 0968-0896/ © 2019 Elsevier Ltd. All rights reserved.

Please cite this article as: Keriann M. Backus, Jian Cao and Sean M. Maddox, Bioorganic & Medicinal Chemistry, https://doi.org/10.1016/j.bmc.2019.05.050

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

blockbuster covalent kinase inhibitors (CKIs)20; the anti-cancer compounds KPT330, which reacts with a conserved cysteine in the nuclear export factor XPO121; and ARS-1620 that inhibits the Gly12Cys mutated oncogenic form of the GTPase KRAS.22 Nearly all human proteins contain at least one cysteine (average 13 cysteines/protein), and recent studies, including ours, indicate that a surprising fraction of cysteines can react with cysteine-reactive compounds.16,23,24 As will be reviewed here, the functions of human immune cells are exquisitely sensitive to oxidative stress, which can have both immunosuppressive and immunostimulatory outcomes. Immune cells are also heavily reliant on dynamic cell signaling cascades that are regulated, in large part, by the catalytic activity of protein kinases, protein phosphatases, and cysteine proteases, which all feature cysteines either as catalytic residues (proteases and phosphatases) or non-catalytic residues (kinases) in their active sites. Despite the widespread adoption of cysteine-reactive compounds as probes, and the direct link between oxidative stress and cysteine function, the utility of cysteine-reactive compounds as immunomodulatory agents remains to be fully realized. Within the burgeoning field of chemical immunology,3 this review aims to lay the groundwork for such studies by providing an overview of technologies available to identify such compounds and by highlighting relevant compounds and their protein targets. By focusing on three well studied and immune-essential protein classes (kinases, phosphatases, and proteases), we aim to provide a focused overview of challenges, opportunities, and lessons for covalent probe development that should prove applicable to additional protein classes.

kinase (IκK)44; phosphatases such as Src homology 2 domain proteintyrosine phosphatase 2 (SHP2)45,46, the phosphatase and tensin homologue (PTEN)47, protein tyrosine phosphatase 1B (PTPN1B)48,49; transcription factors, including nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB),50 the tumor suppressor protein p53,51 forkhead box class O transcription factors (FOXO),52 and nuclear factor erythroid 2-related factor 2 (NRF2)53; proteases such as caspases,54 cathepsins,55 and metalloproteinases56; redox sensors, such as the kelch like ECH associated protein 1 (KEAP1)57; and metabolic enzymes such as prolyl hydroxylases.58 The study of Cys Ox-PTM contributions to immunity should benefit from the numerous chemical tools developed to study cysteine-mediated redox signaling.59 For further information, we recommend the reviews by Nathan, Belikov, and Franchina.27,60,61 As with ROS, human immunity is also sensitive to small molecule electrophiles. For example, the clinically approved treatment for multiple sclerosis, Tecfidera®, which is also known as dimethylfumarate, suppresses T cell activation.27,62,63 The electrophilic metabolite itaconate also has anti-inflammatory activity. Itaconate labels several cysteines in KEAP1, the redox sensor that regulates the abundance of NRF2, the master transcriptional regulator of cellular redox homeostasis. KEAP1 alkylation increases NRF2 activity, which leads to increased expression of anti-inflammatory and anti-oxidant genes.57 Additionally, itaconate has also been shown to have anti-inflammatory activity independent of NRF2.64 Curcumin, an electrophilic component of turmeric, is known for its anti-inflammatory affects, including suppression of NFκB signaling and inhibition of proinflammatory cytokine production.65 Curiously, as with low-level ROS, low levels of curcumin has been shown to be pro-inflammatory, indicating the possible existence of pro-inflammatory cysteines that may act orthogonal to their more well-characterized immunosuppressive counterparts.66 In contrast, the orthogonal activities of ROS may also reflect a dose-dependent effect with modest oxidation of some cysteines as pro-proliferative, whereas more pronounced oxidation as anti-proliferative or even proapoptotic.

2. Identification of immunomodulatory cysteines 2.1. Cysteines as regulators of immune cell signaling During oxidative metabolism or in response to xenobiotics, cytokines, or bacterial invasion, immune cells generate elevated levels of intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can react with the cysteine thiol side chains, resulting in the formation of cysteine oxidative modifications (Cys Ox-PTMs). Oxidative stress imbalances are associated with many immune-related diseases, including autoimmune disorders, cancers and infections.25,26 While the mechanisms by which oxidative stress regulates immunity still have not been fully dissected, it has become clear that the functions of both innate and adaptive immune cells are intimately tied to the modification state of redox-sensitive cysteine thiols. Somewhat counterintuitively, oxidative stress can have both proand anti-inflammatory activity. ROS are required for cellular activation of peripheral T cells, and an absence of ROS is associated with hypoactivation and decreased T cell proliferation.27–29 Conversely, inappropriately high levels of ROS are associated with DNA damage and even cellular death.30,31 Thus, for T cells, an optimal balance of ROS, and presumably of the resulting Cys Ox-PTMs, is required for regulation of immune cell states and prevention of autoimmunity. In innate immune cells, ROS and RNS are associated with phagocytic antimicrobial activity.32–34 ROS has been implicated in macrophage reprogramming, altering the balance between classically activated M(LPS) or M (LPS + IFNγ) macrophages, which have antimicrobial activity, and alternatively activated M(IL-4) macrophages, which have immunosuppressive activity by recruiting TH2 cells.35 Both metabolic reprogramming and engagement of Toll-like receptors have been implicated in ROS production in activated macrophages.36,37 The production of ROS is required for M(IL-4) cell polarization.35,38 Taken together these studies indicate a complex interplay of ROS contributing to both pro- and anti-inflammatory processes. There are numerous immune-relevant proteins with cysteines sensitive to oxidative stress, including the T cell receptor39; tyrosine kinases, such as the epidermal growth factor receptor (EGFR),40 c-Jun Nterminal kinase 2 (JNK2),41 RAC-alpha serine/threonine-protein kinase (AKT),42 zeta-chain associated protein of 70 kDa (ZAP70),43 and IκB

2.2. Chemoproteomic profiling of cysteines With the seeming ubiquity of reactive and redox sensitive cysteines in human immune cell signaling pathways, the development of chemical reagents to label and alter the functions of these residues is an obvious strategy for the development of immunomodulatory compounds. In response to the recent demand for covalent probes, several new approaches that combine quantitative chemoproteomics and activity-based protein profiling (ABPP)67 have emerged to guide the production of covalent compounds.16,23,24,68,69 These strategies can be divided into two general categories, (1) proteome-wide mapping of cysteines using pan-cysteine reactive chemical probes such as iodoacetamide alkyne (IAA) and (2) target identification using custom probes that feature an electrophile, binding group, and an enrichment handle (typically either azide, alkyne, or biotin) to identify only those protein targets labeled by specific chemical probes. We anticipate that when combined with electrophile profiling of immune cells, both methods will uncover new immunomodulatory and ligandable cysteine residues. One example of proteome-wide cysteine profiling is isotopic tandem orthogonal proteolysis-activity-based protein profiling (isoTOPABPP).70 The general isoTOP-ABPP workflow is depicted in Fig. 1, which shows both the method for reactivity profiling (Fig 1a and c) and small molecule screening (Fig 1b and c). IsoTOP-ABPP was first developed as a quantitative method to measure the intrinsic reactivity of cysteine thiols in complex biological settings. In cysteine reactivity profiling, cellular lysates are treated with different concentrations (e.g. 100 µM or 10 µM) of a highly reactive and cysteine-specific probe such as IAA or with equal amounts of probe for two time points (e.g. 6 min or 60 min; Fig. 1a). As shown in Fig. 1c, the samples are then conjugated by copper-catalyzed azide–alkyne cycloaddition (CuAAC) or “click” chemistry71,72 to isotopically differentiated, biotinylated, tobacco etch 2

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

Fig. 1. Chemoproteomic identification of hyper-reactive and small molecule targeted cysteines using isoTOP-ABPP. a-c, show general schemes for isoTOP-ABPP reactivity profiling and compound screening. a,c, To identify hyper-reactive cysteines, samples are first labeled with the pan-cysteine reactive probe iodoacetamide alkyne (IAA), shown in the inset in panel 'a,' at the indicated concentrations (1 × or 10×). Next, as shown in panel 'c,' these samples are conjugated to isotopically differentiated, biotinylated (purple diamonds), tobacco etch virus (TEV) protease cleavable tags by click chemistry. After isotopic labeling, the samples are then combined, enriched, digested, and analyzed by LC-MS/MS. Hyper-reactive cysteines are those that show MS1 chromatographic peak area ratios close to one (shown in box). b,c, To identify cysteines labeled by electrophilic compounds, cells or lysates are treated with compounds or vehicle followed by treatment with equal concentrations of IAA, as shown in 'b.' Blockade of IAA labeling by pre-treatment with compound is detected by conjugation to TEV-cleavable enrichment tags, as shown in 'c,' followed by enrichment and sequential digests, as with reactivity profiling. Compound-labeled cysteines are identified as those that show altered MS1 chromatographic peak area ratios, typically four-fold or greater (shown in box).

3

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

strategies to identify the protein targets of covalent probes.24,69 Fig. 2 shows a general scheme for chemoproteomic experiments using such custom capture probes. For example, alkyne-containing analogues of the FDA-approved drugs afatinib and ibrutinib, two covalent kinase inhibitors (CKIs) that function through inhibition of selected tyrosine kinases, were used to generate proteomic inventories of the proteins targeted by these drugs in human cancer cells, including both on-target and off-target proteins.69 By treating heavy and light cells (stable isotope labeling by amino acids in cell culture [SILAC]83) with the afatinib and ibrutinib probes, respectively, followed by proteomic analysis, probe-probe comparisons were generated and used to identify specific and overlapping targets of each probe. Next, competitive profiling studies, where labeling with alkyne-probe is competed by pre-treatment with the parent compound, were used to delineate high- and low-affinity labeled targets. This strategy has been expanded to identify the target profiles of additional clinically relevant CKIs.68 Marto and coworkers identified that cysteine-reactive probes share common gas-phase dissociation patterns.84 Applying this discovery, the authors developed the method Covalent Inhibitor Target-Site Identification (CITeID), as a general method to identify the protein targets of covalent probes. Exemplifying the utility of CITeID, the authors identified new probes that target the kinase PKN3, which has been shown to promote malignant growth though interactions with the Rho family of small GTPases.24,85,86 Advantages of chemoproteomic studies that use custom probes include improved identification of low abundance proteins and identification of proteins that fail to react with broadly reactive probes, such as IAA.

virus (TEV) protease-cleavable peptide tags. Following isotopic labeling, samples are combined pairwise, enriched on streptavidin resin, and subjected to sequential tryptic and TEV digests. The TEV-eluted peptides are then analyzed by LC-MS/MS and highly reactive or “hyperreactive” cysteines are identified as those residues found in peptides that have integrated MS1 chromatographic peak area ratios near to one. Proteome-wide reactivity measurements can provide a good indication of cysteine functionality as previous studies have reported a strong correlation between residue reactivity and contributions to biological processes, including catalysis, metal coordination, and redox sensitivity.70 While a useful predictor for general accessibility to labeling by covalent probes, cysteine reactivity alone is, however, not sufficient to determine whether an individual cysteine can be labeled by chemical probes, which can be reported by competitive isoTOP-ABPP.16,73 Competitive isoTOP-ABPP is a modified version of isoTOP-ABPP that has been developed to identify cysteines labeled by electrophilic compounds (Fig. 1b and c). In competitive isoTOP-ABPP, cells or lysates are first treated in a pairwise manner with either a cysteine-reactive compound or vehicle, respectively. The treated and control samples are then labeled with equal concentrations of IAA, followed by capture, enrichment and digest as described for reactivity profiling. High affinity compound-cysteine interactions are then identified by the blockade of IAA labeling by pre-treatment with a compound. Compound-labeled cysteines are defined as those residues that exhibit four-fold or greater changes in MS1 chromatographic peak area for the cysteine-containing peptides identified by isoTOP-ABPP, comparing treated and control samples. Proteome-wide implementation of isoTOP-ABPP to screen electrophilic small molecules has successfully identified compounds that label several cysteines required for immune cell function, including probes that label the cysteine proteases caspase-8 and caspase-1016 and cysteines in the kinase PKCΘ, which block T cell activation when modified by the MS drug Tecfidera®62 and cysteines in the kinase IRAK4, which block cytokine production when modified by Tecfidera®.74 Modified versions of isoTOP-ABPP are available,75–79 which all leverage the same general labeling and capture strategy. Competitive isoTOP-ABPP and related methods offer several strengths, including the ability to profile unmodified compounds and compatibility with primary cells and tissues. However, these approaches often fail to identify the protein targets of electrophilic compounds for several reasons, including low protein abundance, incompatible tryptic peptide lengths, and poor peptide ionization efficiency, problems that are shared across peptide-centric proteomics studies.80–82 These limitations can be addressed by custom azide-, alkyne- or biotin-functionalized probes. Complementary to isoTOP-ABPP and related methods, the combination of custom azide-, alkyne- or biotin-containing electrophilic probes with quantitative MS has also yielded tailored chemoproteomic

2.3. Electrophiles Choice of electrophile is an essential consideration for the design of any covalent probe (See Fig. 3 for representative electrophiles, which are clustered based on their mechanism of covalent labeling). Most covalent probes feature either α,β-unsaturated or α-halo carbonyl electrophiles, which are known to react selectively with cysteine thiols.87 While some of these electrophiles are often broadly reactive, including β-unsubstituted vinyl sulfonamides, vinyl sulfones, many αhaloacetamides, and maleimides, others, including acrylamides, βsubstituted acetylenes, and β-substituted haloacetamides, typically exhibit highly attenuated thiol reactivity.16,88,89 Other notable classes of electrophiles with demonstrated cysteine selectivity include 2-chloropropionamides,16,90 metabolically labile fumarates,91 acyloxymethyl ketones (AOMK),92,93 chloro94 and fluoromethyl ketones (cmk and fmk),94–97 aryl halides,98–100 chloromethyl triazoles,101 fluoro and chloroacetamidines,102,103 terminal alkynes,104–107 bromobenzyl phosphonates,108 and covalent reversible compounds, such as nitriles,109,110

Fig. 2. Chemoproteomic identification of proteins that contain targetable cysteines using custom probes, which incorporate an electrophile (star), binding group, and enrichment tag R (biotin, alkyne, or azide). In competition experiments, isotopically differentiated cells (SILAC) are labeled with electrophilic compounds or vehicle, followed by labeling with the azide-, alkyne- or biotin-containing capture probe. The samples are then combined pairwise, enriched, digested, and subjected to LCMS/MS analysis. For probe/probe experiments, light and heavy samples are treated with two different enrichment probes, followed by analysis as with competition experiments. 4

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

Fig. 3. Cysteine-reactive electrophiles incorporated into available tool compounds and chemical probes. Electrophilic groups are shown in red and are grouped based on their specific mechanism of covalent cysteine labeling.

cyanonitriles,111–113 keto heterocycles,114 and chlorofluoroacetamides.115 Several recent studies have reported comprehensive evaluations of a wide range of electrophiles and electrophilic compound libraries, which provide useful guides for the design and optimization of future probes.116–121 For those electrophiles assayed, these studies provide a benchmark of general reactivity. However, no studies have systematically compared all available electrophiles. Given that the electrophile reactivity can be further tuned by additional substituents, as demonstrated by the aforementioned studies, the accurate de novo prediction of the general reactivity of a given compound remains challenging. Electrophile choice will be highlighted in the following discussions of kinase, phosphatase and protease probes.

further subcategorized into tyrosine-specific kinases, serine/threoninespecific kinases, and mixed specificity kinases. Both protein and metabolic kinases play key roles in many human diseases, including immune disorders and cancers.162–164 Kinase-mediated signaling cascades are required for nearly all aspects of immune cell function, including B and T cell receptor activation and signaling,165,166 innate inflammatory response,167 inflammasome activation, regulation of type 1 interferon response,168 Toll-like receptor signaling,169 nuclear factor kappa-lightchain-enhancer of activated B cell (NF-κB) sigaling,170 programmed cell death (PCD-1/PD-L1) immune checkpoint,171 among many other processes. Dysregulated kinase activity has been shown to play a central role in virtually all diseases, including autoimmune disorders and cancers.172,173 As such, there has been a substantial recent effort to develop potent and selective kinase inhibitors.174 Given the high sequence and structural homology of many kinases, the production of selective inhibitors remains challenging. Quite surprisingly, over 200 members of the human protein kinase family have been found to harbor active site cysteines.20,175 The identification of covalent kinase inhibitors (CKIs) that target these residues is an exciting strategy to generate extremely potent and selective ATP competitive kinase inhibitors, which has been the subject to several comprehensive recent reviews.20,176–178 CKIs have received significant attention for

3. Opportunities for immunomodulation by targeting kinases, phosphatases, and cysteine proteases 3.1. Covalent kinase inhibitors (CKIs) Kinases are enzymes that catalyze the transfer of a phosphate group from a high energy donor molecule (typically ATP) to a substrate molecule. Kinases are classified based on the substrate that they phosphorylate, including metabolic kinases and protein kinases, which are 5

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

Table 1 Proteins and cysteines labeled by representative covalent compounds that have potential as immunomodulators. Structures of selected compounds that represent a wide range of protein targets and scaffolds are shown in Figs. 4–6. Target

Compound

Labeled Residue

Catalytic Nucleophile?

Pathway/process

Inhibitor Reference (s)

Afatanib, dacomitinib Neratinib Osimertinib Ibrutinib, zanubrutinib, acalabrutinib Hypothemycin E6201 PF-06651600 Compound 2 JNK-IN-8 MKK7-COV1 Tecfidera® Tecfidera® HNE

Cys797 Cys797, Cys805 Cys797 Cys481 Cys166 Cys207 Cys909 Cys174 Cys154 Cys218 Cys14 Cys17 Cys13 Cys22

No No No No No No No No No No No No No

Growth, proliferation, differentiation Growth, proliferation, differentiation Growth, proliferation, differentiation B cell receptor signaling MAPK/ERK MAPK/ERK JAK/STAT Cytokine and growth factor signaling MAPK/ERK MAPK/ERK T cell activation Cytokine Production Cell Cycle

122–127

4-Isoavenaciolide FIAsH-EDT2 N-(Quinolin-4-yl)prop-2-enamide 9,10-Phenanthrenediones α-Bromoacetophenones INCA1, 2 and 6

Cys124, Cys171 Cys333, Cys367 Cys333 Cys851 Cys435 Cys262

Yes No No No Yes No

MAPK/ERK Cytokine and inhibitory receptor Cytokine and inhibitory receptor T cell activation T cell activation, proliferation T cell activation

146

Multiple

Yes

Apoptosis, inflammation

92,95,96,153 96

Caspase-3 Caspase-8, Caspase-10

zVAD-FMK, Ac-DEVD-CHO, Ac-YVADAOMK DW3 Compound 7

Cys163 Cys360, Cys401

Yes Yes

154

Caspase-8

Compound 63-R

Cys360

Yes

Calpains, Cathepsins Cathepsin K

E-64 Balicatib, osimertinib

Multiple Cys139

Yes Yes

Cathepsin S Cathepsin Z

LHVS MGP140

Cys139 Cys92

Yes Yes

Apoptosis T cell activation, Inflammation, Apoptosis T cell activation, Inflammation, Apoptosis Inflammation, antigen maturation Bone matrix mineralization, inflammation Antigen presentation, T cell selection Immune cell activation, migration and maturation

Kinases EGFR EGFR, HER2 EGFR T790M BTK MAPK1/ERK2 MAP2K1/MEK1 JAK3 TAK1 JNK MKK7 PKCΘ IRAK4 ZAK (MLTK) Phosphatases VHR/DUSP3 SHP2/PTPN11 SHP2/PTPN11 CD45 SHP1/PTPN6 Calcineurin A/PPP3CC Proteases Caspases

their therapeutic efficacy in the treatment of several cancers.179 Despite the clear immune-relevance of many of the targeted kinases, the immunotherapeutic potential of CKIs remains to be fully realized. As, to our knowledge, all CKIs identified to-date target protein kinases, we will limit our discussion here to electrophilic compounds that target protein kinases, focusing particularly on those kinases with immunomodulatory activity. Selected CKIs are shown in Table 1 and Fig. 4. To learn more about recent advances in the development of metabolic kinase inhibitors, we would direct readers to the following recent articles.180–182 The irreversible nature of CKIs offers several key advantages when compared to non-covalent kinase inhibitors. Covalent modification of kinase cysteine residues has been shown to efficiently block kinase activity in an ATP competitive manner, achieving near complete target occupancy and sustained inhibition. Cysteine labeling also provides an additional selectivity filter not available for reversible inhibitors. Michael acceptors are the electrophile of choice for most CKIs20, which can likely be ascribed to their generally attenuated proteome-wide reactivity.16 While most CKIs have been produced by structure guided retrofitting of non-covalent lead compounds with Michael acceptors,129,135 recent studies indicate the synergy between covalent fragment screening and CKI discovery.97 Highlighting the clinical utility of CKIs, to date six CKIs (afatinib, neratinib, osimertinib, dacomitinib, ibrutinib, and acalabrutinib) have received FDA approval for the treatment of several cancers (Fig. 4).178,183,184 Afatinib, neratinib, osimertinib and dacomitinib target Cys797 in the epidermal growth factor receptor (EGFR) kinase and are FDA approved for the treatment of non-small cell lung cancers.20,126,134,185 Neratinib is also approved to label Cys805 of human epidermal growth

128 129,130 131–134 135–139 140 141 142 143 144 145 62 74 73

147 148 149,150 151 152

16 16 155 156–158 159,160 161

factor receptor 2 (HER2), and clinical data indicates that the other EGFR inhibitors will likely also have efficacy at labeling HER2.131,132,186 Ibrutinib labels the conserved Cys481 of Bruton's tyrosine kinase (BTK)135,136 and is approved to treat cancers (chronic lymphocytic leukemia, mantle cell lymphoma, Waldenström's macroglobulinemia) and autoimmune disorders (graft-versus-host disease).187–190 Acquired resistance is one limitation of CKIs that can be addressed by second generation inhibitors. For example, second generation BTK inhibitors, both reversible191,192 and irreversible (e.g. acalabrutinib and zanubrutib),137–139,193 have since been developed to treat tumors with acquired resistance to Ibrutinib.137–139,194,195 One frequent ibrutinib resistance mechanism is the C481S mutation in BTK, which leads to poor patient prognosis.196 Several studies have successfully targeted C481S BTK, using targeted protein degradation197 or conventional non-covalent inhibitors.198 Osimertinib is approved for the treatment of cancers that harbor the T790M afatinib-resistant mutant form of EGFR,133 and osimertinib has recently supplanted afatinib as first-line treatment of EGFR mutated lung cancers, including those that don’t harbor T790M. While generally well-tolerated, off-target affects have been detected for some CKIs. Chemoproteomic studies recently revealed that osimertinib accumulates in lysosomes upon longterm exposure, leading to accumulation of several cathepsin off-targets,68 and afatinib was shown to have a significant number of covalent off-targets at higher concentrations.69 These studies indicate the importance of in cell and in vivo studies for all covalent inhibitors, including chemoproteomic studies that can accurately report the potency and selectivity of lead compounds. The application of CKIs to alter immune cell signaling is an obvious extension of the recent explosion of potent and selective CKIs. In fact, as 6

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

Fig. 4. Structures of selected covalent kinase inhibitors (CKIs) and their respective protein targets, which are shown in parentheses. Electrophilic groups are shown in red.

we alluded to above, CKIs that inhibit BTK are already being harnessed for their immunomodulatory potential in the clinic. BTK acts downstream of the B cell receptor and plays a critical role in B cell maturation and activation.199 Mutations in BTK cause the primary immunodeficiency C-linked agammaglobulinemia.200 BTK inhibition can, somewhat counterintuitively, also have immunostimulatory activity, causing a restoration of T cell-dependent anti-tumor immune responses in pancreas ductal adenocarcinoma (PDA), a particularly devasting form of cancer.201 In contrast with the clear functions of BTK in B cells, the link between EGFR and human immunity is less well established. While FDA-approved EGFR inhibitors, most notably afatinib, are usually associated with the treatment of lung cancer, the well-accepted contributions of EGFR to the mitogen-activated protein kinases and extracellular signal-regulated kinases (MAPK/ERK) signal transduction pathways implicate EGFR in activation of innate and adaptive immune cells.202–204 As EGFR activity has been linked to increased expression of PD1 and PD-L1 and immune evasion, EGFR inhibition has also been identified as an attractive strategy to improve the antigenicity of EGFR positive tumors, particularly non-small cell lung cancers.205,206 Ongoing randomized clinical trials are testing the efficacy of treatment regimens that combine EGFR inhibitors with immunotherapy.207 In addition to targeting EGFR, multiple other kinases in the MAPK/ ERK signaling pathway have been targeted by CKIs. The natural product hypothemycin, a resorcyclic acid lactone, was shown to label Cys166 in human ERK2.140 A hypothemycin analogue, E6201 (targeting MEK),141 was reported in 2009 as a potential treatment for inflammatory diseases such as psoriasis. However, as Cys166 is highly conserved across 46 of

the known protein kinases, hypothemycin has since been demonstrated to be a relatively promiscuous multi-kinase inhibitor.140 Janus kinase 3 (JAK3) is another receptor tyrosine kinase (RTK) that is intimately linked to immune cell activation, which also features a ligandable cysteine. JAK3 mutations produce severe combined immunodeficiency syndrome.208 Because of this human phenotype, JAK3 was prioritized as a target for immunosuppressive therapies to prevent organ transplant rejection as well as autoimmune disorders, such as psoriasis and rheumatoid arthritis. Recently, several reversible JAK3 inhibitors, “Jakinibs,” have become available in the clinic.209 JAK3 has a conserved active site cysteine, Cys909, which can be labeled by CKIs, including cyanoacrylamide-containing covalent reversible compounds210 and the covalent clinical candidate PF-06651600, which was found to exhibit far greater selectivity for JAK3 when compared with related non-covalent inhibitors.142 Other related kinases, including JAK1, JAK2, and TYK2, also harbor conserved redox sensitive cysteines.211 Given the JAK3 precedent, these kinases will likely also be amenable to labeling by covalent probes. CKIs can also inactivate kinases required for NF-κB activation, including transforming growth factor β-activated kinase 1 (TAK1), c-JunN-terminal kinase (JNK), and zeta-chain-associated protein kinase (ZAP70). TAK1 phosphorylation activates both the IκB kinase (IKK) complex, JNK, and p38, which results in NF-κB nuclear translocation and transcription of a pro-inflammatory, pro-proliferative, and prosurvival genes.212 Genetic data indicates that TAK1 is a potential target for the treatment of inflammatory disorders, including cancers.213,214 Structure -guided development of TAK1 inhibitors identified 7

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

irreversible, cysteine-reactive inhibitors that share structural similarities to the reversible inhibitor Imatinib.143 Another example that exploits an imatinib fragment coupled with an electrophilic trap is JNKIN-8, a JNK1, 2, and 3 inhibitor.144 Inhibitors like JNK-IN-8 selectively block the phosphorylation of the JNK substrate, c-Jun, and analogs of these inhibitors have been used in live-cell imaging of JNK.215 More recently, covalent docking in silico led to the development of a cysteinereactive 3-arylindazole derivative (MKK7-COV1) as a potent and selective inhibitor of MAP kinase kinase 7 (MKK7).145 MKK7 is one of two kinases involved in upstream activation of JNK. ZAP70, a master regulator of T cell activation, has been targeted by sulfone-containing heterocyclic cysteine electrophiles.216,217 These compounds label cysteines in the tandem-SH2 domains of ZAP70, domains that are responsible for interactions with the immunoreceptor tyrosine-based activation motifs (ITAMs).218 Labeling of these non-active site residues blocks the kinase ITAM interactions that are essential for T cell activation. That these ZAP70 inhibitors label a non-active site cysteine indicates an opportunity to produce CKIs that target cysteines in exosites as an exciting new strategy to alter kinase function, discussed in greater detail below. In addition to the kinases detailed above, proteome-wide competitive isoTOP-ABPP profiling studies indicated that many additional kinases should be accessible to CKIs, including sterile alpha motif and leucine zipper containing kinase (ZAK), interleukin-1 receptor-associated kinase 1 (IRAK1), serine-threonine-protein kinase 38 (STK38), cyclin-dependent kinases 2 and 5 (CDK2 and CDK5), mitogen-activated protein kinases MAP2K3, MAP2K4, MAP2K7, and nuclear factor kappaB kinase subunit beta (IKBKB), among others.16,62 An intriguing observation from these chemoproteomic studies is that a sizeable number (∼50%) of ligandable cysteines in kinases are not found in the kinase active site, but rather in exosites, which could serve as putative allosteric sites and be coopted for selective kinase inhibition. Such sites could function in a similar manner to the cysteines in the SH2 domain of ZAP70, discussed above. One such example of non-active site cysteines is a pair of cysteines (Cys14 and Cys17) found in protein kinase C theta (PKCΘ). Modification of these residues by the multiple sclerosis drug dimethyl fumarate (Tecfidera®) was shown to block the activation of primary T cells.62 Labeling of Cys13 in the IL-R1-associated kinase (IRAK4) by dimethyl fumarate has been shown to disrupt the IRAK4MYD88 protein complex, which inhibits T cell cytokine production.74 Although significant progress has been made in the optimization of potent and selective CKIs, many kinases still lack suitable chemical probes. Lipid derived electrophiles, including the oxidative stress product 4-hydroxynonenal (HNE), can modify cysteines and can also serve as a good indicator of druggability. HNE has been shown to label cysteines in AKT2,219 AKT3,220 and ZAK,73 indicating a possible axis between oxidative stress and blockade of kinase function. While there has been a recent renaissance in kinase inhibitors, the biological functions and therapeutic potential of many human protein kinases remain poorly annotated.221 As some kinases have proven to be much more promiscuous binders, the production of compounds that target the inactive forms of kinases is an alternative strategy to produce selective kinase inhibitors.222 However, complicating matters, some kinases bind to inhibitors more promiscuously in their inactive conformation.223 Therefore, at least in some cases, it may be more challenging to generate selective inhibitors for inactive kinases. Furthermore, as current phenotypic data, obtained both using genetic approaches and using chemical inhibitors, indicate that most kinase inhibitors will have immunosuppressive activity, it remains to be seen whether CKIs, in addition to ibrutinib, can in some cases have immunostimulatory activity. The development of selective phosphatase inhibitors, discussed in the next section, that can act in a manner orthogonal to CKIs, is another exciting opportunity to produce immunostimulatory compounds.

3.2. Progress towards covalent phosphatase inhibitors Protein phosphatases, which catalyze the dynamic and reversible process of protein dephosphorylation, are essential mediators of intracellular signal transduction that act as the physiological counterparts to protein kinases.224,225 Protein phosphatases can be divided into three broad categories, protein tyrosine phosphatases (PTPs), protein serine/ threonine phosphatases, and dual specificity phosphatases.224,226–228 As the majority of immunomodulatory phosphatases identified to date are PTPs, this review will focus on providing an overview of available covalent probes that label PTPs. Where relevant, we will also highlight several non-tyrosine phosphatases that are particularly amenable for covalent probe development. Nearly half of the 107 PTPs encoded in the human genome229 are expressed in human immune cells.230 Many PTPs have been characterized as essential modulators of immune cell function, including Src homology 1 domain protein-tyrosine phosphatase (SHP1 or PTPN6), 231–233 SHP2 (also referred to as PTPN11),45,46 the receptor-type tyrosine-protein phosphatase C (PTPRC or CD45),234–236 calcineurin (PP2B),237 human vaccinia H1‐related phosphatase (VHR),238,239 among many others. For a comprehensive survey of immune-relevant phosphatases, see the recent review by Mustelin and coworkers.230 In contrast with the immunostimulatory roles of most kinases, the catalytic activity of most PTPs has been linked to immunosuppressive or dampening activity, with several activation-promoting exceptions (e.g. CD45, SHP2, and LMPTP). This essentiality of PTPs in dampening signaling indicates an opportunity for the development of PTP inhibitors as immunostimulatory agents for use in cancer immunotherapy. Considerable recent effort has been dedicated to the pursuit of optimized chemical probes that can selectively inactivate individual phosphatases (See Table 1 and Fig. 5 for examples).240–243 Covalent probes are an obvious choice for PTP inhibitors.241–243 PTP active sites almost universally feature a highly conserved catalytic cysteine found in a signature sequence motif C(X)5R.228 The intrinsic reactivity of these cysteine thiols is high, as hydrogen bonds with surrounding residues perturb the pKa of the cysteine thiol (calculated pKa values ∼4.5–5.5).228 Despite this reactivity, the PTP catalytic cysteines remain stubbornly undruggable.228 Reasons for the absence of potent and selective phosphatase inhibitors include the high sequence and structural homology of phosphatase active sites and the polar and electronegative nature of the pTyr substrate and substrate mimetics founds in many inhibitor pharmacophores, which limit the cellular uptake and in vivo activity of such compounds. In isoTOP-ABPP cysteine reactivity profiling studies, none of the PTP catalytic thiols were identified as hyperreactive.70 In contrast, and quite unexpectedly, several of the conserved non-catalytic cysteine residues found in kinase active sites, as described above, were identified as hyper-reactive, including residues in MAP2K7, NEK9, CDC42BPB, STK24 and CAMK4, among others. Thus, the relatively modest intrinsic reactivity of their catalytic thiols, as measured by chemoproteomics studies of whole cell lysates, may to some extent rationalize the observed undruggability of phosphatases. Despite these challenges, the catalytic cysteines of several immunomodulatory phosphatases have been labeled by covalent molecules. CD45 negatively modulates T-cell receptor mediated signaling through the dynamic phosphorylation status of Src-family protein tyrosine kinases and their substrates.244 A series of 9,10-phenanthrenediones were reported to inhibit CD45 and suppress T cell proliferation. While these compounds were initially proposed to act by labeling the catalytic cysteine (Cys851), forming a hemithioacetal intermediate,149 a more recent study indicated that oxidation of the Cys781 was the likely mechanism of action.150 The electrophilic natural product pulchellalactam has also been shown to inhibit CD45, although the specific mode of action remains unclear.245 In contrast with the proactivation function of CD45, human vaccinia H1‐related phosphatase (VHR), a dual‐specific phosphatase (DSP) acts as a negative regulator of T cell signaling through dephosphorylation of ERK, JNK, and 8

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

Fig. 5. Structures of selected phosphatase inhibitors and their respective protein targets, which are shown in parentheses. Electrophilic groups are shown in red.

STAT5.238,239 The natural product 4-isoavenaciolide, which is an exomethylene containing bicyclic bislactone, inhibits VHR, likely through labeling of the catalytic cysteine (Cys124).146 VHR activity is also blocked by non-specific electrophiles, such as acrolein246 and seleninate.247 SHP1 and SHP2 are two closely related tyrosine phosphatases that are both attractive potential immunotherapeutic targets. SHP1 expression is restricted to hematopoietic cells, spanning all lineages and maturation states,248 whereas SHP2 is ubiquitously expressed.249 Genetic data indicate that SHP1 inhibition should cause increased immune cell activation and decreased sensitivity to regulatory T cells.231–233 However, the utility of available SHP1 inhibitors has been limited by offtarget inhibition of SHP2 and a lack of in vivo activity. Several covalent compounds that label the catalytic cysteine of SHP1 have been identified, including halo-acetophenone derivatives250 and the tyrosine phosphatase inhibitor-1 (TPI-1), which features a quinone electrophile.251 Both of these compounds are, however, unlikely to demonstrate appreciable specificity for SHP1. As with SHP1, SHP2 inhibition is also thought to enhance T cell activation, in part by blocking SHP2 catalyzed dephosphorylation of the co-receptor CD28, which has the effect of suppressing T cell function.252 Complicating matters, a recent study by Rota and coworkers indicates that SHP2 is not required for PD1 signaling, indicating possible redundant or compensatory mechanisms.253 Electrophilic dietary isothiocyanates also inactivate SHP2 through covalent reversible modification of the catalytic cysteine.254 Both the organic biarsenical compound FlAsH-EDT2255 and the small drug-like aminoisoquinoline acrylamide148 block SHP2 phosphatase activity in a cysteine-dependent manner by labeling Cys333, a noncatalytic “backdoor” cysteine. As will be discussed more below, allosteric inhibitors, including compounds that label non-catalytic cysteines in phosphatases is a potential strategy to generate potent and selective phosphatase inhibitors. Covalent compounds that target phosphatase exosites have shown promise as selective phosphatase inhibitors. Quinone-containing electrophiles have been found to allosterically inhibit the phosphatase calcineurin by irreversibly labeling Cys266, modification of which blocks interactions with calcineurin’s substrate NFAT (nuclear factor of activated T cells).152 As calcineurin is required for T cell activation and adhesion,256 this inhibition will have an immunosuppressive effect. Non-catalytic phosphatase cysteines are well represented in our recent isoTOP-ABPP ligandability profiling studies, which includes residues in protein phosphatases such as the threonine-protein phosphatase 2A (PPP2R5D), dual specificity protein phosphatase 12 (DUSP12), serine/ threonine-protein phosphatase 4 regulatory subunit 2 (PPP4R2), and

metabolic phosphatases such as the lysophosphatidic acid phosphatase type 6 (ACP6), phosphoglycolate phosphatase (PGP), and fructose-2,6bisphosphatase (TIGAR).16 The functions of most of these cysteines, and whether covalent probes that label these residues will alter protein function, remains to be determined. Many cysteines in phosphatases are sensitive to redox regulation,257,258 and this redox sensitivity may provide another clue to identify druggable phosphatase cysteines. PTP catalytic cysteines are also known to be highly sensitive to reversible oxidation, including sulfenylation and formation of a reversible sulfenyl amide species.259–261 The development of nucleophilic probes that react with Cys Ox-PTMs—analogous to probes that react with the sulfenylated form of EGFR 40,262–264—may prove widely applicable to altering phosphatase function. In fact, sulfone stabilized carbanions have been shown react with the active-site sulfenyl amide in PTP1B.265 Redox sensitive “backdoor” cysteines have also been identified that regulate phosphatase activity, including residues in SHP1, SHP2,266 CDC25,267 PTEN,268 and low molecular weight phosphotyrosine-protein phosphatases (LMWPTP).269 Compounds that label these backdoor cysteines may provide a new avenue towards the production of selective phosphatase inhibitors. 3.3. Cysteine proteases Cysteine proteases are enzymes that use a catalytic cysteine nucleophile to hydrolyze peptide bonds via a covalent tetrahedral intermediate. By incorporating electrophiles that covalently label the catalytic cysteine thiol, mechanism-based inhibitors and activity-based chemical probes can be designed for cysteine proteases.270 Among the 143 cysteine proteases encoded by the human genome,271 the 12 caspases,272 11 cathepsins,273 and 11 calpains274 are all well studied in the context of immunity and therefore will serve as the focus of this section. Caspase-, calpain-, and cathepsin-mediated proteolysis are all reported to contribute to both innate and adaptive immune cell function, including key contributions to inflammation, activation, proliferation and death.275–278 While proteases and their cognate substrates cleaved during apoptosis have been studied extensively, those involved in most non-apoptotic processes remain less well annotated.279–281 Reasons for this gap include the genetic essentiality of several key proteases, lack of suitable rodent models, high homology and seeming redundancy of many cysteine proteases, multistep proteolytic maturation process for most proteases, and a relative absence of selective inhibitors. Here we will provide a survey of compound classes available to inactivate members of the caspase, calpain and cathepsin families and potential 9

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

caspases,307,308 is an attractive strategy to circumvent the promiscuity of active caspase inhibitors. Pro-caspase inhibitors offer the added potential advantage of more fully preventing downstream caspase activation. We recently identified a chemical probe (Fig. 6, Compound 7) that inactivates the pro-forms of both caspase-8 and caspase-10, which share a high degree of sequence homology. Through medicinal chemistry, we also identified a structurally related probe that selectively labels pro-caspase-8 with no off-target labeling observed for pro-caspase-10 (Fig. 6, Compound 63-R)16 As these compounds irreversibly label the catalytic cysteine residues of caspase-8 and -10, their inhibitory activity is maintained even after intermolecular proteolytic cleavage by other caspases. Inhibition of caspase-8 in immortalized T cells (Jurkat cells) was sufficient to block FasL-induced apoptosis, whereas blockade of both caspase-8 and -10 was required to prevent apoptosis in primary human T cells. These data support that caspase-10 can initiate apoptosis in primary lymphocytes, and together with human genetic data295 indicate that selective chemical inactivation of caspase-10 should efficiently elevate circulating populations of cytotoxic T cells. However, pro-caspase-10 selective inhibitors have so far proven elusive to generate. In addition to caspase-8 and -10, our previous studies indicate that caspase-2, -4, and -5 are also labeled by small electrophilic fragments.16 This labeling also likely occurs in the zymogen form of these proteases, indicating future opportunities for the production of more selective probes that target additional pro-caspases. Calpains are neutral cytosolic cysteine proteases with many substrates, including G-proteins and cytoskeletal proteins. Calpains modulate inflammation, including contributions to T cell activation309 and promotion of neutrophil recruitment.310 The exquisite sensitivity of these calcium-dependent proteases to intracellular calcium levels, is consistent with a model where the calpain-calpastatin (combined protease and inhibitor) system is a feedback mechanism for T cell activation,309 which is mediated by a rapid influx of Ca2+ after TCR stimulation.311–313 Calpains have also been linked to promotion of neutrophil recruitment.310 Despite their intriguing biology, there is a near complete absence of isoform-selective calpain inhibitors. Widely used calpain inhibitors include the nonspecific protease inhibitors EDTA, E-64, leupeptin, and MG-132.314 Among the several non-peptidic calpain inhibitors that are reported include the natural product aclacinomycin A,315 carboxamides,316 chalcones,317 and mercaptoacrylate analogues.318 However, the proteome-wide reactivity profile of these electrophile classes remains to be assessed and noncalpain off-targets are likely. Our studies indicate that several calpain cysteines should be amenable to probes that label non-catalytic cysteines including Cys640 in calpain-2 and Cys197 in calpain-7.16 While the consequences of probe labeling at these cysteines remains to be determined, their absence in other family members indicates the likely selective nature of compounds tailored to react specifically with these residues. Cysteine cathepsins are another highly homologous family of cysteine proteases that, while most well characterized for their essential role in lysosomal protein degradation, have also been implicated as indispensable for immune cell function. Cathepsins regulate antigen presentation by professional antigen presenting cells (APCs, such as dendritic cells, B cells, macrophages and thymic epithelial cells) in two ways, first by stepwise proteolytic trimming of the invariant chain of the MHC-II (cathepsin S)160,319–321 and second by proteolysis of antigens into antigenic peptides (e.g. Cathepsin B, L and H).322,323 Selective cathepsin inhibition may offer a strategy to manipulate antigen presentation by APCs. Cathepsins are highly expressed in tumor associated macrophages (TAMs), which have both pro-inflammatory anti-tumorigenic activity (M[LPS] or M[LPS + IFNγ] classically activated macrophages) and, conversely, the ability to promote cancer progression (M [IL-4] suppressive macrophages).324 Joyce and colleagues demonstrated that TAM protease activity of cathepsin B and cathepsin S enhances pancreatic tumor growth, angiogenesis, and invasion.325 Cathepsin S has been implicated in T cell selection in the thymus.326

applications to address many of these limitations and inform and alter the functions of cysteine proteases in immune cell activation, inflammation, differentiation, and death. Caspase-mediated proteolysis plays a central role in regulation of immune cell function. Inflammatory caspases, including caspase-1, -4, -5, -8, and -12 in humans (caspase-1, -11, -12 in mice), contribute to inflammation by proteolytic activation of cytokines, including IL-1B and IL-18.282–284 Pro-inflammatory caspase activation and subsequent cytokine cleavage occurs upon assembly of the inflammasome, which is a large (700 kDa) multiprotein complex that orchestrates the host response to endogenous and exogenous insults, particularly pathogen infections.285–287 There are several different inflammasomes, including NLRP3, NLRP1, AIM2, and NLRC4, which sense different pathogenassociated molecular patterns (PAMPs) or danger-associated molecular patterns (DAMPs).288 A key outcome of inflammasome activation is pyroptosis,289 a specialized form of inflammatory cell death that is mechanistically distinct from programmed cell death (apoptosis). Dysregulated inflammasome activity has been linked to autoimmune disorders,286 and altering the activity of the NLRP3 inflammasome, for example by inhibiting caspase-1, is under investigation for the treatment of a variety of inflammatory diseases, including Alzheimer's disease, Parkinson's disease, and type 2 diabetes, among others.287,289–291 Apoptotic caspases, including initiator caspases (caspase-2, -8, -9 and -10) and executioner caspases (caspase-3, -6, and -7), are most well characterized for their essential roles in initiating and executing apoptosis.272,292 Paradoxical to its pro-apoptotic activity, caspase-8, but not caspase-10, is also essential for lymphocyte activation293–296 and differentiation.297 Deletion of caspase-8 also reduces the activation of dendritic cells, B cells, and macrophages,298–300 indicating that chemical inhibition of caspase-8 will likely have wide ranging immunosuppressive consequences. Caspase-3, most well known as an executioner of apoptosis, is also linked to T cell activation301 and proliferation.302 It remains unclear whether non-apoptotic caspase mediated processes are driven by cleavage of distinct substrates or by dosedependent cleavage (e.g. a small amount of proteolysis is pro-proliferative, while more cleavage is pro-apoptotic). Studies aimed at distinguishing between these two models would benefit from selective inhibitors of individual caspases. Their essential roles in immune cell function implicate the caspases as exciting candidate proteins for the development of small-molecule immunotherapeutic agents. Irreversible and reversible peptide probes have been extensively investigated as inhibitors of caspases (See Table 1 and Fig. 6 for examples of irreversible inhibitors). These compounds include tetrapeptide aldehydes (e.g. DEVD-CHO),96,153 chloromethyl ketones and fluoromethyl ketones (e.g. YVAD-cfk94 and z-VADfmk,95,96 and acyloxymethyl ketones (AOMK),92,303 which inhibit both pro-inflammatory and pro-apoptotic caspases non-specifically. Wolan and coworkers recently developed selective caspase-3 probes, including DW3, that combine several non-natural amino acids with a thiophene acyloxymethyl ketone electrophile to obtain 40-fold selectivity for caspase-3 over caspase-8154. While peptide inhibitors can, in some cases, inhibit caspases with picomolar affinity, as with most peptidebased inhibitors, these compounds all suffer limited in cell activity and require a huge excess of compound (∼100 µM in many cases) to achieve full caspase inhibition. Caspases are maintained in cells as inactive proenzymes (also called zymogens), which are activated by cleavage after certain aspartate residues. This process is tightly regulated, as aberrant proteolysis can have devastating results, including the proliferation of cancers, neurodegeneration, and immunological disorders.272,304–306 Historically, peptide- and small molecule-based caspase inhibitor discovery campaigns have focused almost exclusively on compounds that target the active caspase conformers. Given the high homology of active caspases, these inhibitors are hampered by narrow selectivity profiles, including both caspase- and non-caspase off-targets. The design of inhibitors that target pro-caspases, which are structurally distinct from active 10

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

Fig. 6. Structures of selected protease inhibitors and activity-based probes and their respective protein targets, which are shown in parentheses. Electrophilic groups are shown in red.

Cathepsin Z (also known as cathepsin X) exopeptidase activity has been linked to activation, migration, and maturation of immune cells (e.g. macrophages, microglia, and dendritic cells),327 and to neuroinflammation.328 While cathepsins C and K are most well known for their essential contributions to regulation of bone density, both can also alter immune cell function. Inactivating mutations in cathepsin C cause defects in neutrophil and natural killer cells, which contribute to the periodontal disease and hyperkeratosis of Papillon-Lefèvre and HaimMunk syndromes.329,330 Cathepsin K activity has also been detected in epithelioid and multinucleated giant cells of macrophage origin,331 and cathepsin K proteolytic activity in macrophages has also been linked to formation of atherosclerotic plaques.332,333 Compared with other classes of cysteine proteases, more progress has been made in the optimization of selective cathepsin inhibitors and activity-based probes, and as such, understanding the trials and tribulations associated with cathepsin inhibition will likely inform the production of inhibitors for other protease classes. As with caspase inhibitors, peptides and peptidomimetics comprise most cathepsin inhibitors, including irreversible, reversible covalent, and reversible

non-covalent compounds. Cathepsin inhibitors have been reviewed previously by Palermo,334 Siklos,335 and Hernandez,336 and ABPs by Verdoes337 and Fonović.338 A large number of electrophiles have been incorporated into cathepsin inhibitors (Fig. 3 and Fig. 6), including vinyl sulfones such as morpholinurea-leucine-homophenylalanine-vinylsulfone-phenyl (LHVS; cathepsin B, S, and L),159,160 peptide aldehydes (cathepsin K, B, and L),339–341 fluoromethyl ketones (cathepsin B) 342 chloromethyl ketones (cathepsin B),343 diazomethyl ketones (cathepsin B, C),344 peptidyl allyl sulfones (cathepsin B and L),345,346 acyloxymethyl ketones (cathepsin B),347 peptidyl sulfonium salts (cathepsin B),346 ketoheterocycles (cathepsin K),348 minimalized fragment arylsulfonyl oxiranes (cathepsin B),349 α-ketoamides (cathepsin S),350 and even latent electrophiles, such as alkynes (cathepsin K).107 The general lack of selectivity of many of these compounds remains problematic. A potential strategy to produce cathepsin inhibitors with improved selectivity is to generate compounds that label non-catalytic cathepsin cysteines. For example, our recent studies indicate that noncatalytic cysteines in cathepsin Z and cathepsin B can be labeled by covalent α-chloroacetamide fragment electrophiles.16 11

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

Production of cathepsin K inhibitors for the treatment of decreased bone density, which until several high profile recent clinical trial failures, was a highly competitive area that has been extensively reviewed.351–353 Cathepsin K inhibitors provide a lesson in the development of extremely potent cysteine protease inhibitors (picomolar Ki,app), including the importance of identification of in vivo off-target activity. Most covalent cathepsin K inhibitors are covalent-reversible compounds, including compounds that feature poorly electrophilic ketones354 and the electrophilic nitriles found in the clinical candidates balicatib and odanacatib.156–158 Although balicatib afforded excellent selectivity in vitro, the compound’s dibasic nature resulted in substantial lysosomal compound accumulation and much greater potency in cells compared with lysates, resulting in off-target labeling of several additional cathepsins, decreased efficacy, and eventual failure in clinical trials due to cutaneous lesions.355,356 Recent studies have revealed similar lysosomotropic behavior of the CKI osimertinib, which labels several cathepsins, including cathepsin C, L1, F, and H.68 The fatty acid amide hydrolase (FAAH) inhibitor BIA 10-2474, another high profile compound that failed clinical trials, was also shown to have increased potency and off-target activity in cells when compared to lysates.357 In contrast, the cathepsin K inhibitor odanacatib is not lysosomotropic.355,358 While clinical trials of odanacatib indicated that inhibition of cathepsin K increased bone matrix mineralization,352 odanacatib ultimately failed phase III trials due to on-target increased risk of stroke.359 Cathepsin K inhibition has also been associated with blockade of inflammation in periodontal diseases360 and rheumatoid arthritis,361 through its contributions to Toll-like receptor 9 signaling and induction of T helper 17 cells.362 Activity-based probes of cathepsin activity have seen widespread adoption. Paulick and coworkers have developed acyloxymethyl ketone-containing peptide-based activity-based probes, including MGP140, that label cathepsin Z with some off-target activity towards cathepsins B, S, and L.161 Notably, a sequence-related epoxide-containing probe, MGP151, exhibited near complete specificity for cathepsin B, indicating that choice of electrophile can prove critical for optimization of the potency and selectivity of covalent probes. Cathepsin probes have shown promise for clinical applications. Tetrapeptide fluorescent cathepsin ABPs have been applied to selectively image the activities of cathepsin L in breast cancer.363 Using ABPs, cathepsin K activity has been linked to atherosclerotic plaque rupture,364,365 which may, in part, rationalize the association between cathepsin K inhibition and stroke. Using a cathepsin-ABP, Joyce and colleagues identified cathepsins B, S, L, and Z as having increased protease activity in solid tumors, contributing to tumor growth, angiogenesis, and invasion,366 supporting future opportunities for cathepsin inhibitors in cancer treatments as well as immunotherapy.

from anti-inflammatory cysteines, which will prove critical for identification of suitable immunomodulatory targets; design of selective covalent phosphatase inhibitors, which may benefit from identification of targetable cysteines in regulatory domains; and concerns regarding cytotoxicity, lack of specificity, or unexpected in vivo promiscuity of covalent compounds, which can be mitigated using ABPP to evaluate in vivo selectivity. Looking beyond kinases, phosphatases, and proteases, we anticipate that the chemistries and methods identified here will translate to other immune-relevant classes of proteins. Many other protein families also feature targetable cysteine residues. While renowned as tough-to-drug, protein interaction domains, including SH2 and phosphotyrosine binding (PTB) domains are central to cell signaling cascades and also contain multiple highly conserved cysteine residues,367,368 including several identified by isoTOP-ABPP screens.16 Enzymes that remodel post-translational modifications (PTMs), including methyl transferases and protein arginine deiminases (PADs) harbor non-catalytic and catalytic cysteines residues, respectively.70,369–371 While there has been considerable success in the development of PAD inhibitors, methyltransferases have remained relatively intractable to covalent probes, which may be due to limited in cell target engagement.372 While the protein arginine methyl transferases (PRMTs) have proven challenging to inhibit, a recent study indicates that alpha-chloroamidines, which were first pioneered as PAD inhibitors, can be converted into PRMT inhibitors.373 Proteins required for ubiquitylation, including E1, E2, and E3 ubiquitin ligases, are another cysteine-rich16 protein class that offer significant opportunities for cysteine reactive compounds, both through direct inhibition374 and in targeted protein degradation. Recent studies have indicated that bifunction covalent compounds can coopt the activities of several E3s, including RNF4,375 RNF114,376 and DCAF16,377 for targeted protein degradation. Proteins involved in epigenetic regulation of transcription, such as bromodomains are also amenable to covalent probes, including labeling at cysteine378 and methionine379 residues. While transcription factors (TFs) have long been considered the holy grail of undruggable proteins, many TFs contain targetable cysteines16 and, at least for some TFs, modification of these cysteines is known to alter transcription. For example, irreversible labeling of a cysteine in the protein YAP1, which is an essential component of the hippo pathway, blocks an interaction with TEAD4.380 YAP has also been implicated in CD4+T cell effector function and it is intriguing to speculate whether targeting Cys367 could enhance proliferation and IL-2 production, consistent with shRNA knockdown of YAP.381 Looking more broadly, the combination of chemoproteomic methods with high-throughput and phenotypic assays likely will uncover new cysteines that can alter immune cell function. For example, we can envision studies where primary immune cells are first treated with libraries of electrophilic compounds and then assayed for a cellular phenotype of interest, such as cytokine production, cellular activation, or proliferation. The protein targets of hit compounds would then be deconvolved using chemoproteomics and lead compound optimized using standard medicinal chemistry methods. Such studies will benefit from judicious use of structurally matched inactive control compounds to facilitate deconvolution of targets and mechanisms of action. Looking beyond cysteine, opportunities to target other amino acids, such as lysine,382 histidine,383 and methionine,379 will likely yield additional future opportunities for covalent immunomodulation, though the chemistries required to label non-thiol amino acid side chains remain less developed.

3.4. Conclusions and future prospects The intersection between covalent probes, activity-based protein profiling, and immunology is an exciting area for synergy. With the redox sensitivity of many immune cell types and the seemingly opposing immunosuppressive and stimulatory effects of ROS, compounds that label cysteine residues are an obvious choice as immunomodulators. By reviewing available covalent compounds that label three protein classes required for immune cell function (kinases, phosphatases, and proteases), we have identified opportunities and challenges to produce covalent immunomodulators. Exciting opportunities include: the potential to repurpose available covalent inhibitors such as potent and selective CKIs and some promising cathepsin and caspase probes as immunomodulators; the design of compounds that label non-catalytic cysteines or inactive/precursor enzymes as a generalizable strategy to identify more potent and selective covalent probes; the identification of compounds with greater structural diversity, both in the electrophile and binding group, which will likely afford improved selectivity. Challenges include: distinguishing pro-

Declaration of Competing Interest The authors declare no conflicts of interest. Acknowledgments This work was supported by startup funds from the David Geffen 12

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

School of Medicine at UCLA and in part by seed funds from the UCLA Jonsson Comprehensive Cancer Center.

[30]

Appendix A. Supplementary data

[31]

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmc.2019.05.050.

[32] [33]

References

[34] [35]

[1] Swaika A, et al. Current state of anti-PD-L1 and anti-PD-1 agents in cancer therapy. Mol Immunol. 2015;67:4–17. https://doi.org/10.1016/j.molimm.2015.02.009. [2] Kontzias A, et al. Jakinibs: a new class of kinase inhibitors in cancer and autoimmune disease. Curr Opin Pharmacol. 2012;12:464–470. https://doi.org/10. 1016/j.coph.2012.06.008. [3] van Kasteren SI, et al. Creating molecules that modulate immune responses. Nat Rev Chem. 2018;2:184–193. https://doi.org/10.1038/s41570-018-0023-9. [4] Klapper JA, et al. High-dose interleukin-2 for the treatment of metastatic renal cell carcinoma : a retrospective analysis of response and survival in patients treated in the surgery branch at the National Cancer Institute between 1986 and 2006. Cancer. 2008;113:293–301. https://doi.org/10.1002/cncr.23552. [5] Smith FO, et al. Treatment of metastatic melanoma using interleukin-2 alone or in conjunction with vaccines. Clin Cancer Res. 2008;14:5610–5618. https://doi.org/ 10.1158/1078-0432.ccr-08-0116. [6] Hodi FS, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Eng J Med. 2010;363:711–723. https://doi.org/10.1056/ NEJMoa1003466. [7] Zou W. Immunosuppressive networks in the tumour environment and their therapeutic relevance. Nat Rev Cancer. 2005;5:263–274. https://doi.org/10.1038/ nrc1586. [8] Zou W, Chen L. Inhibitory B7-family molecules in the tumour microenvironment. Nat Rev Immunol. 2008;8:467–477. https://doi.org/10.1038/nri2326. [9] Alsaab HO, et al. PD-1 and PD-L1 checkpoint signaling inhibition for cancer immunotherapy: mechanism, combinations, and clinical outcome. 561 561 Front Pharmacol. 2017;8. https://doi.org/10.3389/fphar.2017.00561. [10] Szostak B, et al. CTLA4 antagonists in phase I and phase II clinical trials, current status and future perspectives for cancer therapy. Exp Opin Investig Drugs. 2019;28:149–159. https://doi.org/10.1080/13543784.2019.1559297. [11] Dyck L, Mills KHG. Immune checkpoints and their inhibition in cancer and infectious diseases. Eur J Immunol. 2017;47:765–779. https://doi.org/10.1002/eji. 201646875. [12] Backus KM. Applications of reactive cysteine profiling. Curr Top Microbiol Immunol. 2019;420:375–417. https://doi.org/10.1007/82_2018_120. [13] Singh J, et al. The resurgence of covalent drugs. Nat Rev Drug Discov. 2011;10:307–317. https://doi.org/10.1038/nrd3410. [14] Bauer RA. Covalent inhibitors in drug discovery: from accidental discoveries to avoided liabilities and designed therapies. Drug Discovery Today. 2015;20:1061–1073. https://doi.org/10.1016/j.drudis.2015.05.005. [15] Johnson DS, et al. Strategies for discovering and derisking covalent, irreversible enzyme inhibitors. Future Med Chem. 2010;2:949–964. [16] Backus KM, et al. Proteome-wide covalent ligand discovery in native biological systems. Nature. 2016;534:570–574. https://doi.org/10.1038/nature18002. [17] Yun CH, et al. The T790M mutation in EGFR kinase causes drug resistance by increasing the affinity for ATP. Proc Natl Acad Sci USA. 2008;105:2070–2075. https://doi.org/10.1073/pnas.0709662105. [18] Singh J, et al. Targeted covalent drugs of the kinase family. Curr Opin Chem Biol. 2010;14:475–480. https://doi.org/10.1016/j.cbpa.2010.06.168. [19] Bar-Peled L, et al. Chemical proteomics identifies druggable vulnerabilities in a genetically defined cancer. Cell. 2017;171:696–709. https://doi.org/10.1016/j. cell.2017.08.051 e623. [20] Liu Q, et al. Developing irreversible inhibitors of the protein kinase cysteinome. Chem Biol. 2013;20:146–159. https://doi.org/10.1016/j.chembiol.2012.12.006. [21] Lapalombella R, et al. Selective inhibitors of nuclear export show that CRM1/ XPO1 is a target in chronic lymphocytic leukemia. Blood. 2012;120:4621–4634. https://doi.org/10.1182/blood-2012-05-429506. [22] Janes MR, et al. Targeting KRAS mutant cancers with a covalent G12C-Specific Inhibitor. Cell. 2018;172:578–589. https://doi.org/10.1016/j.cell.2018.01.006. [23] Bar-Peled L, et al. Chemical proteomics identifies druggable vulnerabilities in a genetically defined cancer. Cell. 2017;171:696–709. https://doi.org/10.1016/j. cell.2017.08.051. [24] Browne CM, et al. A Chemoproteomic strategy for direct and proteome-wide covalent inhibitor target-site identification. J Am Chem Soc. 2018;141:191–203. https://doi.org/10.1021/jacs.8b07911. [25] Padgett LE, et al. The role of reactive oxygen species and proinflammatory cytokines in type 1 diabetes pathogenesis. Ann N Y Acad Sci. 2013;1281:16–35. https://doi.org/10.1111/j.1749-6632.2012.06826.x. [26] Hultqvist M, et al. The protective role of ROS in autoimmune disease. Trends Immunol. 2009;30:201–208. https://doi.org/10.1016/j.it.2009.03.004. [27] Belikov AV, et al. T cells and reactive oxygen species. J Biomed Sci. 2015;22:85. https://doi.org/10.1186/s12929-015-0194-3. [28] Sena LA, et al. Mitochondria are required for antigen-specific T cell activation through reactive oxygen species signaling. Immunity. 2013;38:225–236. [29] Angelini G, et al. Antigen-presenting dendritic cells provide the reducing

[36] [37] [38] [39]

[40] [41] [42] [43] [44] [45] [46] [47] [48] [49] [50] [51] [52] [53] [54] [55] [56]

[57] [58] [59] [60]

13

extracellular microenvironment required for T lymphocyte activation. J Proceed Natl Acad Sci. 2002;99:1491–1496. https://doi.org/10.1073/pnas.022630299 %. Hildeman DA, et al. T cell apoptosis and reactive oxygen species. J Clin Investig. 2003;111:575–581. https://doi.org/10.1172/JCI18007. Chen X, et al. Reactive oxygen species regulate t cell immune response in the tumor microenvironment. Oxid Med Cell Longevity. 2016;2016:10. https://doi.org/ 10.1155/2016/1580967. Fang FC. Antimicrobial reactive oxygen and nitrogen species: concepts and controversies. Nat Rev Microbiol. 2004;2:820. https://doi.org/10.1038/nrmicro1004. Fang FC. Antimicrobial actions of reactive oxygen species. mBio 2. 2011:e00141–00111. https://doi.org/10.1128/mBio.00141-11. Segal BH, et al. Genetic, biochemical, and clinical features of chronic granulomatous disease. Medicine (Baltimore). 2000;79:170–200. Zhang Y, et al. ROS play a critical role in the differentiation of alternatively activated macrophages and the occurrence of tumor-associated macrophages. Cell Res. 2013;23:898–914. https://doi.org/10.1038/cr.2013.75. West AP, et al. TLR signalling augments macrophage bactericidal activity through mitochondrial ROS. Nature. 2011;472:476. https://doi.org/10.1038/nature09973. Mills EL, et al. Succinate dehydrogenase supports metabolic repurposing of mitochondria to drive inflammatory macrophages. Cell. 2016;167:457–470.e413. https://doi.org/10.1016/j.cell.2016.08.064. Xu Q, et al. NADPH oxidases are essential for macrophage differentiation. J Biol Chem. 2016;291:20030–20041. https://doi.org/10.1074/jbc.M116.731216. Cemerski S, et al. Oxidative-stress-induced T lymphocyte hyporesponsiveness is caused by structural modification rather than proteasomal degradation of crucial TCR signaling molecules. Eur J Immunol. 2003;33:2178–2185. https://doi.org/10. 1002/eji.200323898. Paulsen CE, et al. Peroxide-dependent sulfenylation of the EGFR catalytic site enhances kinase activity. Nat Chem Biol. 2011;8:57–64. https://doi.org/10.1038/ nchembio.736. Nelson KJ, et al. H2O2 oxidation of cysteine residues in c-Jun N-terminal kinase 2 (JNK2) contributes to redox regulation in human articular chondrocytes. J Biol Chem. 2018:pp. https://doi.org/10.1074/jbc.RA118.004613. Wani R, et al. Isoform-specific regulation of Akt by PDGF-induced reactive oxygen species. Proc Natl Acad Sci USA. 2011;108:10550–10555. https://doi.org/10. 1073/pnas.1011665108. Visperas PR, et al. Modification by covalent reaction or oxidation of cysteine residues in the tandem-SH2 domains of ZAP-70 and Syk can block phosphopeptide binding. Biochem J. 2015;465:149–161. https://doi.org/10.1042/BJ20140793. Reynaert NL, et al. Dynamic redox control of NF-kappaB through glutaredoxinregulated S-glutathionylation of inhibitory kappaB kinase beta. PNAS. 2006;103:13086–13091. https://doi.org/10.1073/pnas.0603290103. Salmond RJ, Alexander DR. SHP2 forecast for the immune system: fog gradually clearing. TRENDS in Immunol. 2006;27. https://doi.org/10.1016/j.it.2006.01.007. Tartaglia M, et al. Somatic mutations in PTPN11 in juvenile myelomonocytic leukemia, myelodysplastic syndromes and acute myeloid leukemia. Nat Genet. 2003;34:148–150. https://doi.org/10.1038/ng1156. Kwon J, et al. Reversible oxidation and inactivation of the tumor suppressor PTEN in cells stimulated with peptide growth factors. PNAS. 2004;101:16419. https:// doi.org/10.1073/pnas.0407396101. Sivaramakrishnan S, et al. A chemical model for redox regulation of protein tyrosine phosphatase 1B (PTP1B) activity. J Am Chem Soc. 2005;127:10830–10831. https://doi.org/10.1021/ja052599e. Zhou H, et al. The biological buffer bicarbonate/CO2 potentiates H2O2-mediated inactivation of protein tyrosine phosphatases. J Am Chem Soc. 2011;133:15803–15805. https://doi.org/10.1021/ja2077137. Morgan MJ, Liu Z-g. Crosstalk of reactive oxygen species and NF-κB signaling. Cell Res. 2011;21:103–115. https://doi.org/10.1038/cr.2010.178. Scotcher J, et al. Redox regulation of tumour suppressor protein p53: identification of the sites of hydrogen peroxide oxidation and glutathionylation. Chem Sci. 2013;4:1257–1269. https://doi.org/10.1039/C2SC21702C. de Keizer PL, et al. Forkhead box o as a sensor, mediator, and regulator of redox signaling. Antioxid Redox Signal. 2011;14:1093–1106. https://doi.org/10.1089/ ars.2010.3403. Deshmukh P, et al. The Keap1-Nrf2 pathway: promising therapeutic target to counteract ROS-mediated damage in cancers and neurodegenerative diseases. Biophys Rev. 2017;9:41–56. https://doi.org/10.1007/s12551-016-0244-4. Borutaite V, Brown GC. Caspases are reversibly inactivated by hydrogen peroxide. FEBS Lett. 2001;500:114–118. https://doi.org/10.1016/S0014-5793(01)02593-5. Zhou Y, et al. Chemoproteomic strategy to quantitatively monitor transnitrosation uncovers functionally relevant S-nitrosation sites on cathepsin D and HADH2. Cell Chem Biol. 2016;23:727–737. https://doi.org/10.1016/j.chembiol.2016.05.008. Fu X, et al. Hypochlorous acid oxygenates the cysteine switch domain of promatrilysin (MMP-7). A mechanism for matrix metalloproteinase activation and atherosclerotic plaque rupture by myeloperoxidase. J Biol Chem. 2001;276:41279–41287. https://doi.org/10.1074/jbc.M106958200. Mills EL, et al. Itaconate is an anti-inflammatory metabolite that activates Nrf2 via alkylation of KEAP1. Nature. 2018;556:113–117. https://doi.org/10.1038/ nature25986. Briggs KJ, et al. Paracrine induction of HIF by glutamate in breast cancer: EglN1 senses cysteine. Cell. 2016;166:126–139. https://doi.org/10.1016/j.cell.2016.05. 042. Paulsen CE, Carroll KS. Cysteine-mediated redox signaling: chemistry, biology, and tools for discovery. Chem Rev. 2013;113:4633–4679. https://doi.org/10.1021/ cr300163e. Nathan C, Cunningham-Bussel A. Beyond oxidative stress: an immunologist's guide

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

[61] [62] [63] [64] [65] [66] [67] [68] [69] [70] [71] [72] [73] [74] [75] [76] [77] [78] [79] [80] [81] [82] [83] [84] [85] [86] [87] [88] [89]

[90]

to reactive oxygen species. Nat Rev Immunol. 2013;13:349–361. https://doi.org/ 10.1038/nri3423. Franchina DG, et al. Reactive oxygen species: involvement in T cell signaling and metabolism. Trends Immunol. 2018;39:489–502. https://doi.org/10.1016/j.it. 2018.01.005. Blewett M, et al. Chemical proteomic map of dimethylfumarate-sensitive cysteine in primary human T cells. Sci Signal. 2016;9:rs10. https://doi.org/10.1126/ scisignal.aaf7694. Kornberg MD, et al. Dimethyl fumarate targets GAPDH and aerobic glycolysis to modulate immunity. Science. 2018;360:449–453. https://doi.org/10.1126/ science.aan4665. Bambouskova M, et al. Electrophilic properties of itaconate and derivatives regulate the IkappaBzeta-ATF3 inflammatory axis. Nature. 2018;556:501–504. https://doi.org/10.1038/s41586-018-0052-z. Bose S, et al. Curcumin and tumor immune-editing: resurrecting the immune system. 6 6 Cell Division. 2015;10. https://doi.org/10.1186/s13008-015-0012-z. Antony S, et al. Immunomodulatory activity of curcumin. Immunol Invest. 1999;28:291–303. https://doi.org/10.3109/08820139909062263. Moellering RE, Cravatt BF. How chemoproteomics can enable drug discovery and development. Chem Biol. 2012;19:11–22. https://doi.org/10.1016/j.chembiol. 2012.01.001. Niessen S, et al. .Proteome-wide map of targets of T790M-EGFR-directed covalent inhibitors. Cell Chem Biol. 2017;24:1388–1400.e1387. https://doi.org/10.1016/j. chembiol.2017.08.017. Lanning BR, et al. A road map to evaluate the proteome-wide selectivity of covalent kinase inhibitors. Nat Chem Biol. 2014;10:760–767. https://doi.org/10. 1038/nchembio.1582. Weerapana E, et al. Quantitative reactivity profiling predicts functional cysteines in proteomes. Nature. 2010;468:790–795. https://doi.org/10.1038/nature09472. Kolb HC, et al. Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed Engl. 2001;40:2004–2021. https://doi.org/10.1002/ 1521-3773(20010601)40:11%3C2004::AID-ANIE2004%3E3.0.CO;2-5. Speers AE, Cravatt BF. Profiling enzyme activities in vivo using click chemistry methods. Chem Biol. 2004;11:535–546. https://doi.org/10.1016/j.chembiol.2004. 03.012. Wang C, et al. A chemoproteomic platform to quantitatively map targets of lipidderived electrophiles. Nat Meth. 2014;11:79–85. https://doi.org/10.1038/nmeth. 2759. Zaro BW, et al. Dimethyl fumarate disrupts human innate immune signaling by targeting the IRAK4–MyD88 complex. J Immunol. 2019:ji1801627. https://doi. org/10.4049/jimmunol.1801627%. Qian Y, et al. An isotopically tagged azobenzene-based cleavable linker for quantitative proteomics. ChemBioChem. 2013;14:1410–1414. https://doi.org/10. 1002/cbic.201300396. Abo M, et al. Isotopically-labeled iodoacetamide-alkyne probes for quantitative cysteine-reactivity profiling. Mol Pharm. 2017:pp. https://doi.org/10.1021/acs. molpharmaceut.7b00832. Pierce A, et al. Eight-channel iTRAQ enables comparison of the activity of six leukemogenic tyrosine kinases. Mol Cell Proteom. 2008;7:853–863. https://doi. org/10.1074/mcp.M700251-MCP200. Abegg D, et al. Proteome-wide profiling of targets of cysteine reactive small molecules by using ethynyl benziodoxolone reagents. Angew Chem Int Ed Engl. 2015;54:10852–10857. https://doi.org/10.1002/anie.201505641. Tian C, et al. Multiplexed Thiol reactivity profiling for target discovery of electrophilic natural products. Cell Chem Biol. 2017;24:1416–1427. https://doi.org/ 10.1016/j.chembiol.2017.08.022 e1415. Tsiatsiani L, Heck AJR. Proteomics beyond trypsin. FEBS J. 2015;282:2612–2626. https://doi.org/10.1111/febs.13287. Liigand P, et al. Influence of the amino acid composition on the ionization efficiencies of small peptides. J Mass Spectrom. 2019;54:481–487. https://doi.org/10. 1002/jms.4348. Fonslow BR, et al. Improvements in proteomic metrics of low abundance proteins through proteome equalization using ProteoMiner prior to MudPIT. J Proteome Res. 2011;10:3690–3700. https://doi.org/10.1021/pr200304u. Ong SE, Mann M. Stable isotope labeling by amino acids in cell culture for quantitative proteomics. Methods Mol Biol. 2007;359:37–52. https://doi.org/10. 1007/978-1-59745-255-7_3. Ficarro SB, et al. Leveraging gas-phase fragmentation pathways for improved identification and selective detection of targets modified by covalent probes. Anal Chem. 2016;88:12248–12254. https://doi.org/10.1021/acs.analchem.6b03394. Unsal-Kacmaz K, et al. The interaction of PKN3 with RhoC promotes malignant growth. Mol Oncol. 2012;6:284–298. https://doi.org/10.1016/j.molonc.2011.12. 001. Mukai H, et al. PKN3 is the major regulator of angiogenesis and tumor metastasis in mice. 18979 18979 Sci Rep. 2016;6. https://doi.org/10.1038/srep18979. Weerapana E, et al. Disparate proteome reactivity profiles of carbon electrophiles. Nat Chem Biol. 2008;4:405–407. https://doi.org/10.1038/nchembio.91. Flanagan ME, et al. Chemical and computational methods for the characterization of covalent reactive groups for the prospective design of irreversible inhibitors. J Med Chem. 2014;57:10072–10079. https://doi.org/10.1021/jm501412a. Ward RA, et al. Structure- and reactivity-based development of covalent inhibitors of the activating and gatekeeper mutant forms of the epidermal growth factor receptor (EGFR). J Med Chem. 2013;56:7025–7048. https://doi.org/10.1021/ jm400822z. Allimuthu D, Adams DJ. 2-Chloropropionamide as a low-reactivity electrophile for irreversible small-molecule probe identification. ACS Chem Biol.

2017;12:2124–2131. https://doi.org/10.1021/acschembio.7b00424. [91] Zaro BW, et al. Metabolically labile fumarate esters impart kinetic selectivity to irreversible inhibitors. J Am Chem Soc. 2016;138:15841–15844. https://doi.org/ 10.1021/jacs.6b10589. [92] Thornberry NA, et al. Inactivation of interleukin-1 beta converting enzyme by peptide (acyloxy)methyl ketones. Biochemistry. 1994;33:3934–3940. [93] Pliura DH, et al. Comparative behaviour of calpain and cathepsin B toward peptidyl acyloxymethyl ketones, sulphonium methyl ketones and other potential inhibitors of cysteine proteinases. Biochem J. 1992;288:759–762. https://doi.org/10. 1042/bj2880759. [94] Enari M, et al. Involvement of an ICE-like protease in Fas-mediated apoptosis. Nature. 1995;375:78–81. https://doi.org/10.1038/375078a0. [95] Slee EA, et al. Benzyloxycarbonyl-Val-Ala-Asp (OMe) fluoromethylketone (ZVAD.FMK) inhibits apoptosis by blocking the processing of CPP32. Biochem J. 1996;315(Pt 1):21–24. https://doi.org/10.1042/bj3150021. [96] Garcia-Calvo M, et al. Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem. 1998;273:32608–32613. https://doi.org/10. 1074/jbc.273.49.32608. [97] Miller RM, Taunton J. Targeting protein kinases with selective and semipromiscuous covalent inhibitors. Methods Enzymol. 2014;548:93–116. https://doi.org/10. 1016/B978-0-12-397918-6.00004-5. [98] Banerjee R, et al. 1,3,5-Triazine as a modular scaffold for covalent inhibitors with streamlined target identification. J Am Chem Soc. 2013;135:2497–2500. https:// doi.org/10.1021/ja400427e. [99] Shannon DA, et al. Investigating the proteome reactivity and selectivity of aryl halides. J Am Chem Soc. 2014;136:3330–3333. https://doi.org/10.1021/ ja4116204. [100] Spokoyny AM, et al. A perfluoroaryl-cysteine S(N)Ar chemistry approach to unprotected peptide stapling. J Am Chem Soc. 2013;135:5946–5949. https://doi.org/ 10.1021/ja400119t. [101] Wang C, et al. Chemoproteomics-enabled discovery of a potent and selective inhibitor of the DNA repair protein MGMT. Angew Chem Int Ed Engl. 2016;55:2911–2915. https://doi.org/10.1002/anie.201511301. [102] Knuckley B, et al. Substrate specificity and kinetic studies of PADs 1, 3, and 4 identify potent and selective inhibitors of protein arginine deiminase 3. Biochemistry. 2010;49:4852–4863. https://doi.org/10.1021/bi100363t. [103] Muth A, et al. Development of a selective inhibitor of protein arginine deiminase 2. J Med Chem. 2017;60:3198–3211. https://doi.org/10.1021/acs.jmedchem. 7b00274. [104] Ekkebus R, et al. On terminal alkynes that can react with active-site cysteine nucleophiles in proteases. J Am Chem Soc. 2013;135:2867–2870. https://doi.org/ 10.1021/ja309802n. [105] Sommer S, et al. Covalent inhibition of SUMO and ubiquitin-specific cysteine proteases by an in situ thiol-alkyne addition. Bioorg Med Chem. 2013;21:2511–2517. https://doi.org/10.1016/j.bmc.2013.02.039. [106] Krysiak JM, et al. Activity-based probes for studying the activity of flavin-dependent oxidases and for the protein target profiling of monoamine oxidase inhibitors. Angew Chem Int Ed Engl. 2012;51:7035–7040. https://doi.org/10.1002/ anie.201201955. [107] Mons E, et al. The alkyne moiety as a latent electrophile in irreversible covalent small molecule inhibitors of cathepsin K. J Am Chem Soc. 2019:pp. https://doi. org/10.1021/jacs.8b11027. [108] Kumar S, et al. Activity-based probes for protein tyrosine phosphatases. Proc Natl Acad Sci USA. 2004;101:7943–7948. https://doi.org/10.1073/pnas.0402323101. [109] Berteotti A, et al. Predicting the reactivity of nitrile-carrying compounds with cysteine: a combined computational and experimental study. ACS Med Chem Lett. 2014;5:501–505. https://doi.org/10.1021/ml400489b. [110] Sinha S, et al. Electrophilicity of pyridazine-3-carbonitrile, pyrimidine-2-carbonitrile, and pyridine-carbonitrile derivatives: a chemical model to describe the formation of thiazoline derivatives in human liver microsomes. Chem Res Toxicol. 2014;27:2052–2061. https://doi.org/10.1021/tx500256j. [111] Bradshaw JM, et al. Prolonged and tunable residence time using reversible covalent kinase inhibitors. Nat Chem Biol. 2015;11:525–531. https://doi.org/10. 1038/nchembio.1817. [112] Krishnan S, et al. Design of reversible, cysteine-targeted Michael acceptors guided by kinetic and computational analysis. J Am Chem Soc. 2014;136:12624–12630. https://doi.org/10.1021/ja505194w. [113] Miller RM, et al. Electrophilic fragment-based design of reversible covalent kinase inhibitors. J Am Chem Soc. 2013;135:5298–5301. https://doi.org/10.1021/ ja401221b. [114] Palmer JT, et al. Keto-1,3,4-oxadiazoles as cathepsin K inhibitors. Bioorg Med Chem Lett. 2006;16:2909–2914. https://doi.org/10.1016/j.bmcl.2006.03.001. [115] Shindo N, et al. Selective and reversible modification of kinase cysteines with chlorofluoroacetamides. Nat Chem Biol. 2019. https://doi.org/10.1038/s41589018-0204-3. [116] Ábrányi-Balogh P, et al. A road map for prioritizing warheads for cysteine targeting covalent inhibitors. Eur J Med Chem. 2018;160:94–107. https://doi.org/10. 1016/j.ejmech.2018.10.010. [117] Gehringer M, Laufer SA. Emerging and re-emerging warheads for targeted covalent inhibitors: applications in medicinal chemistry and chemical biology. J Med Chem. 2018:pp. https://doi.org/10.1021/acs.jmedchem.8b01153. [118] Resnick E, et al. Rapid covalent-probe discovery by electrophile-fragment screening. J Am Chem Soc. 2019. https://doi.org/10.1021/jacs.9b02822. [119] Keeley A, et al. Design and characterization of a heterocyclic electrophilic fragment library for the discovery of cysteine-targeted covalent inhibitors. MedChemComm. 2019;10:263–267. https://doi.org/10.1039/C8MD00327K.

14

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al. [120] Kathman SG, et al. A fragment-based method to discover irreversible covalent inhibitors of cysteine proteases. J Med Chem. 2014;57:4969–4974. https://doi. org/10.1021/jm500345q. [121] Cee VJ, et al. Systematic study of the glutathione (GSH) reactivity of N-arylacrylamides: 1. Effects of aryl substitution. J Med Chem. 2015;58:9171–9178. https://doi.org/10.1021/acs.jmedchem.5b01018. [122] Li D, et al. BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models. Oncogene. 2008;27:4702–4711. https://doi.org/ 10.1038/onc.2008.109. [123] Carmi C, et al. Epidermal growth factor receptor irreversible inhibitors: chemical exploration of the cysteine-trap portion. Mini Rev Med Chem. 2011;11:1019–1030. https://doi.org/10.2174/138955711797247725. [124] Solca F, et al. Target binding properties and cellular activity of afatinib (BIBW 2992), an irreversible ErbB family blocker. J Pharmacol Exp Ther. 2012;343:342–350. https://doi.org/10.1124/jpet.112.197756. [125] Engelman JA, et al. PF00299804, an irreversible pan-ERBB inhibitor, is effective in lung cancer models with EGFR and ERBB2 mutations that are resistant to gefitinib. Cancer Res. 2007;67:11924–11932. https://doi.org/10.1158/0008-5472.CAN-071885. [126] Gonzales AJ, et al. Antitumor activity and pharmacokinetic properties of PF00299804, a second-generation irreversible pan-erbB receptor tyrosine kinase inhibitor. Mol Cancer Ther. 2008;7:1880–1889. https://doi.org/10.1158/15357163.MCT-07-2232. [127] Reed, JE, Smaill, JB. Comprehensive Accounts of Pharmaceutical Research and Development: From Discovery to Late-Stage Process Development Volume 1 Vol. 1239 ACS Symposium Series Ch. 8, (American Chemical Society, 2016), pp. 207–233, doi: 10.1021/bk-2016-1239.ch008. [128] Tsou HR, et al. Optimization of 6,7-disubstituted-4-(arylamino)quinoline-3-carbonitriles as orally active, irreversible inhibitors of human epidermal growth factor receptor-2 kinase activity. J Med Chem. 2005;48:1107–1131. https://doi. org/10.1021/jm040159c. [129] Wissner A, et al. Synthesis and structure-activity relationships of 6,7-disubstituted 4-anilinoquinoline-3-carbonitriles. The design of an orally active, irreversible inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) and the human epidermal growth factor receptor-2 (HER-2). J Med Chem. 2003;46:49–63. https://doi.org/10.1021/jm020241c. [130] Minami Y, et al. The major lung cancer-derived mutants of ERBB2 are oncogenic and are associated with sensitivity to the irreversible EGFR/ERBB2 inhibitor HKI272. Oncogene. 2007;26:5023. https://doi.org/10.1038/sj.onc.1210292. [131] Zhou W, et al. Novel mutant-selective EGFR kinase inhibitors against EGFR T790M. Nature. 2009;462:1070–1074. https://doi.org/10.1038/nature08622. [132] Yver A. Osimertinib (AZD9291)-a science-driven, collaborative approach to rapid drug design and development. Ann Oncol. 2016;27:1165–1170. https://doi.org/ 10.1093/annonc/mdw129. [133] Finlay MRV, et al. Discovery of a potent and selective EGFR inhibitor (AZD9291) of both sensitizing and T790M resistance mutations that spares the wild type form of the receptor. J Med Chem. 2014;57:8249–8267. https://doi.org/10.1021/ jm500973a. [134] Jänne PA, et al. AZD9291 in EGFR inhibitor-resistant non-small-cell lung cancer. N Engl J Med. 2015;372:1689–1699. https://doi.org/10.1056/NEJMoa1411817. [135] Pan Z, et al. Discovery of selective irreversible inhibitors for Bruton’s tyrosine kinase. ChemMedChem. 2007;2:58–61. https://doi.org/10.1002/cmdc. 200600221. [136] Honigberg LA, et al. The Bruton tyrosine kinase inhibitor PCI-32765 blocks B-cell activation and is efficacious in models of autoimmune disease and B-cell malignancy. Proc Natl Acad Sci USA. 2010;107:13075–13080. https://doi.org/10.1073/ pnas.1004594107. [137] Tam C, et al. The BTK inhibitor, Bgb-3111, Is safe, tolerable, and highly active in patients with relapsed/refractory B-cell malignancies: initial report of a phase 1 first-in-human trial. Blood. 2015;126:832. [138] Tam CS, et al. Twice daily dosing with the highly specific BTK inhibitor, Bgb-3111, achieves complete and continuous BTK occupancy in lymph nodes, and is associated with durable responses in patients (pts) with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL). Blood. 2016:128642. [139] Wu J, et al. Acalabrutinib (ACP-196): a selective second-generation BTK inhibitor. J Hematol Oncol. 2016;9:21. https://doi.org/10.1186/s13045-016-0250-9. [140] Schirmer A, et al. Targeted covalent inactivation of protein kinases by reorcylic acid lactone polyketides. Proc Natl Acad Sci. 2006;103:4234–4239. https://doi. org/10.1073/pnas.0600445103. [141] Goto M, et al. E6201 [(3S,4R,5Z,8S,9S,11E)-14-(Ethylamino)-8, 9,16-trihydroxy3,4-dimethyl-3,4,9,19-tetrahydro-1H-2-benzoxacyclotetradecine- 1,7(8H)-dione], a novel kinase inhibitor of mitogen-activated protein kinase/extracellular signalregulated kinase kinase (MEK). J Pharmacol Exp Therapeut. 2009;331:485–495. https://doi.org/10.1124/jpet.109.156554. [142] Telliez JB, et al. Discovery of a JAK3-selective inhibitor: functional differentiation of JAK3-selective inhibition over pan-JAK or JAK1-selective inhibition. ACS Chem Biol. 2016;11:3442–3451. https://doi.org/10.1021/acschembio.6b00677. [143] Tan L, et al. Structure-guided development of covalent TAK1 inhibitors. Bioorg Med Chem. 2017;25:838–846. https://doi.org/10.1016/j.bmc.2016.11.035. [144] Zhang T, et al. Discovery of potent and selective covalent inhibitors of JNK. Chem Biol. 2012;19:140–154. https://doi.org/10.1016/j.chembiol.2011.11.010. [145] Shraga A, et al. Covalent docking identifies a potent and selective MKK7 inhibitor. Cell Chem Biol. 2019;26:98–108.e105. https://doi.org/10.1016/j.chembiol.2018. 10.011. [146] Ueda K, et al. 4-Isoavenaciolide covalently binds and inhibits VHR, a dual-specificity phosphatase. FEBS Lett. 2002;525:48–52. https://doi.org/10.1016/S0014-

5793(02)03065-X. [147] Rask-Andersen M, et al. The druggable genome: evaluation of drug targets in clinical trials suggests major shifts in molecular class and indication. Annu Rev Pharmacol Toxicol. 2014;54:9–26. https://doi.org/10.1146/annurev-pharmtox011613-135943. [148] Marsh-Armstrong B, et al. The allosteric site on SHP2’s protein tyrosine phosphatase domain is targetable with druglike small molecules. ACS Omega. 2018;3:15763–15770. https://doi.org/10.1021/acsomega.8b02200. [149] Urbanek RA, et al. Potent reversible inhibitors of the protein tyrosine phosphatase CD45. J Med Chem. 2001;44:1777–1793. https://doi.org/10.1021/jm000447i. [150] Wang Q, et al. Catalytic inactivation of protein tyrosine phosphatase CD45 and protein tyrosine phosphatase 1B by polyaromatic quinones. Biochemistry. 2004;43:4294–4303. https://doi.org/10.1021/bi035986e. [151] Arabaci G, et al. alpha-bromoacetophenone derivatives as neutral protein tyrosine phosphatase inhibitors: structure-Activity relationship. Bioorg Med Chem Lett. 2002;12:3047–3050. https://doi.org/10.1016/S0960-894X(02)00681-9. [152] Kang S, et al. Inhibition of the calcineurin-NFAT interaction by small organic molecules reflects binding at an allosteric site. J Biol Chem. 2005;280:37698–37706. https://doi.org/10.1074/jbc.M502247200. [153] Nicholson DW, et al. Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis. Nature. 1995;376:37–43. https://doi.org/10. 1038/376037a0. [154] Vickers CJ, et al. Selective detection and inhibition of active caspase-3 in cells with optimized peptides. J Am Chem Soc. 2013;135:12869–12876. https://doi.org/10. 1021/ja406399r. [155] Barrett AJ, et al. L-trans-Epoxysuccinyl-leucylamido(4-guanidino)butane (E-64) and its analogues as inhibitors of cysteine proteinases including cathepsins B, H and L. Biochem J. 1982;201:189–198. https://doi.org/10.1042/bj2010189. [156] Jerome C, et al. .Balicatib, a cathepsin K inhibitor, stimulates periosteal bone formation in monkeys. Osteoporos Int. 2011;22:3001–3011. https://doi.org/10. 1007/s00198-011-1529-x. [157] Gauthier JY, et al. The discovery of odanacatib (MK-0822), a selective inhibitor of cathepsin K. Bioorg Med Chem Lett. 2008;18:923–928. https://doi.org/10.1016/j. bmcl.2007.12.047. [158] Palmer JT, et al. Design and Synthesis of tri-ring P3 benzamide-containing aminonitriles as potent, selective, orally effective inhibitors of cathepsin K. J Med Chem. 2005;48:7520–7534. https://doi.org/10.1021/jm058198r. [159] Palmer JT, et al. Vinyl sulfones as mechanism-based cysteine protease inhibitors. J Med Chem. 1995;38:3193–3196. https://doi.org/10.1021/jm00017a002. [160] Riese RJ, et al. Essential role for cathepsin S in MHC class II-associated invariant chain processing and peptide loading. Immunity. 1996;4:357–366. https://doi.org/ 10.1016/S1074-7613(00)80249-6. [161] Paulick MG, Bogyo M. Development of activity-based probes for cathepsin X. ACS Chem Biol. 2011;6:563–572. https://doi.org/10.1021/cb100392r. [162] Wymann MP, Schneiter R. Lipid signalling in disease. Nat Rev Mol Cell Biol. 2008;9:162. https://doi.org/10.1038/nrm2335. [163] Bhullar KS, et al. Kinase-targeted cancer therapies: progress, challenges and future directions. 48 48 Mol Cancer. 2018;17. https://doi.org/10.1186/s12943-0180804-2. [164] Duong-Ly KC, Peterson JR. The human kinome and kinase inhibition. Curr Prot Pharmacol. 2013:1–14. https://doi.org/10.1002/0471141755.ph0209s60. [165] Cantrell DA. T-cell antigen receptor signal transduction. Immunology. 2002;105:369–374. https://doi.org/10.1046/j.1365-2567.2002.01391.x. [166] Dal Porto JM, et al. B cell antigen receptor signaling 101. Mol Immunol. 2004;41:599–613. https://doi.org/10.1016/j.molimm.2004.04.008. [167] Arthur JSC, Ley SC. Mitogen-activated protein kinases in innate immunity. Nat Rev Immunol. 2013;13:679. https://doi.org/10.1038/nri3495. [168] Ivashkiv LB, Donlin LT. Regulation of type I interferon responses. Nat Rev Immunol. 2014;14:36–49. https://doi.org/10.1038/nri3581. [169] Kawasaki T, Kawai T. T. toll-like receptor signaling pathways. Front Immunol. 2014;5. https://doi.org/10.3389/fimmu.2014.00461. [170] Liu T, et al. NF-κB signaling in inflammation. Signal Trans Target Ther. 2017;2:17023. https://doi.org/10.1038/sigtrans.2017.23. [171] Bardhan K, et al. The PD1:PD-L1/2 Pathway from discovery to clinical implementation. Front Immunol. 2016;7. https://doi.org/10.3389/fimmu.2016. 00550. [172] Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature. 2001;411:355–365. https://doi.org/10.1038/35077225. [173] Cohen P. Immune diseases caused by mutations in kinases and components of the ubiquitin system. Nat Immunol. 2014;15:521–529. https://doi.org/10.1038/ni. 2892. [174] Ferguson FM, Gray NS. Kinase inhibitors: the road ahead. Nat Rev Drug Discovery. 2018;17:353. https://doi.org/10.1038/nrd.2018.21. [175] Zhang J, et al. Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer. 2009;9:28–39. https://doi.org/10.1038/nrc2559. [176] Chaikuad A, et al. The cysteinome of protein kinases as a target in drug development. Angew Chem Int Ed. 2018;57:4372–4385. https://doi.org/10.1002/anie. 201707875. [177] Barf T, Kaptein A. Irreversible protein kinase inhibitors: balancing the benefits and risks. J Med Chem. 2012;55:6243–6262. https://doi.org/10.1021/jm3003203. [178] Zhao Z, Bourne PE. Progress with covalent small-molecule kinase inhibitors. Drug Discovery Today. 2018;23:727–735. https://doi.org/10.1016/j.drudis.2018.01. 035. [179] Engel J, et al. Hope and disappointment: covalent inhibitors to overcome drug resistance in non-small cell lung cancer. ACS Med Chem Lett. 2015;7:2–5. https:// doi.org/10.1021/acsmedchemlett.5b00475.

15

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al. [180] McCloud RL, et al. Deconstructing lipid kinase inhibitors by chemical proteomics. Biochemistry. 2018;57:231–236. https://doi.org/10.1021/acs.biochem.7b00962. [181] Borne AL. Activity-Based Protein Profiling. Springer International Publishing; 2019:175–210. https://doi.org/10.1007/82_2018_124. [182] Stark A-K, et al. PI3K inhibitors in inflammation, autoimmunity and cancer. Curr Opin Pharmacol. 2015;23:82–91. https://doi.org/10.1016/j.coph.2015.05.017. [183] Shirley M. Dacomitinib: First Global Approval. Drugs. 2018;78:1947–1953. https://doi.org/10.1007/s40265-018-1028-x. [184] Markham A, Dhillon S. Acalabrutinib: first global approval. Drugs. 2018;78:139–145. https://doi.org/10.1007/s40265-017-0852-8. [185] Carmi C, et al. Clinical perspectives for irreversible tyrosine kinase inhibitors in cancer. Biochem Pharmacol. 2012;84:1388–1399. https://doi.org/10.1016/j.bcp. 2012.07.031. [186] Soria J-C, et al. Osimertinib in untreated EGFR-mutated advanced non–small-cell lung cancer. N Engl J Med. 2017;378:113–125. https://doi.org/10.1056/ NEJMoa1713137. [187] Ponader S, et al. The Bruton tyrosine kinase inhibitor PCI-32765 thwarts chronic lymphocytic leukemia cell survival and tissue homing in vitro and in vivo. Blood. 2012;119:1182–1189. https://doi.org/10.1182/blood-2011-10-386417. [188] Wilson WH, et al. Targeting B cell receptor signaling with ibrutinib in diffuse large B cell lymphoma. Nat Med. 2015;21:922. https://doi.org/10.1038/nm.3884. [189] Treon SP, et al. Ibrutinib is highly active as first line therapy in symptomatic waldenstrom's macroglobulinemia. Blood. 2017;130:2767. [190] Miklos D, et al. Ibrutinib for chronic graft-versus-host disease after failure of prior therapy. Blood. 2017;130:2243–2250. https://doi.org/10.1182/blood-2017-07793786. [191] Watterson SH, et al. Discovery of 6-fluoro-5-(R)-(3-(S)-(8-fluoro-1-methyl-2,4dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (BMS-986142): a reversible inhibitor of Bruton's Tyrosine Kinase (BTK) conformationally constrained by two locked atropisomers. J Med Chem. 2016;59:9173–9200. https://doi.org/10.1021/ acs.jmedchem.6b01088. [192] Crawford JJ, et al. Discovery of GDC-0853: a potent, selective, and noncovalent bruton's tyrosine kinase inhibitor in early clinical development. J Med Chem. 2018;61:2227–2245. https://doi.org/10.1021/acs.jmedchem.7b01712. [193] Tam CS, et al. A head-to-head Phase III study comparing zanubrutinib versus ibrutinib in patients with Waldenström macroglobulinemia. Future Oncol. 2018;14:2229–2237. https://doi.org/10.2217/fon-2018-0163. [194] Woyach JA, et al. Resistance mechanisms for the Bruton's Tyrosine kinase inhibitor ibrutinib. N Engl J Med. 2014;370:2286–2294. https://doi.org/10.1056/ NEJMoa1400029. [195] Furman RR, et al. Ibrutinib resistance in chronic lymphocytic leukemia. N Engl J Med. 2014;370:2352–2354. https://doi.org/10.1056/NEJMc1402716. [196] Martin P, et al. Postibrutinib outcomes in patients with mantle cell lymphoma. J Blood. 2016;127:1559–1563. https://doi.org/10.1182/blood-2015-10-673145 %. [197] Buhimschi AD, et al. Targeting the C481S ibrutinib-resistance mutation in bruton’s Tyrosine kinase using PROTAC-mediated degradation. Biochemistry. 2018;57:3564–3575. https://doi.org/10.1021/acs.biochem.8b00391. [198] Johnson AR, et al. Battling Btk mutants with noncovalent inhibitors that overcome Cys481 and Thr474 mutations. ACS Chem Biol. 2016;11:2897–2907. https://doi. org/10.1021/acschembio.6b00480. [199] Rawlings DJ, Witte ON. Bruton's tyrosine kinase is a key regulator in B-cell development. Immunol Rev. 1994;138:105–119. https://doi.org/10.1111/j.1600065X.1994.tb00849.x. [200] Satterthwaite AB, Witte ON. The role of Bruton’s tyrosine kinase in B-cell development and function: a genetic perspective. Immunol Rev. 2000;175:120–127. https://doi.org/10.1034/j.1600-065X.2000.017504.x. [201] Gunderson AJ, et al. Bruton tyrosine kinase-dependent immune cell cross-talk drives pancreas cancer. Cancer Discovery. 2016;6:270–285. https://doi.org/10. 1158/2159-8290.CD-15-0827. [202] Schlessinger J. Cell signalling by receptor tyrosine kinases. Cell. 2000;103:211–225. https://doi.org/10.1016/j.cell.2010.06.011. [203] Marmor MD, et al. Signal transduction and oncogenesis by ErbB/HER receptors. Int J Radiat Oncol Biol Phys. 2004;58:903–913. https://doi.org/10.1016/j.ijrobp. 2003.06.002. [204] Minutti CM, et al. Epidermal growth factor receptor expression licenses Type-2 helper T cells to function in a T cell receptor-independent fashion. Immunity. 2017;47:710–722. https://doi.org/10.1016/j.immuni.2017.09.013. [205] Chen N, et al. Upregulation of PD-L1 by EGFR activation mediates the immune escape in EGFR-Driven NSCLC: implication for optional immune targeted therapy for NSCLC patients with EGFR mutation. J Thorac Oncol. 2015;10:910–923. https://doi.org/10.1097/jto.0000000000000500. [206] Akbay EA, et al. Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer Discov. 2013;3:1355–1363. https://doi.org/10. 1158/2159-8290.Cd-13-0310. [207] Liang H, et al. Immunotherapy combined with epidermal growth factor receptortyrosine kinase inhibitors in non-small-cell lung cancer treatment. OncoTarget Ther. 2018;11:6189–6196. https://doi.org/10.2147/OTT.S178497. [208] Pesu M, et al. Jak3, severe combined immunodeficiency, and a new class of immunosuppressive drugs. Immunol Rev. 2005;203:127–142. https://doi.org/10. 1111/j.0105-2896.2005.00220.x. [209] Schwartz DM, et al. Type I/II cytokines, JAKs, and new strategies for treating autoimmune diseases. Nat Rev Rheumatol. 2015;12:25. https://doi.org/10.1038/ nrrheum.2015.167. [210] Forster M, et al. Selective JAK3 inhibitors with a covalent reversible binding mode targeting a new induced fit binding pocket. Cell Chem Biol. 2016;23:1335–1340.

https://doi.org/10.1016/j.chembiol.2016.10.008. [211] Duhé RJ. Redox regulation of Janus kinase. JAK-STAT. 2013;2. https://doi.org/ 10.4161/jkst.26141. [212] Totzke J, et al. Takinib, a selective TAK1 inhibitor, broadens the therapeutic efficacy of TNF-alpha inhibition for cancer and autoimmune disease. Cell Chem Biol. 2017;24:1029–1039.e1027. https://doi.org/10.1016/j.chembiol.2017.07.011. [213] Sato S, et al. TAK1 is indispensable for development of T cells and prevention of colitis by the generation of regulatory T cells. Int Immunol. 2006;18:1405–1411. https://doi.org/10.1093/intimm/dxl082. [214] Sakurai H. Targeting of TAK1 in inflammatory disorders and cancer. Trends Pharmacol Sci. 2012;33:522–530. https://doi.org/10.1016/j.tips.2012.06.007. [215] Qian L, et al. Live-cell imaging and profiling of c-Jun N-terminal kinases using covalent inhibitor-derived probes. Chem Commun. 2019;55:1092–1095. https:// doi.org/10.1039/C8CC09558B. [216] Visperas PR, et al. Modification by covalent reaction or oxidation of cysteine residues in the tandem-SH2 domains of ZAP-70 and Syk can block phosphopeptide binding. Biochem J. 2015;465:149–161. https://doi.org/10.1042/bj20140793. [217] Visperas PR, et al. Identification of inhibitors of the association of ZAP-70 with the T cell receptor by high-throughput screen. SLAS Discov. 2017;22:324–331. https:// doi.org/10.1177/1087057116681407. [218] Love PE, Hayes SM. ITAM-mediated signaling by the T-cell antigen receptor. a002485 a2485 Cold Spring Harbor Perspect Biol. 2010;2. https://doi.org/10.1101/ cshperspect.a002485. [219] Shearn CT, et al. Modification of Akt2 by 4-hydroxynonenal inhibits insulin-dependent Akt signaling in HepG2 cells. Biochemistry. 2011;50:3984–3996. https:// doi.org/10.1021/bi200029w. [220] Long MJC, et al. Akt3 is a privileged first responder in isozyme-specific electrophile response. Nat Chem Biol. 2017;13:333. https://doi.org/10.1038/nchembio. 2284. [221] Drewry DH, et al. Progress towards a public chemogenomic set for protein kinases and a call for contributions. PLoS ONE. 2017;12. https://doi.org/10.1371/journal. pone.0181585. [222] Wang W, et al. Discovery of inactive conformation-selective kinase inhibitors by utilizing cascade assays. Biochemistry. 2017;56:4449–4456. https://doi.org/10. 1021/acs.biochem.7b00521. [223] Hanson SM, et al. What makes a kinase promiscuous for inhibitors? cell. Chem Biol. 2018:pp. https://doi.org/10.1016/j.chembiol.2018.11.005. [224] Cohen P. The structures and regulation of protein phosphatases. Annu Rev Biochem. 1989;58:453–508. https://doi.org/10.1146/annurev.bi.58.070189. 002321. [225] Hunter T. The genesis of tyrosine phosphorylation. Cold Spring Harbor Perspect Biol. 2014;6. https://doi.org/10.1101/cshperspect.a020644. [226] Camps M, et al. Dual specificity phosphatases: a gene family for control of MAP kinase function. Faseb J. 2000;14:6–16. [227] Patterson KI, et al. Dual-specificity phosphatases: critical regulators with diverse cellular targets. Biochem J. 2009;418:475–489. [228] Zhang Z-Y. Progress in nucleic acid research and molecular biology. Academic Press; 2003:171–220 doi: 10.1016/S0079-6603(03)01006-7. [229] Alonso A, et al. Protein tyrosine phosphatases in the human genome. Cell. 2004;117:699–711. https://doi.org/10.1016/j.cell.2004.05.018. [230] Mustelin T, et al. Protein tyrosine phosphatases and the immune response. Nat Rev Immunol. 2005;5:43–57. https://doi.org/10.1038/nri1530. [231] Zhang J, et al. Roles of the SHP-1 tyrosine phosphatase in the negative regulation of cell signalling SHP-1 and the motheaten phenotype. seminars. Immunology. 2000;12:361–378. https://doi.org/10.1006/smim.2000.0223. [232] Pao LI, et al. Nonreceptor protein-tyrosine phosphatases in immune cell signaling. Annu Rev Immunol. 2007;25:473–523. https://doi.org/10.1146/annurev. immunol.23.021704.115647. [233] Mercadante ER, Lorenz UM. T cells deficient in the tyrosine phosphatase SHP-1 resist suppression by regulatory T cells. J Immunol. 2017;199:129–137. https:// doi.org/10.4049/jimmunol.1602171. [234] Volarević S, et al. Intimate association of Thy-1 and the T-cell antigen receptor with the CD45 tyrosine phosphatase. Proc Natl Acad Sci USA. 1990;87:7085–7089. https://doi.org/10.1073/pnas.87.18.7085. [235] Hermiston ML, et al. CD45: a critical regulator of signaling thresholds in immune cells. Annu Rev Immunol. 2003;21:107–137. https://doi.org/10.1146/annurev. immunol.21.120601.140946. [236] Saunders AE, Johnson P. Modulation of immune cell signalling by the leukocyte common tyrosine phosphatase, CD45. Cell Signal. 2010;22:339–348. https://doi. org/10.1016/j.cellsig.2009.10.003. [237] Baba Y, et al. Structure-based design of a highly selective catalytic site-directed inhibitor of Ser/Thr protein phosphatase 2B (calcineurin). J Am Chem Soc. 2003;125:9740–9749. https://doi.org/10.1021/ja034694y. [238] Todd JL, et al. Extracellular regulated kinases (ERK) 1 and ERK2 are authentic substrates for the dual-specificity protein-tyrosine phosphatase VHR. A novel role in down-regulating the ERK pathway. J Biol Chem. 1999;274:13271–13280. https://doi.org/10.1074/jbc.274.19.13271. [239] Kang TH, Kim KT. Negative regulation of ERK activity by VRK3-mediated activation of VHR phosphatase. Nat Cell Biol. 2006;8:863–869. https://doi.org/10. 1038/ncb1447. [240] Stanford SM, Bottini N. Targeting tyrosine phosphatases: time to end the stigma. Trends Pharmacol Sci. 2017;38:524–540. https://doi.org/10.1016/j.tips.2017.03. 004. [241] Ruddraraju KV, Zhang ZY. Covalent inhibition of protein tyrosine phosphatases. Mol BioSyst. 2017;13:1257–1279. https://doi.org/10.1039/c7mb00151g. [242] Lee S, Wang Q. Recent development of small molecular specific inhibitor of

16

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

[243] [244] [245] [246] [247] [248]

[249] [250] [251]

[252] [253] [254] [255] [256] [257] [258] [259] [260] [261] [262] [263] [264] [265]

[266] [267] [268] [269] [270] [271] [272]

protein tyrosine phosphatase 1B. Med Res Rev. 2007;27:553–573. https://doi.org/ 10.1002/med.20079. Bialy L, Waldmann H. Inhibitors of protein tyrosine phosphatases: Next-generation drugs? Angew Chem – Int Ed. 2005;44:3814–3839. https://doi.org/10.1002/anie. 200461517. Falahati R, Leitenberg D. Selective regulation of TCR signaling pathways by the CD45 protein tyrosine phosphatase during thymocyte development. J Immunol (Baltimore, Md. 1950). 1950;181(2008):6082–6091. Alvi KA, et al. Pulchellalactam: A CD45 protein tyrosine phosphatase inhibitor from the marine fungus corollospora pulchella. J Antibiot. 1997;51:515–517. https://doi.org/10.7164/antibiotics.51.515. Seiner DR, et al. Kinetics and mechanism of protein tyrosine phosphatase 1B inactivation by acrolein. Chem Res Toxicol. 2007;20:1315–1320. https://doi.org/10. 1021/tx700213s. Abdo M, et al. Seleninate in place of phosphate: irreversible inhibition of protein tyrosine phosphatases. J Am Chem Soc. 2008;130:13196–13197. https://doi.org/ 10.1021/ja804489m. Yi TL, et al. Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13. Mol Cell Biol. 1992;12:836–846. https://doi.org/10. 1128/MCB.12.2.836. Freeman Jr RM, et al. Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew. PNAS. 1992;89:11239–11243. https://doi.org/10.1073/pnas.89.23.11239. Arabaci G, et al. α-haloacetophenone derivatives as photoreversible covalent inhibitors of protein tyrosine phosphatases. J Am Chem Soc. 1999;121:5085–5086. https://doi.org/10.1021/ja9906756. Kundu S Novel, et al. SHP-1 inhibitors tyrosine phosphatase inhibitor-1 and analogs with preclinical anti-tumor activities as tolerated oral agents. J Immunol (Baltimore, Md. 1950). 1950;184(2010):6529–6536. https://doi.org/10.4049/ jimmunol.0903562. Hui E, et al. T cell costimulatory receptor CD28 is a primary target for PD-1mediated inhibition. Science. 2017;355:1428–1433. https://doi.org/10.1126/ science.aaf1292. Rota G, et al. Shp-2 Is dispensable for establishing T cell exhaustion and for PD-1 signaling in vivo. Cell Rep. 2018;23:39–49. https://doi.org/10.1016/j.celrep.2018. 03.026. Lewis SM, et al. Inactivation of protein tyrosine phosphatases by dietary isothiocyanates. Bioorg Med Chem Lett. 2015;25:4549–4552. https://doi.org/10. 1016/j.bmcl.2015.08.065. Chio CM, et al. Targeting a cryptic allosteric site for selective inhibition of the oncogenic protein tyrosine phosphatase Shp2. Biochemistry. 2015;54:497–504. https://doi.org/10.1021/bi5013595. Dutta D, et al. Recruitment of calcineurin to the TCR positively regulates T cell activation. Nat Immunol. 2016;18:196. https://doi.org/10.1038/ni.3640. den Hertog J, et al. Redox regulation of protein-tyrosine phosphatases. Arch Biochem Biophys. 2005;434:11–15. https://doi.org/10.1016/j.abb.2004.05.024. Salmeen A, Barford D. Functions and mechanisms of redox regulation of cysteinebased phosphatases. Antioxid Redox Signal. 2005;7:560–577. https://doi.org/10. 1089/ars.2005.7.560. Yang J, et al. Site-specific mapping and quantification of protein S-sulphenylation in cells. Nat Commun. 2014;5:4776. https://doi.org/10.1038/ncomms5776. van Montfort RL, et al. Oxidation state of the active-site cysteine in protein tyrosine phosphatase 1B. Nature. 2003;423:773–777. https://doi.org/10.1038/ nature01681. Salmeen A, et al. Redox regulation of protein tyrosine phosphatase 1B involves a sulphenyl-amide intermediate. Nature. 2003;423:769–773. https://doi.org/10. 1038/nature01680. Gupta V, Carroll KS. Rational design of reversible and irreversible cysteine sulfenic acid-targeted linear C-nucleophiles. Chem Commun (Camb). 2016;52:3414–3417. https://doi.org/10.1039/c6cc00228e. Yang J, et al. Global, in situ, site-specific analysis of protein S-sulfenylation. Nat Protoc. 2015;10:1022–1037. https://doi.org/10.1038/nprot.2015.062. Truong TH, et al. Molecular basis for redox activation of epidermal growth factor receptor kinase. Cell Chem Biol. 2016;23:837–848. https://doi.org/10.1016/j. chembiol.2016.05.017. Parsons ZD, et al. Sulfone-stabilized carbanions for the reversible covalent capture of a posttranslationally-generated cysteine oxoform found in protein tyrosine phosphatase 1B (PTP1B). Bioorg Med Chem. 2016;24:2631–2640. https://doi.org/ 10.1016/j.bmc.2016.03.054. Chen CY, et al. Redox regulation of SH2-domain-containing protein tyrosine phosphatases by two backdoor cysteines. Biochemistry. 2009;48:1399–1409. https://doi.org/10.1021/bi801973z. Sohn J, Rudolph J. Catalytic and chemical competence of regulation of cdc25 phosphatase by oxidation/reduction. Biochemistry. 2003;42:10060–10070. https://doi.org/10.1021/bi0345081. Lee SR, et al. Reversible inactivation of the tumor suppressor PTEN by H2O2. J Biol Chem. 2002;277:20336–20342. https://doi.org/10.1074/jbc.M111899200. Souza ACS, et al. Review From immune response to cancer: a spot on the low molecular weight protein tyrosine phosphatase. Cell Mol Life Sci. 2009;66:1140–1153. https://doi.org/10.1007/s00018-008-8501-8. Kato D, et al. Activity-based probes that target diverse cysteine protease families. Nat Chem Biol. 2005;1:33–38. https://doi.org/10.1038/nchembio707. Puente XS, et al. Human and mouse proteases: a comparative genomic approach. Nat Rev Genet. 2003;4:544–558. https://doi.org/10.1038/nrg1111. McIlwain DR, et al. Caspase functions in cell death and disease. Cold Spring Harb

Perspect Biol. 2013;5. https://doi.org/10.1101/cshperspect.a008656. [273] Rossi A, et al. Comprehensive search for cysteine cathepsins in the human genome. Biol Chem. 2004;385:363–372. https://doi.org/10.1515/bc.2004.040. [274] Ji J, et al. Critical role of calpain in inflammation. Biomed Rep. 2016;5:647–652. https://doi.org/10.3892/br.2016.785. [275] Hailfinger S, et al. Adapter and enzymatic functions of proteases in T-cell activation. Immunol Rev. 2009;232:334–347. https://doi.org/10.1111/j.1600-065X. 2009.00830.x. [276] van Kasteren SI, Overkleeft HS. Endo-lysosomal proteases in antigen presentation. Curr Opin Chem Biol. 2014;23:8–15. https://doi.org/10.1016/j.cbpa.2014.08.011. [277] McArthur K, Kile BT. Apoptotic caspases: multiple or mistaken identities? Trends Cell Biol. 2018;28:475–493. https://doi.org/10.1016/j.tcb.2018.02.003. [278] Nakajima Y-i, Kuranaga E. Caspase-dependent non-apoptotic processes in development. Cell Death Different. 2017;24:1422. https://doi.org/10.1038/cdd. 2017.36. [279] Crawford ED, et al. The DegraBase: a database of proteolysis in healthy and apoptotic human cells. Mol Cell Proteom. 2013;12:813–824. [280] Dix MM, et al. Functional interplay between caspase cleavage and phosphorylation sculpts the apoptotic proteome. Cell. 2012;150:426–440. https://doi.org/10. 1016/j.cell.2012.05.040. [281] Turowec JP, et al. An unbiased proteomic screen reveals caspase cleavage is positively and negatively regulated by substrate phosphorylation. Mol Cell Proteom: MCP. 2014;13:1184–1197. https://doi.org/10.1074/mcp.M113.037374. [282] Sollberger G, et al. Caspase-1: the inflammasome and beyond. Innate Immun. 2014;20:115–125. https://doi.org/10.1177/1753425913484374. [283] Man SM, et al. Molecular mechanisms and functions of pyroptosis, inflammatory caspases and inflammasomes in infectious diseases. Immunol Rev. 2017;277:61–75. https://doi.org/10.1111/imr.12534. [284] Jimenez Fernandez D, Lamkanfi M. Inflammatory caspases: key regulators of inflammation and cell death. Biol Chem. 2015;396:193–203. https://doi.org/10. 1515/hsz-2014-0253. [285] Broz P, Dixit VM. Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol. 2016;16:407. https://doi.org/10.1038/nri.2016.58. [286] Lamkanfi M, et al. Deregulated inflammasome signaling in disease. Immunol Rev. 2011;243:163–173. https://doi.org/10.1111/j.1600-065X.2011.01042.x. [287] Yang Y, et al. Recent advances in the mechanisms of NLRP3 inflammasome activation and its inhibitors. Cell Death Dis. 2019;10:128. https://doi.org/10.1038/ s41419-019-1413-8. [288] Guo H, et al. Inflammasomes: mechanism of action, role in disease, and therapeutics. Nat Med. 2015;21:677–687. https://doi.org/10.1038/nm.3893. [289] Bergsbaken T, et al. Pyroptosis: host cell death and inflammation. Nat Rev Microbiol. 2009;7:99–109. https://doi.org/10.1038/nrmicro2070. [290] Voet S, et al. Inflammasomes in neuroinflammatory and neurodegenerative diseases. J EMBO Mol Med. 2019. https://doi.org/10.15252/emmm.201810248% e10248. [291] Venegas C, et al. Microglia-derived ASC specks cross-seed amyloid-beta in Alzheimer's disease. Nature. 2017;552:355–361. https://doi.org/10.1038/ nature25158. [292] Elmore S. Apoptosis: a review of programmed cell death. Toxicol Pathol. 2007;35:495–516. https://doi.org/10.1080/01926230701320337. [293] Chun HJ, et al. Pleiotropic defects in lymphocyte activation caused by caspase-8 mutations lead to human immunodeficiency. Nature. 2002;419:395–399. https:// doi.org/10.1038/nature01063. [294] Salmena L, et al. Essential role for caspase 8 in T-cell homeostasis and T-cellmediated immunity. Genes Dev. 2003;17:883–895. https://doi.org/10.1101/gad. 1063703. [295] Wang J, et al. Inherited human Caspase 10 mutations underlie defective lymphocyte and dendritic cell apoptosis in autoimmune lymphoproliferative syndrome type II. Cell. 1999;98:47–58. https://doi.org/10.1016/s0092-8674(00) 80605-4. [296] Zhu S, et al. Genetic alterations in caspase-10 may be causative or protective in autoimmune lymphoproliferative syndrome. Hum Genet. 2006;119:284–294. https://doi.org/10.1007/s00439-006-0138-9. [297] Yi F, et al. Non-apoptotic signaling through Fas activates Akt and promotes T cell differentiation through Caspase-8. J Immunol. 2017;198 217.215. [298] Cuda CM, et al. Caspase-8 acts as a molecular rheostat to limit RIPK1- and MyD88mediated dendritic cell activation. J Immunol. 2014;192:5548–5560. https://doi. org/10.4049/jimmunol.1400122. [299] Beisner DR, et al. Cutting edge: innate immunity conferred by B cells is regulated by caspase-8. J Immunol. 2005;175:3469–3473. https://doi.org/10.4049/ jimmunol.175.6.3469. [300] Cuda CM, et al. Conditional deletion of caspase-8 in macrophages alters macrophage activation in a RIPK-dependent manner. Arthritis Res Ther. 2015;17:291. https://doi.org/10.1186/s13075-015-0794-z. [301] McComb S, et al. Caspase-3 is transiently activated without Cell Death during early antigen driven expansion of CD8+ T cells in vivo. PLoS ONE. 2010;5. https://doi. org/10.1371/journal.pone.0015328. [302] Koenig A, et al. Proliferating gammadelta T cells manifest high and spatially confined caspase-3 activity. Immunology. 2012;135:276–286. https://doi.org/10. 1111/j.1365-2567.2011.03540.x. [303] Kato D, et al. Activity-based probes that target diverse cysteine protease families. Nat Chem Biol. 2005;1:33. https://doi.org/10.1038/nchembio707. [304] Troy CM, Shelanski ML. Caspase-2 and tau-a toxic partnership? Nat Med. 2016;22:1207–1208. https://doi.org/10.1038/nm.4227. [305] Strowig T, et al. Inflammasomes in health and disease. Nature. 2012;481:278–286. https://doi.org/10.1038/nature10759.

17

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al. [306] Olsson M, Zhivotovsky B. Caspases and cancer. Cell Death Differ. 2011;18:1441–1449. https://doi.org/10.1038/cdd.2011.30. [307] S.J. Riedl et al. Structural basis for the activation of human procaspase-7 Proceed Natl Acad Sci USA 98 2001 14790 14795 10.1073/pnas.221580098. [308] Thomsen ND, et al. Structural snapshots reveal distinct mechanisms of procaspase3 and -7 activation. PNAS. 2013;110:8477–8482. https://doi.org/10.1073/pnas. 1306759110. [309] Mikosik A, et al. Roles of calpain-calpastatin system (CCS) in human T cell activation. Oncotarget. 2016;7:76479–76495. https://doi.org/10.18632/oncotarget. 13259. [310] Kumar V, et al. Calpains promote neutrophil recruitment and bacterial clearance in an acute bacterial peritonitis model. Eur J Immunol. 2014;44:831–841. https:// doi.org/10.1002/eji.201343757. [311] Oh-hora M, Rao A. Calcium signaling in lymphocytes. Curr Opin Immunol. 2008;20:250–258. https://doi.org/10.1016/j.coi.2008.04.004. [312] Feske S. ORAI1 and STIM1 deficiency in human and mice: roles of store-operated Ca2+ entry in the immune system and beyond. Immunol Rev. 2009;231:189–209. https://doi.org/10.1111/j.1600-065X.2009.00818.x. [313] Feske S. Calcium signalling in lymphocyte activation and disease. Nat Rev Immunol. 2007;7:690–702. https://doi.org/10.1038/nri2152. [314] Donkor IO. An updated patent review of calpain inhibitors (2012–2014) AU Donkor, Isaac O. Exp Opin Ther Pat. 2015;25:17–31. https://doi.org/10.1517/ 13543776.2014.982534. [315] Figueiredo-Pereira ME, et al. The antitumor drug aclacinomycin a, which inhibits the degradation of ubiquitinated proteins, shows selectivity for the chymotrypsinlike activity of the bovine pituitary 20 S proteasome. J Biol Chem. 1996;271:16455–16459. https://doi.org/10.1074/jbc.271.28.16455. [316] Nam DH, et al. Design and synthesis of 4-quinolinone 2-carboxamides as calpain inhibitors. Bioorg Med Chem Lett. 2008;18:205–209. https://doi.org/10.1016/j. bmcl.2007.10.097. [317] Baek KH, et al. Synthesis and investigation of dihydroxychalcones as calpain and cathepsin inhibitors. Bioorg Chem. 2013;51:24–30. https://doi.org/10.1016/j. bioorg.2013.09.002. [318] Adams SE, et al. The structural basis of differential inhibition of human calpain by indole and phenyl alpha-mercaptoacrylic acids. J Struct Biol. 2014;187:236–241. https://doi.org/10.1016/j.jsb.2014.07.004. [319] Laufer TM, et al. Unopposed positive selection and autoreactivity in mice expressing class II MHC only on thymic cortex. Nature. 1996;383:81. https://doi. org/10.1038/383081a0. [320] Shi G-P, et al. Role for cathepsin F in invariant chain processing and major histocompatibility complex class II peptide loading by macrophages. J Exp Med. 2000;191:1177–1186. https://doi.org/10.1084/jem.191.7.1177. [321] Villadangos JA, Ploegh HL. Proteolysis in MHC class II antigen presentation: Who's in charge? Immunity. 2000;12:233–239. https://doi.org/10.1016/S1074-7613(00) 80176-4. [322] Delamarre L, et al. Differential lysosomal proteolysis in antigen-presenting cells determines antigen fate. Science. 2005;307:1630–1634. https://doi.org/10.1126/ science.1108003. [323] McCurley N, Mellman I. Monocyte-derived dendritic cells exhibit increased levels of lysosomal proteolysis as compared to other human dendritic cell populations. PLoS ONE. 2010;5. https://doi.org/10.1371/journal.pone.0011949. [324] Heusinkveld M, van der Burg SH. Identification and manipulation of tumor associated macrophages in human cancers. J Transl Med. 2011;9:216. https://doi. org/10.1186/1479-5876-9-216. [325] Gocheva V, et al. IL-4 induces cathepsin protease activity in tumor-associated macrophages to promote cancer growth and invasion. Genes Dev. 2010;24:241–255. https://doi.org/10.1101/gad.1874010. [326] Shi GP, et al. Cathepsin S required for normal MHC class II peptide loading and germinal center development. Immunity. 1999;10:197–206. https://doi.org/10. 1016/S1074-7613(00)80020-5. [327] Akkari L, et al. Distinct functions of macrophage-derived and cancer cell-derived cathepsin Z combine to promote tumor malignancy via interactions with the extracellular matrix. Genes Dev. 2014;28:2134–2150. https://doi.org/10.1101/gad. 249599.114. [328] Allan ERO, et al. A role for cathepsin Z in neuroinflammation provides mechanistic support for an epigenetic risk factor in multiple sclerosis. J Neuroinflamm. 2017;14. https://doi.org/10.1186/s12974-017-0874-x. [329] Toomes C, et al. Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis. Nat Genet. 1999;23:421–424. https://doi.org/10.1038/70525. [330] Meade JL, et al. A family with Papillon-Lefevre syndrome reveals a requirement for cathepsin C in granzyme B activation and NK cell cytolytic activity. Blood. 2006;107:3665–3668. https://doi.org/10.1182/blood-2005-03-1140. [331] Diaz A, et al. Cathepsin K expression in epithelioid and multinucleated giant cells. J Pathol. 2002;197:690. https://doi.org/10.1002/path.1143. [332] Jaffer FA, et al. Optical visualization of cathepsin K activity in atherosclerosis with a novel, protease-activatable fluorescence sensor. Circulation. 2007;115:2292–2298. https://doi.org/10.1161/circulationaha.106.660340. [333] Lutgens E, et al. Disruption of the cathepsin K gene reduces atherosclerosis progression and induces plaque fibrosis but accelerates macrophage foam cell formation. Circulation. 2006;113:98–107. https://doi.org/10.1161/circulationaha. 105.561449. [334] Palermo C, Joyce JA. Cysteine cathepsin proteases as pharmacological targets in cancer. Trends Pharmacol Sci. 2008;29:22–28. https://doi.org/10.1016/j.tips. 2007.10.011. [335] Siklos M, et al. Cysteine proteases as therapeutic targets: does selectivity matter? A

[336] [337] [338] [339]

[340] [341] [342] [343] [344] [345] [346] [347] [348] [349] [350] [351] [352] [353] [354] [355] [356]

[357] [358] [359] [360] [361] [362] [363] [364]

18

systematic review of calpain and cathepsin inhibitors. Acta Pharmaceut Sin B. 2015;5:506–519. https://doi.org/10.1016/j.apsb.2015.08.001. Hernandez AA, Roush WR. Recent advances in the synthesis, design and selection of cysteine protease inhibitors. Curr Opin Chem Biol. 2002;6:459–465. https://doi. org/10.1016/S1367-5931(02)00345-9. Verdoes M, Verhelst SH. Detection of protease activity in cells and animals. Biochim Biophys Acta. 1864;2016:130–142. https://doi.org/10.1016/j.bbapap. 2015.04.029. Fonović M, Bogyo M. Activity-based probes as a tool for functional proteomic analysis of proteases. Exp Rev Proteom. 2008;5:721–730. https://doi.org/10.1586/ 14789450.5.5.721. Watanabe M, et al. Inhibition of rat liver cathepsins B and L by the peptide aldehyde benzyloxycarbonyl-leucyl-leucyl-leucinal and its analogues AU - Ito, Hisashi. J Enzyme Inhib Med Chem. 2009;24:279–286. https://doi.org/10.1080/ 14756360802166921. Guo CJ, et al. Discovery of reactive microbiota-derived metabolites that inhibit host proteases. Cell. 2017;168:517–526.e518. https://doi.org/10.1016/j.cell. 2016.12.021. Votta BJ, et al. Peptide aldehyde inhibitors of cathepsin K inhibit bone resorption both in vitro and in vivo. J Bone Miner Res. 1997;12:1396–1406. https://doi.org/ 10.1359/jbmr.1997.12.9.1396. Rasnick D. Synthesis of peptide fluoromethyl ketones and the inhibition of human cathepsin B. Anal Biochem. 1985;149:461–465. https://doi.org/10.1016/00032697(85)90598-6. Gray J, et al. Evidence that inhibition of cathepsin-B contributes to the neuroprotective properties of caspase inhibitor Tyr-Val-Ala-Asp-chloromethyl ketone. J Biol Chem. 2001;276:32750–32755. https://doi.org/10.1074/jbc.M103150200. Green GD, Shaw E. Peptidyl diazomethyl ketones are specific inactivators of thiol proteinases. J Biol Chem. 1981;256:1923–1928. Fennell BD, et al. Optimization of peptidyl allyl sulfones as clan CA cysteine protease inhibitors. J Enzyme Inhib Med Chem. 2013;28:468–478. https://doi.org/ 10.3109/14756366.2011.651466. Shaw E. Peptidyl sulfonium salts. A new class of protease inhibitors. J Biol Chem. 1988;263:2768–2772. Edem PE, et al. Synthesis and evaluation of radioiodinated acyloxymethyl ketones as activity-based probes for cathepsin B. J Med Chem. 2014;57:9564–9577. https://doi.org/10.1021/jm501357r. Tavares FX, et al. Ketoheterocycle-based inhibitors of cathepsin K: a novel entry into the synthesis of peptidic ketoheterocycles. Bioorg Med Chem Lett. 2005;15:3891–3895. https://doi.org/10.1016/j.bmcl.2005.05.091. Dana D, et al. Development of cell-active non-peptidyl inhibitors of cysteine cathepsins. Bioorg Med Chem. 2013;21:2975–2987. https://doi.org/10.1016/j.bmc. 2013.03.062. Lee M-J, et al. Effects of novel human cathepsin S inhibitors on cell migration in human cancer cells AU - Tsai, Ju-Ying. J Enzyme Inhib Med Chem. 2014;29:538–546. https://doi.org/10.3109/14756366.2013.823957. Brömme D, Lecaille F. Cathepsin K inhibitors for osteoporosis and potential offtarget effects. Exp Opin Investig Drugs. 2009;18:585–600. https://doi.org/10.1517/ 13543780902832661. Chapurlat RD. Odanacatib: a review of its potential in the management of osteoporosis in postmenopausal women. Therap Adv Musculoskel Dis. 2015;7:103–109. https://doi.org/10.1177/1759720X15580903. Lu J, et al. Advances in the discovery of cathepsin K inhibitors on bone resorption. J Enzyme Inhib Med Chem. 2018;33:890–904. https://doi.org/10.1080/14756366. 2018.1465417. Yamashita DS, et al. Structure and design of potent and selective cathepsin K inhibitors. J Am Chem Soc. 1997;119:11351–11352. https://doi.org/10.1021/ ja972204u. Desmarais S, et al. Effect of cathepsin k inhibitor basicity on in vivo off-target activities. Mol Pharmacol. 2008;73:147–156. https://doi.org/10.1124/mol.107. 039511. Falgueyret JP, et al. Lysosomotropism of basic cathepsin K inhibitors contributes to increased cellular potencies against off-target cathepsins and reduced functional selectivity. J Med Chem. 2005;48:7535–7543. https://doi.org/10.1021/ jm0504961. van Esbroeck ACM, et al. Activity-based protein profiling reveals off-target proteins of the FAAH inhibitor BIA 10–2474. Science. 2017;356:1084–1087. https:// doi.org/10.1126/science.aaf7497. Desmarais S, et al. Pharmacological inhibitors to identify roles of cathepsin K in cell-based studies: a comparison of available tools. Biol Chem. 2009;390:941–948. https://doi.org/10.1515/bc.2009.092. Mullard A, Merck & Co. drops osteoporosis drug odanacatib. Nat Rev Drug Discov. 2016;15:669. https://doi.org/10.1038/nrd.2016.207. Hao L, et al. A small molecule, odanacatib, inhibits inflammation and bone loss caused by endodontic disease. Infect Immun. 2015;83:1235–1245. https://doi.org/ 10.1128/iai.01713-14. Hao L, et al. Deficiency of cathepsin K prevents inflammation and bone erosion in rheumatoid arthritis and periodontitis and reveals its shared osteoimmune role. FEBS Lett. 2015;589:1331–1339. https://doi.org/10.1016/j.febslet.2015.04.008. Asagiri M, et al. Cathepsin K-dependent toll-like receptor 9 signaling revealed in experimental arthritis. Science. 2008;319:624–627. https://doi.org/10.1126/ science.1150110. Poreba M, et al. Selective imaging of cathepsin L in breast cancer by fluorescent activity-based probes. Chem Sci. 2018;9:2113–2129. https://doi.org/10.1039/ c7sc04303a. Abd-Elrahman I, et al. Characterizing cathepsin activity and macrophage subtypes

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx

K.M. Backus, et al.

[365] [366] [367] [368] [369] [370] [371] [372] [373]

in excised human carotid plaques. Stroke. 2016;47:1101–1108. https://doi.org/ 10.1161/strokeaha.115.011573. Bouhlel MA, et al. PPARγ activation primes human monocytes into alternative M2 macrophages with anti-inflammatory properties. Cell Metab. 2007;6:137–143. https://doi.org/10.1016/j.cmet.2007.06.010. Joyce JA, et al. Cathepsin cysteine proteases are effectors of invasive growth and angiogenesis during multistage tumorigenesis. Cancer Cell. 2004;5:443–453. https://doi.org/10.1016/S1535-6108(04)00111-4. Mills JE, et al. A novel disulfide bond in the SH2 Domain of the C-terminal Src kinase controls catalytic activity. J Mol Biol. 2007;365:1460–1468. https://doi. org/10.1016/j.jmb.2006.10.076. Evans JV, et al. Src binds cortactin through an SH2 domain cystine-mediated linkage. J Cell Sci. 2012;125:6185–6197. https://doi.org/10.1242/jcs.121046. Bicker KL, Thompson PR. The protein arginine deiminases: Structure, function, inhibition, and disease. Biopolymers. 2013;99:155–163. https://doi.org/10.1002/ bip.22127. Nemmara VV, et al. The development of benzimidazole-based clickable probes for the efficient labeling of cellular protein arginine deiminases (PADs). ACS Chem Biol. 2018;13:712–722. https://doi.org/10.1021/acschembio.7b00957. Dillon MB, et al. Novel inhibitors for PRMT1 discovered by high-throughput screening using activity-based fluorescence polarization. ACS Chem Biol. 2012;7:1198–1204. https://doi.org/10.1021/cb300024c. Lee H-Y, et al. Covalent inhibitors of nicotinamide N-methyltransferase (NNMT) provide evidence for target engagement challenges in situ. Bioorg Med Chem Lett. 2018;28:2682–2687. https://doi.org/10.1016/j.bmcl.2018.04.017. Mann SA, et al. The development and characterization of a chemical probe targeting PRMT1 over PRMT5. Bioorg Med Chem. 2019;27:224–229. https://doi.org/

10.1016/j.bmc.2018.12.001. [374] Wertz IE, Wang X From. Discovery to bedside: targeting the ubiquitin system. Cell Chem Biol. 2018:pp. https://doi.org/10.1016/j.chembiol.2018.10.022. [375] Ward CC, et al. Covalent ligand screening uncovers a RNF4 E3 ligase recruiter for targeted protein degradation applications. ACS Chem Biol. 2019:pp. https://doi. org/10.1021/acschembio.8b01083. [376] Spradlin JN, et al. Harnessing the anti-cancer natural product nimbolide for targeted protein degradation. bioRxiv. 2018. https://doi.org/10.1101/436998. [377] Zhang X, et al. Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16. bioRxiv. 2018. https://doi.org/10.1101/443804. [378] Daguer J-P, et al. Identification of covalent bromodomain binders through DNA display of small molecules. Angew Chem Int Ed. 2015;54:6057–6061. https://doi. org/10.1002/anie.201412276. [379] Kharenko OA, et al. Design and characterization of novel covalent bromodomain and extra-terminal domain (BET) inhibitors targeting a methionine. J Med Chem. 2018;61:8202–8211. https://doi.org/10.1021/acs.jmedchem.8b00666. [380] Bum-Erdene, K et al. Small-Molecule Covalent Modification of Conserved Cysteine Leads to Allosteric Inhibition of the TEAD-Yap Protein-Protein Interaction. Cell Chemical Biology. doi: 10.1016/j.chembiol.2018.11.010. [381] Meng K, Butte MJ. Exploring the role of YAP signaling in CD4+ T cell biology. J Immunol. 2016;196 55.29. [382] Hacker SM, et al. Global profiling of lysine reactivity and ligandability in the human proteome. Nat Chem. 2017;9:1181–1190. https://doi.org/10.1038/nchem. 2826. [383] Jia S, et al. Bioinspired thiophosphorodichloridate reagents for chemoselective histidine bioconjugation. J Am Chem Soc. 2019;141:7294–7301. https://doi.org/ 10.1021/jacs.8b11912.

19