Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery

Please cite this article in press as: Burslem and Crews, Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery, Cell (2020), https://doi.org/10.1016/j.cell.2019.11.031

Leading Edge

Primer Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery George M. Burslem1 and Craig M. Crews1,2,* 1Department

of Molecular, Cellular and Developmental Biology, Yale University, New Haven, CT, USA of Chemistry and Pharmacology, Yale University, New Haven, CT, USA *Correspondence: [email protected] https://doi.org/10.1016/j.cell.2019.11.031 2Departments

New biological tools provide new techniques to probe fundamental biological processes. Here we describe the burgeoning field of proteolysis-targeting chimeras (PROTACs), which are capable of modulating protein concentrations at a post-translational level by co-opting the ubiquitin-proteasome system. We describe the PROTAC technology and its application to drug discovery and provide examples where PROTACs have enabled novel biological insights. Furthermore, we provide a workflow for PROTAC development and use and discuss the benefits and issues associated with PROTACs. Finally, we compare PROTAC-mediated protein-level modulation with other technologies, such as RNAi and genome editing. Introduction Technological advances often lead to major biological discoveries (Botstein, 2010; Editorial, 2000; Fields, 2001; van Steensel, 2015), which, in turn, drive new technology development. The search for new technologies to answer biological questions has given rise to the field of chemical biology (Altmann et al., 2009), and perhaps one of the most exciting technologies to arise is targeted protein degradation by proteolysis-targeting chimeras (PROTACs). In this Primer, we will review this technology, its applications to drug discovery, and its use in the exploration of fundamental biology. Additionally, we will outline the workflow involved in successfully employing PROTACs for experimentation and compare PROTACs with other techniques. Overview of the Technology The Ubiquitin-Proteasome System The ubiquitin-proteasome system (UPS) is the primary intracellular mechanism for destruction of damaged proteins or those no longer required (Amm et al., 2014). This has been extensively reviewed elsewhere (Kleiger and Mayor, 2014); however, a brief explanation of the UPS is provided here as it pertains to PROTACs. The 76-residue protein ubiquitin is attached to proteins via a lysine isopeptide bond as a post-translational modification (PTM) via a cascade of three enzymes: an E1 activating enzyme, an E2 conjugating enzyme, and an E3 ligase (Figure 1). Free ubiquitin is activated by an E1 in an ATP-dependent process during which it is converted to a C-terminal thioester (Figure 1). Trans-thioesterification passes the ubiquitin from the E1 unto an E2. Finally, an E3 complex facilitates transfer of ubiquitin, either directly or indirectly, to a substrate protein lysine. Ubiquitin itself can, in turn, be ubiquitinated on one or more of its seven surface lysine residues. These PTMs have a multi-

tude of biological functions that are still being investigated (Komander and Rape, 2012; Yau and Rape, 2016), but the canonical role of K48 polyubiquitin is to signal a protein for destruction via the proteasome (Grice and Nathan, 2016). Enzymes involved in protein ubiquitination are present in the human genome in increasing numbers as they progress from E1, of which there are only 2, to E3, with more than 600 postulated E3 family members (Ardley and Robinson, 2005). E3 ligases serve to recruit substrates and facilitate transfer of ubiquitin from an E2 conjugating enzyme to the target protein. When a protein is tagged with poly-K48 ubiquitination and recognized by the proteasome, the ubiquitin chains are removed by proteasome-associated deubiquitinating enzymes (DUBs) and recycled, whereas the protein substrate is unfolded and degraded (Figure 1). Hijacking the UPS System The PROTAC technology allows the UPS system to be chemically co-opted and aimed to degrade a specific target protein. This approach employs E3 ligase ligands, fused via a flexible chemical linker to a targeting element for the protein of interest, to elicit ectopic ubiquitination, resulting in protein degradation (Figure 1). Beginning with proof-of-concept experiments in cell lysates with peptidic ligands (Sakamoto et al., 2001), the technology has matured and is routinely used in cultured cells and in vivo and has even entered clinical trials. Importantly, the technology is now comprised of all-synthetic modular compounds (Bondeson et al., 2015; Winter et al., 2015) that function against a wide range of protein classes and in different subcellular locations, including the cytosol, the nucleus, and the plasma membrane (Burslem et al., 2018b). As we come to better understand this new technology, it is clear that protein-protein contacts between the neo-substrate and E3 complex are a crucial determinant of PROTAC success, as shown in Figure 2 (Bondeson et al., 2018; Gadd et al., 2017; Nowak et al., 2018; Smith Cell 181, April 2, 2020 ª 2019 Elsevier Inc. 1

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Figure 1. Schematic Representation of the Ubiquitin Cycle and How It Can Be Co-opted by PROTACs Shown are ubiquitin-E1 thioester (UBA1 Ub complex, PDB: 3CMM), ubiquitin-E2 thioester (UBE2D3-UbDha complex, PDB: 5IFR), the ubiquitin-E2-E3-substrate complex (model composed of PDB: 1LQB, 5N4W, and 5IFR), the ubiquitin-E2E3-PROTAC-neosubstrate complex (model composed of PDB: 5T35, 5N4W, and 5IFR), and the proteasome (PDB: 5I4G).

available for recruitment provide a greater chance of successful PROTAC development. Applications PROTAC technology has applications in both biological discovery and drug discovery. In many ways, PROTACs represent the chemical equivalent of small interfering RNA (siRNA), albeit allowing removal of a protein at a post-translational level rather than silencing at a post-transcriptional level. Therefore, they are a useful tool for studying the roles of a protein in biological systems in the laboratory. Additionally, the small-molecule nature of PROTACs circumvents issues associated with delivery and biodistribution that hinder clinical applications of siRNA, eliciting great interest from the pharmaceutical industry.

et al., 2019; Zorba et al., 2018). Farnaby et al. (2019) and Roy et al. (2019) have been instrumental in the structural and biophysical characterization of co-operativity in this area. Only a handful of E3 ligases have been employed in the PROTAC technology (Table 1), with most of the reported compounds using either cereblon (CRBN) or Von Hippel-Lindau (VHL) because of the availability of drug-like small molecules that recruit them (Buckley et al., 2012; Winter et al., 2015). cellular inhibitor of apoptosis (cIAP) ligands have also been used but often lead to auto-ubiquitination and degradation of the E3 ligase itself, making them less attractive (Sekine et al., 2008). The use of nutlin compounds to recruit MDM2 played a key role in the development of the first all-small-molecule PROTAC (Schneekloth et al., 2008) and has recently re-emerged as a potent method for targeted protein degradation (Hines et al., 2019). With more than 600 postulated members of the E3 superfamily, it is exciting to watch ligands emerge for new E3 ligases and their application to targeted protein degradation (Ottis et al., 2017; Spradlin et al., 2019). Indeed, some of these new ligands allow degradation only in specific cellular compartments because of their restricted localization (Zhang et al., 2019b). Given the importance of E3/target protein-protein interactions in PROTAC-mediated degradation, more E3s

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PROTACs in Drug Discovery Unsurprisingly, given its small-molecule nature, PROTAC technology is progressing into the clinic for numerous indications. Initial studies have focused on degradation of hormone receptors (Flanagan and Neklesa, 2019), specifically androgen receptors (Neklesa et al., 2019) and estrogen receptors (Flanagan et al., 2019). Notably, these trials are for orally bioavailable PROTACs, highlighting the benefits of PROTACs over therapeutic RNAi (Setten et al., 2019) or traditional selective estrogen downregulators (SERDs) (Patel and Bihani, 2018). Androgen Receptor Androgen receptor (AR) antagonists, such as enzalutamide (Rathkopf and Scher, 2013), have been used therapeutically with great benefits for prostate cancer patients; however, resistance often arises. AR PROTACs have been shown repeatedly to outperform enzalutamide, particularly in resistance contexts of increased androgen levels or mutations in AR that convert antagonists into agonists (Han et al., 2019; Salami et al., 2018). The AR PROTAC ARV-110 is currently in clinical trials for metastatic castration-resistant prostate cancer. Estrogen Receptor The SERDs validated induced estrogen receptor (ER) degradation as a therapeutic strategy, with fulvestrant being Food and Drug Administration (FDA) approved to treat breast cancer (Howell et al., 2004). However, ER PROTACs induce more

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Figure 2. Composition of a PROTAC for BRD-4 Left: structure of VHL ligand bound to VHL (PBD: 6FMI). Center: PROTAC (MZ-1)-induced ternary complex between VHL and BRD-4 (PDB: 5T35). Right: structure of JQ-1 bound to BRD-4 (PDB: 3MXF).

efficient degradation than fulvestrant and possess improved pharmacological properties (Flanagan et al., 2019; Hu et al., 2019). The ER PROTAC ARV-471 is currently in clinical trials for advanced or metastatic ER+/Her2 breast cancer. BRD4 The bromodomain and extraterminal (BET) domain epigenetic reader protein BRD4 is perhaps the most commonly PROTACtargeted protein (reviewed in Yang et al., 2019) and is often used as a test substrate for technological advances in protein degradation (Martin et al., 2019; Spradlin et al., 2019; Ward et al., 2019; Zhang et al., 2019b). Targeting BRD4 using PROTACs is particularly attractive because its degradation results in more robust loss of c-Myc relative to simple BRD4 inhibition, resulting in enhanced apoptosis of various cancer cells both in vitro and in vivo (Lu et al., 2015; Piya et al., 2019; Winter et al., 2015). Indeed, BRD4-targeting PROTACs appear to be potential therapeutics for secondary acute myeloid leukemia (AML) (Saenz et al., 2017, 2019), multiple myeloma (Zhang et al., 2018), mantle cell lymphoma (Sun et al., 2018), diffuse large B cell lymphoma (Jain et al., 2019), and castration-resistant prostate cancer (Raina et al., 2016) in preclinical models. Interestingly, BRD4 PROTACs have also been employed as a test bed for PROTAC resistance mechanisms, revealing that resistance to VHL-recruiting PROTACs can arise from loss of Cul2 and that CRBN-recruiting PROTAC-treated cells can lose CRBN or its cognate E2, UBE2G1 (Ottis et al., 2019; Zhang et al., 2019a). These mechanisms of resistance should be considered when developing PROTACs into therapeutics. Targeting Aggregated Proteins A challenging but potentially very exciting class of targets for protein degradation are aggregation-prone proteins such as alpha-synuclein, transthyretin, and tau (Iadanza et al., 2018). Degradation of these proteins, either in their monomeric or

aggregated state, via direct recruitment to E3 ligases by PROTACs is an enticing approach to treating protein aggregation diseases. Several PROTAC studies have begun to address this challenge by inducing tau degradation (Chu et al., 2016; Silva et al., 2019). Biological Discoveries Enabled by PROTACs Beyond serving as potential drugs, PROTACs are unique tools for the study of protein function, combining the advantages of small molecules with those of other gene silencing and/or editing techniques. Perhaps most importantly, they enable the study of acute protein loss rather than the gradual selection of cells that survive without a given protein, avoiding issues arising from compensatory pathways or reprogramming. PROTACs can also be employed in contexts, such as patient samples, where other techniques may not be amenable. Below are examples where PROTACs enabled discovery or confirmation of biological insights. BCR-ABL-Independent CML Stem Cells The development of imatinib, followed by other BCR-ABL kinase inhibitors, has transformed the lives of chronic myeloid leukemia (CML) patients. However, kinase inhibition is not curative in most patients (Druker, 2009). A population of CML stem cells survives despite inhibition of oncogenic kinase activity (Corbin et al., 2011), and it has been postulated that they depend on kinase-independent roles of BCR-ABL for survival. Unfortunately, this has proven to be challenging to study, especially in patient samples, because of the inherent selection requirements following genetic knockdown. Our laboratory and others have developed PROTACs capable of inducing degradation of BCRABL (Lai et al., 2016; Shibata et al., 2017, 2019). Recently, a collaborative study with Brian Druker (Burslem et al., 2019), employing allosteric BCR-ABL PROTACs, revealed that CML

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Table 1. E3 Ligases Currently Used in PROTACs E3 Adaptor

Native Substrate

Ligand

Key References

VHL

hydroxylated HIF-1a

VHL peptidomimetics

Bondeson et al., 2015; Buckley et al., 2012; Schneekloth et al., 2004

CRBN

glutamine synthetase MEIS2

IMiDs

Lu et al., 2015; Winter et al., 2015

MDM2

p53

idasanutlin

Hines et al., 2019; Schneekloth et al., 2008

b-TRCP

b-catenin

phosphorylated peptide

Sakamoto et al., 2001

cIAP

second mitochondrion-derived activator of caspases (SMAC)

bestatin esters MV-1 LCL161 (SMAC mimetics)

Itoh et al., 2010

RNF4

poly-sumoylated proteins

CCW-16 (covalent fragment)

Ward et al., 2019

RNF14

p21

nimbolide (natural product)

Spradlin et al., 2019

DCAF16

unknown

KB02 (covalent fragment)

Zhang et al., 2019b

patient stem cells are not dependent on the presence of BCR-ABL protein, suggesting that another mechanism drives their proliferation and that alternative treatments should be explored. FLT-3 ITD Plays Non-kinase Roles in AML Internal tandem duplication (ITD) of key FLT-3 subdomains are validated driver mutations in acute myeloid leukemia, but clinical trials have demonstrated that constant and complete kinase inhibition is required for therapeutic benefits (Grunwald and Levis, 2013; Pratz et al., 2009; Smith et al., 2012). We postulated that degradation of mutant FLT-3 ITD could provide a potential therapeutic approach because degradation results in complete and sustained lack of signaling. Conversion of the phase 3 clinical candidate quizartinib (AC220) (Zarrinkar et al., 2009) into a PROTAC resulted in a compound with enhanced antiproliferative effects relative to quizartinib despite being a less potent inhibitor in vitro and in cellulo, revealing non-kinase roles of FLT-3 ITD in AML (Burslem et al., 2018c). TRIM24 as a Key Dependency in AML Similar to BRD4 PROTAC development, PROTACs have also been used to study the bromodomain-containing transcriptional coactivating protein TRIM24. Conversion of the TRIM24 ligand IACS9571 (Palmer et al., 2016) into a PROTAC (Gechijian et al., 2018) generated a potent TRIM24 degrader. Interestingly, although IACS9571 inhibits TRIM24 binding to hyperacetylated chromatin, it fails to induce a strong transcriptional response. However, TRIM24 degradation via PROTAC-mediated VHL recruitment significantly upregulates tumor suppressor genes in AML cells, demonstrating the TRIM24 dependency of acute leukemias. BRD9 Is Critical for Synovial Sarcoma Having developed exquisitely selective BRD9 PROTACs (Remillard et al., 2017) to study this target’s role in the BRG1/ BRM-associated factor (BAF) complex, the degrader was employed to confirm the dependence of synovial sarcoma on the non-canonical BAF complex associated with expression of the SS18-SSX oncogenic fusion protein and/or loss of SNF5 (Brien et al., 2018). Interestingly, malignant rhabdoid tumors also lack SNF-5 and rely on BRD9-containing non-canonical BAF complexes, as evidenced by PROTAC-mediated BRD9 depletion (Michel et al., 2018). This demonstrated that, upon loss of SNF-5, cancer cells remodel the BAF com-

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plex. PROTACs for other components of the BAF complex have also been reported recently (Farnaby et al., 2019). FAK Scaffolding Roles Are Crucial for Cell Migration but Not Proliferation Given that patient studies with focal adhesion kinase (FAK) inhibitors have failed to live up to their preclinical promise, Cromm et al. (2018) incorporated the FAK inhibitor defactinib into a PROTAC to assess the effect of FAK degradation versus inhibition (Cromm et al., 2018). Cell culture studies revealed that, although FAK kinase inhibition is unable to prevent cell migration in wound healing assays and invasion in trans-well assays, FAK degradation was efficacious, underscoring the kinase-independent functions of FAK. Interestingly, both this work and a concurrent study from Boehringer Ingelheim (Popow et al., 2019) failed to phenocopy the antiproliferative effect of short hairpin RNA (shRNA)-mediated FAK depletion (McDonald et al., 2017). Tag-Based Systems Development of a bespoke PROTAC for a potential target may be beyond the means of many academic laboratories; therefore, tag-based methods have been developed to combine genetic modification with the power of PROTAC technology. The two most commonly used PROTAC systems are discussed below. HaloPROTACs The HaloPROTAC system utilizes the HaloTag protein, an engineered bacterial dehalogenase that allows orthogonal, covalent conjugation of a chloroalkane-labeled molecule to a fusion protein of interest (Los et al., 2008). Both VHL-recruiting (Buckley et al., 2015; Tovell et al., 2019) and cIAP-recruiting HaloPROTACs (Tomoshige et al., 2015, 2016) have been reported to induce degradation of various HaloTag fusion substrates, including cytosolic (ERK, MEK, and GFP), endosomal (VPS34 and SGK3) and nuclear-localized (CREB1) proteins. The HaloPROTAC system has also afforded biological insights into multiple systems, as detailed below. The Role of PNPLA3 in Fatty Liver Disease BasuRay et al. (2019) used HaloPROTAC3 to degrade HaloTag fused with patatin-like phospholipase domain-containing protein 3 (PNPLA3) in vivo. An I148M mutation in PNPLA3 is associated with non-alcoholic fatty liver disease, which leads to steatosis via accumulation of triglycerides into lipid droplets

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(Smagris et al., 2015). The I148M mutation results in an approximately 80% reduction in triglyceride hydrolase activity, but, surprisingly, the presence of the reduced or non-catalytic PNPLA3 protein is required for development of hepatic steatosis. PROTAC-mediated in vivo degradation of I148M PNPLA3 returned liver triglycerides in mice to normal, providing additional evidence that mutant PNPLA3 accumulation is responsible for hepatic steatosis. Kinetics of WASH Complex Formation The HaloPROTAC system has also been used to confirm that heat shock factor binding protein 1 (HSBP1) is an assembly factor for the Wiskott–Aldrich syndrome protein and SCAR homolog (WASH) complex, which plays a key role in endosomal sorting. Degradation of a HaloTag:WASH fusion protein and subsequent siRNA knockdown of HSBP1 during PROTAC washout demonstrated that HSBP1 is the assembly factor required for remodeling of CCDC53 homotrimers into the WASH complex (Visweshwaran et al., 2018). This approach highlights the use of PROTACs to enable temporal control of protein levels, enabling pulse-chase experimentation that may otherwise be challenging. dTAG The dTAG system works in a similar way as HaloPROTACs, but instead of tagging protein with the HaloTag protein, the F36V FKBP mutant protein (Clackson et al., 1998) is fused to the protein of interest. The dTAG system uses an F36V-selective ‘‘bump’’ ligand, tethered to a immunomodulatory imide drug (IMiD) derivative to recruit CRBN and subsequently degrade the FKBP fusion protein (Nabet et al., 2018). This system has been shown to be amenable to in vivo experimentation via intraperitoneal (i.p.) injection at 25 mg/kg. Proof-of-principle studies have shown dTAG to be amenable to a wide variety of proteins, including HDAC1, MYC, EZH2, PLK1, and KRASG12V, and the system has been employed to address biological questions, as exemplified below. Basal-like Breast Cancer Cells Are Not MELK Dependent Previous studies using shRNA silencing suggested that basallike breast cancer (BBC) cells depend on maternal embryonic leucine zipper kinase (MELK) expression for survival (Hebbard et al., 2010; Toure´ et al., 2016). However, a thorough study employing selective MELK inhibitors, CRISPR, and PROTAC-mediated degradation discovered that BBC cells are agnostic to MELK levels (Huang et al., 2017). Huang et al. (2017) employed CRISPR gene editing to knock out MELK and observed no effect on proliferation. Similarly, MELK inhibitors exhibited no antiproliferative activity. Concerned that compensatory signaling arose during the selection and/or cloning procedures to explain their observations, they also introduced a dTAG version of MELK prior to knockout of endogenous MELK so that MELK was constantly expressed, circumventing any impetus for compensation to arise. Employment of a dTAG PROTAC to rapidly and selectively induce dTAG-MELK degradation showed no effect on proliferation despite acute MELK loss, confirming that BBC cells are not MELK dependent. Cytosolic Mutant Nucleophosmin Is Crucial for Leukemogenesis Brunetti et al. (2018) used CRISPR/Cas9 gene editing to demonstrate that mutant nucleophosmin (NPM1) relocalization, via nuclear export sequence disruption, could suppress cell prolifera-

tion and induce differentiation of hematopoietic stem cells. This occurs via disruption of the HOX/MEIS1 transcriptional program, consistent with the hypothesis that HOX/MEIS1 genes are master regulators of the hematopoietic lineage (Argiropoulos et al., 2007). To further confirm that lack of cytosolic NPM1 induces this phenotype, Brunetti et. al. (2018) employed the dTAG system to rapidly deplete NPM1 (>85% loss in 4 h). This resulted in the same phenotype, confirming direct correlation between HOX/MEIS1 expression downregulation and lack of cytosolic NPM1. The dTAG system has also been used to confirm the addiction of acute myeloid leukemia cells to ENL(YEATS) domain-containing protein 1 following its identification in a genome wide CRISPR screen (Erb et al., 2017) and to demonstrate that degradation of SNF5 allows re-association of cMyc with chromatin (Weissmiller et al., 2019). It has also been used to demonstrate that YY1 has no direct role in the modulation of replication timing (Sima et al., 2019) and to demonstrate that OCT4 is crucial for localization of Mediator condensates at super-enhancers in embryonic stem cells (Boija et al., 2018). HaloPROTAC versus dTAG Both HaloPROTAC and dTAG are powerful approaches and could conceivably be used simultaneously because of their orthogonality. However, there are subtle differences between the two systems that may determine which system is most suitable for each application. For example, dTAG requires a smaller tag (FKBP12F36V, 12 kDa) to be incorporated into the target protein than HaloTag (33 kDa), which may favor the use of dTAG in congested systems where tags may perturb protein-protein interactions. However, the commercially availability and multitude of other uses for HaloTag fusions (England et al., 2015) makes it the perfect choice when, for example, one wishes to look at subcellular localization of a protein as well as induce its degradation. Furthermore, the HaloPROTAC system does not exhibit the ‘‘hook effect’’ observed with dTAG. Additionally, the use of VHL ligands in the HaloPROTAC approach enables the use of diastereomer controls and avoids issues associated with the use of IMiD-based PROTACs, discussed below. Workflow To use the PROTAC system, it is necessary to either develop de novo a PROTAC capable of targeting the protein of interest or to modify the latter with a tag (described above) to enable degradation via HaloPROTAC or dTAG degrader molecule. Indeed, it may be advantageous to perform preliminary studies with the tagged target protein to establish proof of concept prior to developing a PROTAC to target the endogenous protein. Given the recent advances in genome editing, it is relatively easy to tag a protein of interest at an endogenous locus to enable its ligand-induced degradation without the necessities of target overexpression or developing novel ligands (Brand and Winter, 2019). Displayed in Figure 3 is a typical workflow to develop a PROTAC. The workflow begins, obviously, with selecting a target protein to degrade. Target Selection Some ideal PROTAC targets are (1) proteins without enzymatic function that cannot be modulated with traditional small

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Figure 3. PROTAC Development Workflow

molecules, (2) proteins that have additional roles beyond enzymatic activity (e.g., scaffolding roles of FAK or BCR-ABL), or (3) proteins that, upon inhibition, undergo compensatory upregulation, making it difficult to achieve maximal loss of protein function. It may be useful to look at previous knockdown experiments (e.g., phenotypic genome-wide CRISPR screens or RNAi) or for cell type-specific dependencies during target selection to gather information about the target protein (Tsherniak et al., 2017). There are some proteins for which inhibition is sufficient to abrogate all function and degradation adds little in terms of biology, but these proteins are likely few and far between. In these cases, PROTACs can nevertheless be beneficial in terms of reduced dosage and duration of effect. More excitingly, PROTACs can extend the druggable proteome to any protein that is ligandable. Of the approximately 17,470 observed proteins (Omenn et al., 2018), only 10%–15% are considered druggable (Hopkins and Groom, 2002), and many more have been shown to be ligandable (Backus et al., 2016; Parker et al., 2017), but many of these small-molecule binding events enact no function. The ability to impart activity to otherwise biologically inert ligands via PROTAC conversion greatly expands the druggable proteome.

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PROTAC Development Subsequently, a thorough literature investigation for known target ligands should be performed. If a ligand is available, then inspection of either a co-crystal structure or known structure-activity relationships may reveal a suitable location for linker attachment. At this point, PROTAC conversion can begin, employing synthetic and medicinal chemistry to append a linker and E3 recruiting element to the targeting ligand. If no ligand has been reported previously, or if the reported compounds have questionable structures, then a campaign to identify a ligand for the target protein may be initiated; alternatively, it may be simpler to initially use the HaloPROTAC or dTAG approach. We recommend use of multiple linker lengths and compositions when developing PROTACs, especially when the synthetic approach allows modularity. Linker length and composition can have profound effects; for example, HaloPROTACs with an ethylene glycol unit removed have no ability to induce degradation (Buckley et al., 2015), BCR-ABL PROTACs with an ether replacing an amide demonstrate more cell permeability (Burslem et al., 2019), and lapatinibbased PROTACs can be either dual EGFR/Her2 degraders or selective EGFR degraders, depending on linker length (Burslem et al., 2018b). When the PROTAC candidate molecules or cell line expressing tagged proteins are available, it is crucial to confirm first that they do indeed induce target degradation. Most commonly this is achieved by immunoblotting, although mass spectrometry or flow cytometry can be used, depending on the target protein properties (Buckley et al., 2015). Iterative rounds of screening may be necessary to synthesize a sufficiently potent PROTAC, or it may be necessary to explore several tagging approaches and/or sites of tag incorporation into the target for maximal degradation. We generally recommend an initial PROTAC treatment time of 24 h, but kinetic characterization often reveals that degradation occurs much more rapidly. Recently, bespoke technologies for the study of PROTACs in cells have been developed that may prove to be instrumental in guiding their development (Riching et al., 2018). The synthesis and assay of candidates is normally the rate-limiting step for use of a PROTAC and depends on the quality of the chemical matter available for the target. Starting with a wellcharacterized small molecule provides a distinct advantage, and as more become available via the Target 2035 initiative (Carter et al., 2019), the rate and ease of PROTAC development will no doubt increase. PROTAC Validation When a PROTAC is identified, it is crucial to carefully characterize the degradation event via control experiments—e.g., via qPCR confirmation that protein loss occurs at a post-translational level and not by decreases in mRNA (Bondeson et al., 2015)—prior to its use as a tool. Some small molecules induce apparent degradation of their target protein but actually act at the transcriptional level (Field et al., 2017). It is also crucial to confirm that degradation is occurring via the UPS. To do this, cells can be co-treated with pharmacological modulators of the UPS. We specifically recommend co-treatment with a proteasome inhibitor (epoxomicin [Kim and Crews, 2013] or bortezomib [Paramore and Frantz, 2003]), which should preserve

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protein levels at untreated levels. Additionally, co-treatment with MLN-4924 (pevonedistat), a NEDD8-activating enzyme inhibitor, will confirm that an active E3 ligase is involved in degradation (Cullin ligases require neddylation for activity) (Brownell et al., 2010). Because of the toxicity of these control compounds, it is often necessary to use the shortest incubation time required for significant degradation (e.g., 5 h versus 24 h). Importantly, degradation by application of a small molecule enables use of an inactive analog as a control compound. This is crucial for discovery of novel biology when employing a PROTAC derived from an inhibitor. Typically, an inactive PROTAC can be created by discrete modification of the E3 ligase ligand structure to compromise its activity; the resulting molecule is incapable of inducing degradation but equally potent as the PROTAC at inhibiting the target. We favor use of the VHL-recruiting ligand for careful interrogation of inhibition versus degradation because the simple inversion of stereocenters on the VHL ligand yields a compound with equal inhibitory activity and pharmacological properties, such as cell permeability, enabling it to function as a true control (Burslem et al., 2018b). Inversion of stereocenters can also be employed to generate inactive compounds as controls for MDM2-recruiting PROTACs (Hines et al., 2019). It is possible to compromise the CRBN-recruiting IMiD ligands via omission of a carbonyl or by methylation of the imide functionality; however, this does subtly modulate its physicochemical properties (Burslem et al., 2018a; Huang et al., 2018). Care must be taken when using IMiD analogs because they may still induce degradation of zinc-finger neo-substrates associated with IMiD pathophysiology (Ishoey et al., 2018). Inactive PROTACs are important experimental controls that can confirm or disprove the PROTAC mechanism of degradation, given that ligand binding alone could destabilize the target without necessarily recruiting an E3 ligase (Huang et al., 2017), such as is the case with hydrophobic tagging or SERDs (Gustafson et al., 2015; Neklesa et al., 2011; Wardell et al., 2011). An additional, optional experiment to conduct at this point is a proteomic characterization of the effect induced by PROTAC treatment. Mass spectrometry or reverse-phase protein arrays, for example, can enable the user to acquire selectivity data for the PROTAC. Quantitative proteomics is a powerful tool to identify other proteins, beyond that targeted, that are downregulated by PROTAC treatment. This may, of course, be a biological result of loss of the target protein, such as with c-Myc loss when BRD4 is degraded (Lu et al., 2015; Winter et al., 2015), or a pharmacological result, such as the unexpected degradation of p38a by foretinib-based PROTACs (Bondeson et al., 2018; Smith et al., 2019). Advantages and Disadvantages of PROTACs Advantages Catalytic Mechanism of Action. Because PROTACs operate via an event-driven rather than an occupancy-driven mechanism, they act as catalysts for selective protein degradation in that one molecule of PROTAC induces the ubiquitination of multiple molecules of target protein (Bondeson et al., 2015). Indeed, recent studies demonstrate that as little as 10% E3 ligase occupancy is able to efficiently induce target degradation

(Zhang et al., 2019b), albeit with covalent modification of the E3, creating a permanently reprogrammed E3 ligase. Limited occupancy on the targeting ligand side has also been demonstrated to be sufficient via use of low-affinity ligands (Crew et al., 2018; Smith et al., 2019) or co-treatment with a competitive agonist (Salami et al., 2018). This catalytic nature of PROTACs mimics RNAi. Small-Molecule Nature. Their small-molecule nature makes PROTACs as simple to use in experiments as inhibitors. No specialized transfection reagents, culture conditions, or viruses are required for PROTAC application. Indeed, with the exception of tag-based systems, use of PROTACs requires no genetic modification of the model system, allowing interrogation of completely endogenous systems. This allows direct comparison of degradation versus inhibition with the same methodology, resulting in an enhanced biological understanding of protein function and abrogating the risks of perturbing complex biological systems with, for example, transfection reagents that may either induce effects of their own or mask subtle effects of RNAi (Jacobsen et al., 2009). Another advantage arising from the small-molecule nature of PROTACs is control over the concentration of compound, enabling quantitative control of protein levels. Rather than employing a ‘‘digital’’ on/off switch, such as observed with gene editing, a PROTAC can be used in an ‘‘analog’’ fashion to modulate protein levels between 0%–100%. This may be particularly powerful to address proteins that are overexpressed in a disease state but nevertheless essential in a normal or healthy state. Following a dose-response experiment, it is possible to treat at a dose that induces sub-maximal degradation, potentially restoring overexpressed protein levels down to normal. Temporal Control. PROTACs allow exquisite temporal control of protein levels (degradation can be observed in as little as 1 h), allowing study of acute protein loss in a way not possible with many other techniques. This prevents biological compensation from arising upon deletion of a key protein during selection, as happens with various RNAi techniques as well as CRISPR/ Cas9. A common CRISPR technique is to employ homologydirected repair (HDR) to insert GFP or resistance genes at the perturbed loci (Leonetti et al., 2016), allowing selection of cells that were successfully edited. Although undoubtedly powerful, this multi-day selection could conceivably select a cellular population with an alternative pathway driving proliferation or circumnavigation of issues arising from loss of a particular protein. PROTACs are able to rapidly deplete a protein in an entire cellular population, providing a more accurate method to interrogate functions of a protein in a native context. Additionally, PROTAC withdrawal allows rapid restoration of target protein levels as fast as protein re-synthesis permits. This provides a reversibility enabling elegant experiments, such as those described above for the WASH complex. Although some PROTACs may survive in cells after washout of compound and continue to enact degradation, concurrent addition of excess E3 ligase ligand can competitively prevent any additional degradation (Burslem et al., 2018b). Portability. A further benefit of the PROTAC approach lies in its portability. Generally, if a PROTAC induces ubiquitination and degradation of a protein within one system, then it can be applied

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more broadly, provided the new system has the required machinery (E3 ligase, etc.). This enables rapid screening of protein roles in different cell types without requiring their genetic modification. Furthermore, it enables study of proteins in contexts not amenable to other techniques, such as unculturable patient cells; e.g., CML stem cells, discussed above (Burslem et al., 2019). Another advantage of portability lies in the transition to in vivo experiments and, potentially, clinical translation. Application of PROTACs in vivo requires no genetic manipulation of the animals, enabling more rapid progression of experimentation and the ability to probe unperturbed systems. Although PROTACs may need optimization of their physico-chemical properties for in vivo work, there are many examples of PROTACs functioning in vivo, including in mammals (Bondeson et al., 2015; Burslem et al., 2018c; Jain et al., 2019; Nabet et al., 2018; Winter et al., 2015) and non-human primates (Sun et al., 2019). Despite any potential pharmacokinetic challenges with PROTACs, their small-molecule nature and portability make them readily and directly translational in a way other techniques are not. It is important to note that PROTACs often exhibit better pharmacokinetic properties than would be anticipated from their molecular weight. In fact, is has been possible to develop PROTACs with oral bioavailability in humans, as evidenced by recent reports of phase 1 clinical trials of ARV-110 and ARV-471, which achieved exposures in the efficacious range observed in preclinical studies when administered orally once per day. Disadvantages Discovery Phase. Despite all of the advantages outlined above for PROTACs, they are not without associated challenges. One major disadvantage is the lead time for PROTAC development, which can be relatively lengthy compared with designing and ordering an siRNA or guide RNA. As outlined in Figure 3, PROTACs require not only a ligand for the protein of interest but also its conversion into a PROTAC. This can be a timeconsuming process involving substantial synthetic and medicinal chemistry. The tag-based systems (HaloPROTACs and dTAG) bypass this discovery phase but also abrogate some of the advantages of the PROTAC system concerning portability and lack of genetic manipulation. Off-Target Effects. As with other techniques, off-target effects with PROTACs are possible. Following PROTAC treatment, the use of proteomics to quantify proteins, including those expressed at low levels, is a powerful tool to assess the off-target effects of PROTACs (Savitski et al., 2018). Even when the recruiting element selectivity is known, proteomics can reveal surprising new PROTAC substrates, likely resulting from the additive effects of protein-protein interactions between the protein in question and the E3 ligase (Bondeson et al., 2018). In theory, ligand binding to an E3 ligase could perturb binding of endogenous substrates. Fortuitously, much higher ligand concentrations than typically employed with VHL-recruiting PROTACs (Frost et al., 2016) are required to stabilize its endogenous target, HIF-1a (Burslem et al., 2017); however, off-target effects could be more problematic with other E3 ligases for which the native substrates currently are poorly characterized or unknown. The off-target effects of CRBN-recruiting PROTACs must be very carefully assessed because the IMiD components, alone or

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incorporated into PROTACs, can induce degradation of zincfinger CRBN neo-substrates (Fischer et al., 2014; Ishoey et al., 2018; Kro¨nke et al., 2014; Matyskiela et al., 2016). Hook Effect. PROTACs and other bifunctional molecules exhibit an initially curious but entirely rational phenomenon whereby higher concentrations do not always result in more effect (Douglass et al., 2013). This so-called hook effect results from formation of unproductive dimers at high PROTAC concentration rather than the productive trimeric complex required for degradation. This leads to concerns surrounding pharmacokinetic/pharmacodynamic (PK/PD) relationships and dosing regimens. However, it should be noted that favorable protein-protein interactions between the E3 and target protein appear to increase the maximum concentration that can be used before the hook effect is observed (Buckley et al., 2015; Tovell et al., 2019). Not All Proteins or Subcellular Locations Are Amenable Yet. Finally, because PROTACs are still an emerging technology, they have not yet been demonstrated to function against every protein class or in every subcellular compartment (Figure 4). Cytosolic and nuclear proteins can routinely be degraded (Bondeson et al., 2015); indeed, some E3 ligases allow selective nuclear degradation (Zhang et al., 2019b). Several examples of receptor tyrosine kinase degradation have been reported (Burslem et al., 2018b, 2018c), but no examples of more than single-pass transmembrane proteins have been reported. HaloPROTACs have been employed to degrade proteins localized to the membranes of endosomes (Tovell et al., 2019). Despite hydrophobic tagging experiments having validated unfolded protein responses in both the Golgi and the endoplasmic reticulum (ER) (Hellerschmied et al., 2019; Raina et al., 2014; Serebrenik et al., 2018), degradation of proteins via PROTAC has yet to be reported. Bespoke ligands for ligases localized to these organelles will likely be required to enact PROTAC-mediated degradation in these subcellular locations (Smith et al., 2011). Comparison with Alternatives In this section we will briefly compare PROTACs with other current technologies that enable the study of protein function and discuss applications where one technique may be preferable. RNAi RNAi has become ubiquitous in modern biological research and is an excellent tool to study protein function (Agrawal et al., 2003; Setten et al., 2019). PROTACs in many ways function as chemical siRNA equivalents, although they do differ slightly. PROTACs have the advantage of being experimentally less complex than siRNA because no transfection reagents are required. However, the ease of PROTAC use is juxtaposed with their rigor of development. Thus, for large target libraries, siRNA may be advantageous, whereas, for study of one particular protein, PROTACs have the advantage. Both techniques are ‘‘knockdown’’ approaches and suffer from potential off-target effects, but given the common use of proteomics to characterize PROTACs, the off-targets may be better known. Indeed, PROTACs have been used to invalidate hits from siRNA experiments (Huang et al., 2017; Nunes et al., 2019; Popow et al., 2019). It may also be possible to achieve greater overall reduction in protein levels, particularly for long-lived

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Figure 4. Diagram of a Typical Mammalian Cell, Denoting Locations of Proteins that Are Susceptible to PROTAC-Mediated Degradation Solid lines represent locations that have been targeted with PROTACs. Dashed lines represent locations that have not yet been targeted with PROTACs.

proteins, with PROTACs compared with RNAi because PROTAC activity targets the protein rather than preventing additional expression. Gene Editing CRISPR/Cas9 and related gene editing techniques are certainly efficient in experiments where long-term protein depletion is required. However, the requisite selection is both time consuming and allows biological compensation of the protein loss. PROTACs provide an advantage in terms of kinetics, allowing study of acute protein loss to expedite workflow and potentially provide more relevant details about the endogenous system. Gene editing provides protein knockout as long as each copy of the gene is edited. This is challenging in polyploid cells (e.g., HeLa cells), but because PROTACs act post-transcriptionally, this is not a concern with their use. Post-Translational Protein Degradation There are many other methods for small-molecule-induced post-translational protein degradation (for example, auxininduced degradation and the Shield-1 system), which we have reviewed in detail elsewhere (Burslem and Crews, 2017). However, these rely on the expression of tagged proteins and/or expression of other endogenous proteins; PROTACs do not. Recently, the Trim-Away system was reported (Clift et al., 2017), employing antibodies to target the E3 ligase TRIM21 to proteins, leading to their degradation. However, this requires the expression of TRIM21 protein and for the antibody to be microinjected or electroporated into the cell, thus lacking the small-molecule advantages of PROTACs. Conclusions In this Primer, we demonstrate, with examples and an explanation of the technology, the power of PROTACs as tools to probe biological systems as well as to be potential therapeu-

tics. This targeted protein degradation approach adds a new tool to the experimental biology toolbox, combining the benefits of RNAi and small-molecule inhibitors and providing complementary orthogonality to the pre-existing tools. We hope that PROTACs will become a mainstay in protein function investigation, and we believe that they enable us, as a community, to address biological questions that are currently intractable. For example, the reversibility and kinetic advantages of PROTACs provide tools to temporally control protein levels with much higher resolution that other approaches. This provides the opportunity to compare acute versus chronic loss of a protein and to study the effect of reintroducing that protein. The ability to deplete a protein for a defined time and then restore it to endogenous levels as fast as transcription allows may prove to be useful in the study of protein complex assembly, particularly with respect to order of association. As the PROTAC technology continues to mature, the disadvantages described above will become less significant. For example, currently the largest challenge in the use of PROTACs is the length of the discovery phase. As we continue to develop enhanced knowledge of the UPS, of the requirements for potent PROTACs, and of ligands for protein targets, the discovery phase will be significantly shorter. Similarly, as ligands for additional E3 ligases become available, the likelihood of successful PROTAC development increases, and more cellular locations become available. ACKNOWLEDGMENTS G.M.B. and C.M.C. thank John Hines, PhD, for editing. G.M.B. is a Fellow of The Leukemia and Lymphoma Society. C.M.C. gratefully acknowledges the American Cancer Society and the NIH for support (R35CA197589). DECLARATION OF INTERESTS C.M.C. is a founder, consultant, and shareholder in Arvinas, which supports research in his lab. C.M.C. is an inventor of the following patents: 9500653 (Small-Molecule Hydrophobic Tagging of Fusion Proteins and Induced Degradation of Same), 9632089 (Small-Molecule Hydrophobic Tagging of Fusion Proteins and Induced Degradation of Same), 10145848 (Small-Molecule Hydrophobic Tagging of Fusion Proteins and Induced Degradation of Same), 9938264 (Proteolysis-Targeting Chimera Compounds and Methods of Preparing and Using Same), 7041298 (Proteolysis-Targeting Chimeric Pharmaceutical), 7208157 (Proteolysis-Targeting Chimeric Pharmaceutical), and

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10071164 (Estrogen-Related Receptor Alpha-Based PROTAC Compounds and Associated Methods of Use).

Small Molecule PROTACs to Induce Degradation of HaloTag Fusion Proteins. ACS Chem. Biol. 10, 1831–1837.

REFERENCES

Burslem, G.M., and Crews, C.M. (2017). Small-Molecule Modulation of Protein Homeostasis. Chem. Rev. 117, 11269–11301.

Agrawal, N., Dasaradhi, P.V.N., Mohmmed, A., Malhotra, P., Bhatnagar, R.K., and Mukherjee, S.K. (2003). RNA interference: biology, mechanism, and applications. Microbiol. Mol. Biol. Rev. 67, 657–685. Altmann, K.-H., Buchner, J., Kessler, H., Diederich, F., Kra¨utler, B., Lippard, S., Liskamp, R., Mu¨ller, K., Nolan, E.M., Samori, B., et al. (2009). The state of the art of chemical biology. ChemBioChem 10, 16–29. Amm, I., Sommer, T., and Wolf, D.H. (2014). Protein quality control and elimination of protein waste: the role of the ubiquitin-proteasome system. Biochim. Biophys. Acta 1843, 182–196. Ardley, H.C., and Robinson, P.A. (2005). E3 ubiquitin ligases. Essays Biochem. 41, 15–30. Argiropoulos, B., Yung, E., and Humphries, R.K. (2007). Unraveling the crucial roles of Meis1 in leukemogenesis and normal hematopoiesis. Genes Dev. 21, 2845–2849. Backus, K.M., Correia, B.E., Lum, K.M., Forli, S., Horning, B.D., Gonza´lezPa´ez, G.E., Chatterjee, S., Lanning, B.R., Teijaro, J.R., Olson, A.J., et al. (2016). Proteome-wide covalent ligand discovery in native biological systems. Nature 534, 570–574. BasuRay, S., Wang, Y., Smagris, E., Cohen, J.C., and Hobbs, H.H. (2019). Accumulation of PNPLA3 on lipid droplets is the basis of associated hepatic steatosis. Proc. Natl. Acad. Sci. U.S.A. 116, 9521–9526. Boija, A., Klein, I.A., Sabari, B.R., Dall’Agnese, A., Coffey, E.L., Zamudio, A.V., Li, C.H., Shrinivas, K., Manteiga, J.C., Hannett, N.M., et al. (2018). Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. Cell 175, 1842–1855.e16. Bondeson, D.P., Mares, A., Smith, I.E., Ko, E., Campos, S., Miah, A.H., Mulholland, K.E., Routly, N., Buckley, D.L., Gustafson, J.L., et al. (2015). Catalytic in vivo protein knockdown by small-molecule PROTACs. Nat. Chem. Biol. 11, 611–617. Bondeson, D.P., Smith, B.E., Burslem, G.M., Buhimschi, A.D., Hines, J., Jaime-Figueroa, S., Wang, J., Hamman, B.D., Ishchenko, A., and Crews, C.M. (2018). Lessons in PROTAC Design from Selective Degradation with a Promiscuous Warhead. Cell Chem. Biol. 25, 78–87.e5. Botstein, D. (2010). Technological innovation leads to fundamental understanding in cell biology. Mol. Biol. Cell 21, 3791–3792. Brand, M., and Winter, G.E. (2019). Locus-Specific Knock-In of a Degradable Tag for Target Validation Studies. In Target Identification and Validation in Drug Discovery: Methods and Protocols, J. Moll and S. Carotta, eds. (Springer New York), pp. 105–119. Brien, G.L., Remillard, D., Shi, J., Hemming, M.L., Chabon, J., Wynne, K., Dillon, E.T., Cagney, G., Van Mierlo, G., Baltissen, M.P., et al. (2018). Targeted degradation of BRD9 reverses oncogenic gene expression in synovial sarcoma. eLife 7, e41305. Brownell, J.E., Sintchak, M.D., Gavin, J.M., Liao, H., Bruzzese, F.J., Bump, N.J., Soucy, T.A., Milhollen, M.A., Yang, X., Burkhardt, A.L., et al. (2010). Substrate-assisted inhibition of ubiquitin-like protein-activating enzymes: the NEDD8 E1 inhibitor MLN4924 forms a NEDD8-AMP mimetic in situ. Mol. Cell 37, 102–111. Brunetti, L., Gundry, M.C., Sorcini, D., Guzman, A.G., Huang, Y.-H., Ramabadran, R., Gionfriddo, I., Mezzasoma, F., Milano, F., Nabet, B., et al. (2018). Mutant NPM1 Maintains the Leukemic State through HOX Expression. Cancer Cell 34, 499–512.e9. Buckley, D.L., Van Molle, I., Gareiss, P.C., Tae, H.S., Michel, J., Noblin, D.J., Jorgensen, W.L., Ciulli, A., and Crews, C.M. (2012). Targeting the von Hippel-Lindau E3 ubiquitin ligase using small molecules to disrupt the VHL/HIF1a interaction. J. Am. Chem. Soc. 134, 4465–4468. Buckley, D.L., Raina, K., Darricarrere, N., Hines, J., Gustafson, J.L., Smith, I.E., Miah, A.H., Harling, J.D., and Crews, C.M. (2015). HaloPROTACS: Use of

10 Cell 181, April 2, 2020

Burslem, G.M., Kyle, H.F., Nelson, A., Edwards, T.A., and Wilson, A.J. (2017). Hypoxia inducible factor (HIF) as a model for studying inhibition of protein-protein interactions. Chem. Sci. (Camb.) 8, 4188–4202. Burslem, G.M., Ottis, P., Jaime-Figueroa, S., Morgan, A., Cromm, P.M., Toure, M., and Crews, C.M. (2018a). Efficient Synthesis of Immunomodulatory Drug Analogues Enables Exploration of Structure-Degradation Relationships. ChemMedChem 13, 1508–1512. Burslem, G.M., Smith, B.E., Lai, A.C., Jaime-Figueroa, S., McQuaid, D.C., Bondeson, D.P., Toure, M., Dong, H., Qian, Y., Wang, J., et al. (2018b). The Advantages of Targeted Protein Degradation Over Inhibition: An RTK Case Study. Cell Chem. Biol. 25, 67–77.e3. Burslem, G.M., Song, J., Chen, X., Hines, J., and Crews, C.M. (2018c). Enhancing Antiproliferative Activity and Selectivity of a FLT-3 Inhibitor by Proteolysis Targeting Chimera Conversion. J. Am. Chem. Soc. 140, 16428–16432. Burslem, G.M., Schultz, A.R., Bondeson, D.P., Eide, C.A., Savage Stevens, S.L., Druker, B.J., and Crews, C.M. (2019). Targeting BCR-ABL1 in Chronic Myeloid Leukemia by PROTAC-Mediated Targeted Protein Degradation. Cancer Res. 79, 4744–4753. Carter, A.J., Kraemer, O., Zwick, M., Mueller-Fahrnow, A., Arrowsmith, C.H., and Edwards, A.M. (2019). Target 2035: probing the human proteome. Drug Discov. Today 24, 2111–2115. Chu, T.-T., Gao, N., Li, Q.-Q., Chen, P.-G., Yang, X.-F., Chen, Y.-X., Zhao, Y.F., and Li, Y.-M. (2016). Specific Knockdown of Endogenous Tau Protein by Peptide-Directed Ubiquitin-Proteasome Degradation. Cell Chem. Biol. 23, 453–461. Clackson, T., Yang, W., Rozamus, L.W., Hatada, M., Amara, J.F., Rollins, C.T., Stevenson, L.F., Magari, S.R., Wood, S.A., Courage, N.L., et al. (1998). Redesigning an FKBP-ligand interface to generate chemical dimerizers with novel specificity. Proc. Natl. Acad. Sci. USA 95, 10437–10442. Clift, D., McEwan, W.A., Labzin, L.I., Konieczny, V., Mogessie, B., James, L.C., and Schuh, M. (2017). A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell 171, 1692–1706.e18. Corbin, A.S., Agarwal, A., Loriaux, M., Cortes, J., Deininger, M.W., and Druker, B.J. (2011). Human chronic myeloid leukemia stem cells are insensitive to imatinib despite inhibition of BCR-ABL activity. J. Clin. Invest. 121, 396–409. Crew, A.P., Raina, K., Dong, H., Qian, Y., Wang, J., Vigil, D., Serebrenik, Y.V., Hamman, B.D., Morgan, A., Ferraro, C., et al. (2018). Identification and Characterization of Von Hippel-Lindau-Recruiting Proteolysis Targeting Chimeras (PROTACs) of TANK-Binding Kinase 1. J. Med. Chem. 61, 583–598. Cromm, P.M., Samarasinghe, K.T.G., Hines, J., and Crews, C.M. (2018). Addressing Kinase-Independent Functions of Fak via PROTAC-Mediated Degradation. J. Am. Chem. Soc. 140, 17019–17026. Douglass, E.F., Jr., Miller, C.J., Sparer, G., Shapiro, H., and Spiegel, D.A. (2013). A comprehensive mathematical model for three-body binding equilibria. J. Am. Chem. Soc. 135, 6092–6099. Druker, B.J. (2009). Perspectives on the development of imatinib and the future of cancer research. Nat. Med. 15, 1149–1152. Editorial. (2000). The importance of technological advances. Nat. Cell Biol. 2, E37. England, C.G., Luo, H., and Cai, W. (2015). HaloTag technology: a versatile platform for biomedical applications. Bioconjug. Chem. 26, 975–986. Erb, M.A., Scott, T.G., Li, B.E., Xie, H., Paulk, J., Seo, H.-S., Souza, A., Roberts, J.M., Dastjerdi, S., Buckley, D.L., et al. (2017). Transcription control by the ENL YEATS domain in acute leukaemia. Nature 543, 270–274. Farnaby, W., Koegl, M., Roy, M.J., Whitworth, C., Diers, E., Trainor, N., Zollman, D., Steurer, S., Karolyi-Oezguer, J., Riedmueller, C., et al. (2019). BAF complex vulnerabilities in cancer demonstrated via structure-based PROTAC design. Nat. Chem. Biol. 15, 672–680.

Please cite this article in press as: Burslem and Crews, Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery, Cell (2020), https://doi.org/10.1016/j.cell.2019.11.031

Field, S.D., Arkin, J., Li, J., and Jones, L.H. (2017). Selective Downregulation of JAK2 and JAK3 by an ATP-Competitive pan-JAK Inhibitor. ACS Chem. Biol. 12, 1183–1187. Fields, S. (2001). The interplay of biology and technology. Proc. Natl. Acad. Sci. USA 98, 10051–10054. Fischer, E.S., Bo¨hm, K., Lydeard, J.R., Yang, H., Stadler, M.B., Cavadini, S., Nagel, J., Serluca, F., Acker, V., Lingaraju, G.M., et al. (2014). Structure of the DDB1-CRBN E3 ubiquitin ligase in complex with thalidomide. Nature 512, 49–53.

Huang, H.-T., Dobrovolsky, D., Paulk, J., Yang, G., Weisberg, E.L., Doctor, Z.M., Buckley, D.L., Cho, J.-H., Ko, E., Jang, J., et al. (2018). A Chemoproteomic Approach to Query the Degradable Kinome Using a Multi-kinase Degrader. Cell Chem. Biol. 25, 88–99.e6. Iadanza, M.G., Jackson, M.P., Hewitt, E.W., Ranson, N.A., and Radford, S.E. (2018). A new era for understanding amyloid structures and disease. Nat. Rev. Mol. Cell Biol. 19, 755–773.

Flanagan, J.J., and Neklesa, T.K. (2019). Targeting Nuclear Receptors with PROTAC degraders. Mol. Cell. Endocrinol. 493, 110452.

Ishoey, M., Chorn, S., Singh, N., Jaeger, M.G., Brand, M., Paulk, J., Bauer, S., Erb, M.A., Parapatics, K., Mu¨ller, A.C., et al. (2018). Translation Termination Factor GSPT1 Is a Phenotypically Relevant Off-Target of Heterobifunctional Phthalimide Degraders. ACS Chem. Biol. 13, 553–560.

Flanagan, J., Qian, Y., Gough, S., Andreoli, M., Bookbinder, M., Cadelina, G., Bradley, J., Rousseau, E., Willard, R., Pizzano, J., et al. (2019). ARV-471, an oral estrogen receptor PROTAC degrader for breast cancer. Cancer Research 79, P5-04-18.

Itoh, Y., Ishikawa, M., Naito, M., and Hashimoto, Y. (2010). Protein knockdown using methyl bestatin-ligand hybrid molecules: design and synthesis of inducers of ubiquitination-mediated degradation of cellular retinoic acid-binding proteins. J. Am. Chem. Soc. 132, 5820–5826.

Frost, J., Galdeano, C., Soares, P., Gadd, M.S., Grzes, K.M., Ellis, L., Epemolu, O., Shimamura, S., Bantscheff, M., Grandi, P., et al. (2016). Potent and selective chemical probe of hypoxic signalling downstream of HIF-a hydroxylation via VHL inhibition. Nat. Commun. 7, 13312.

Jacobsen, L., Calvin, S., and Lobenhofer, E. (2009). Transcriptional effects of transfection: the potential for misinterpretation of gene expression data generated from transiently transfected cells. Biotechniques 47, 617–624.

Gadd, M.S., Testa, A., Lucas, X., Chan, K.-H., Chen, W., Lamont, D.J., Zengerle, M., and Ciulli, A. (2017). Structural basis of PROTAC cooperative recognition for selective protein degradation. Nat. Chem. Biol. 13, 514–521.

Jain, N., Hartert, K., Tadros, S., Fiskus, W., Havranek, O., Ma, M.C.J., Bouska, A., Heavican, T., Kumar, D., Deng, Q., et al. (2019). Targetable genetic alterations of TCF4 (E2-2) drive immunoglobulin expression in diffuse large B cell lymphoma. Sci. Transl. Med. 11, eaav5599.

Gechijian, L.N., Buckley, D.L., Lawlor, M.A., Reyes, J.M., Paulk, J., Ott, C.J., Winter, G.E., Erb, M.A., Scott, T.G., Xu, M., et al. (2018). Functional TRIM24 degrader via conjugation of ineffectual bromodomain and VHL ligands. Nat. Chem. Biol. 14, 405–412.

Kim, K.B., and Crews, C.M. (2013). From epoxomicin to carfilzomib: chemistry, biology, and medical outcomes. Nat. Prod. Rep. 30, 600–604.

Grice, G.L., and Nathan, J.A. (2016). The recognition of ubiquitinated proteins by the proteasome. Cell. Mol. Life Sci. 73, 3497–3506.

Komander, D., and Rape, M. (2012). The ubiquitin code. Annu. Rev. Biochem. 81, 203–229.

Grunwald, M.R., and Levis, M.J. (2013). FLT3 inhibitors for acute myeloid leukemia: a review of their efficacy and mechanisms of resistance. Int. J. Hematol. 97, 683–694.

Kro¨nke, J., Udeshi, N.D., Narla, A., Grauman, P., Hurst, S.N., McConkey, M., Svinkina, T., Heckl, D., Comer, E., Li, X., et al. (2014). Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science 343, 301–305.

Gustafson, J.L., Neklesa, T.K., Cox, C.S., Roth, A.G., Buckley, D.L., Tae, H.S., Sundberg, T.B., Stagg, D.B., Hines, J., McDonnell, D.P., et al. (2015). SmallMolecule-Mediated Degradation of the Androgen Receptor through Hydrophobic Tagging. Angew. Chem. Int. Ed. Engl. 54, 9659–9662. Han, X., Wang, C., Qin, C., Xiang, W., Fernandez-Salas, E., Yang, C.-Y., Wang, M., Zhao, L., Xu, T., Chinnaswamy, K., et al. (2019). Discovery of ARD-69 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Androgen Receptor (AR) for the Treatment of Prostate Cancer. J. Med. Chem. 62, 941–964. Hebbard, L.W., Maurer, J., Miller, A., Lesperance, J., Hassell, J., Oshima, R.G., and Terskikh, A.V. (2010). Maternal embryonic leucine zipper kinase is upregulated and required in mammary tumor-initiating cells in vivo. Cancer Res. 70, 8863–8873. Hellerschmied, D., Serebrenik, Y.V., Shao, L., Burslem, G.M., and Crews, C.M. (2019). Protein Folding State-dependent Sorting at the Golgi Apparatus. Mol. Biol. Cell 30, 2296–2308. Hines, J., Lartigue, S., Dong, H., Qian, Y., and Crews, C.M. (2019). MDM2-Recruiting PROTAC Offers Superior, Synergistic Antiproliferative Activity via Simultaneous Degradation of BRD4 and Stabilization of p53. Cancer Res. 79, 251–262. Hopkins, A.L., and Groom, C.R. (2002). The druggable genome. Nat. Rev. Drug Discov. 1, 727–730. Howell, S.J., Johnston, S.R.D., and Howell, A. (2004). The use of selective estrogen receptor modulators and selective estrogen receptor down-regulators in breast cancer. Best Pract. Res. Clin. Endocrinol. Metab. 18, 47–66. Hu, J., Hu, B., Wang, M., Xu, F., Miao, B., Yang, C.-Y., Wang, M., Liu, Z., Hayes, D.F., Chinnaswamy, K., et al. (2019). Discovery of ERD-308 as a Highly Potent Proteolysis Targeting Chimera (PROTAC) Degrader of Estrogen Receptor (ER). J. Med. Chem. 62, 1420–1442. Huang, H.-T., Seo, H.-S., Zhang, T., Wang, Y., Jiang, B., Li, Q., Buckley, D.L., Nabet, B., Roberts, J.M., Paulk, J., et al. (2017). MELK is not necessary for the proliferation of basal-like breast cancer cells. eLife 6, e26693.

Kleiger, G., and Mayor, T. (2014). Perilous journey: a tour of the ubiquitin-proteasome system. Trends Cell Biol. 24, 352–359.

Lai, A.C., Toure, M., Hellerschmied, D., Salami, J., Jaime-Figueroa, S., Ko, E., Hines, J., and Crews, C.M. (2016). Modular PROTAC Design for the Degradation of Oncogenic BCR-ABL. Angew. Chem. Int. Ed. Engl. 55, 807–810. Leonetti, M.D., Sekine, S., Kamiyama, D., Weissman, J.S., and Huang, B. (2016). A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Proc. Natl. Acad. Sci. USA 113, E3501–E3508. Los, G.V., Encell, L.P., McDougall, M.G., Hartzell, D.D., Karassina, N., Zimprich, C., Wood, M.G., Learish, R., Ohana, R.F., Urh, M., et al. (2008). HaloTag: a novel protein labeling technology for cell imaging and protein analysis. ACS Chem. Biol. 3, 373–382. Lu, J., Qian, Y., Altieri, M., Dong, H., Wang, J., Raina, K., Hines, J., Winkler, J.D., Crew, A.P., Coleman, K., and Crews, C.M. (2015). Hijacking the E3 Ubiquitin Ligase Cereblon to Efficiently Target BRD4. Chem. Biol. 22, 755–763. Martin, R., Bryan, M., Marleen, B., Daniele, S., Antonio, M., Michele, P., and Dirk, T. (2019). PHOTACs Enable Optical Control of Protein Degradation. ChemRxiv, 10.26434chemrxiv.8206688.v2. Matyskiela, M.E., Lu, G., Ito, T., Pagarigan, B., Lu, C.C., Miller, K., Fang, W., Wang, N.Y., Nguyen, D., Houston, J., et al. (2016). A novel cereblon modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase. Nature 535, 252–257. McDonald, E.R., 3rd, de Weck, A., Schlabach, M.R., Billy, E., Mavrakis, K.J., Hoffman, G.R., Belur, D., Castelletti, D., Frias, E., Gampa, K., et al. (2017). Project DRIVE: A Compendium of Cancer Dependencies and Synthetic Lethal Relationships Uncovered by Large-Scale, Deep RNAi Screening. Cell 170, 577– 592.e10. Michel, B.C., D’Avino, A.R., Cassel, S.H., Mashtalir, N., McKenzie, Z.M., McBride, M.J., Valencia, A.M., Zhou, Q., Bocker, M., Soares, L.M.M., et al. (2018). A non-canonical SWI/SNF complex is a synthetic lethal target in cancers driven by BAF complex perturbation. Nat. Cell Biol. 20, 1410–1420. Nabet, B., Roberts, J.M., Buckley, D.L., Paulk, J., Dastjerdi, S., Yang, A., Leggett, A.L., Erb, M.A., Lawlor, M.A., Souza, A., et al. (2018). The dTAG system for

Cell 181, April 2, 2020 11

Please cite this article in press as: Burslem and Crews, Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery, Cell (2020), https://doi.org/10.1016/j.cell.2019.11.031

immediate and target-specific protein degradation. Nat. Chem. Biol. 14, 431–441.

Rathkopf, D., and Scher, H.I. (2013). Androgen receptor antagonists in castration-resistant prostate cancer. Cancer J. 19, 43–49.

Neklesa, T.K., Tae, H.S., Schneekloth, A.R., Stulberg, M.J., Corson, T.W., Sundberg, T.B., Raina, K., Holley, S.A., and Crews, C.M. (2011). Small-molecule hydrophobic tagging-induced degradation of HaloTag fusion proteins. Nat. Chem. Biol. 7, 538–543.

Remillard, D., Buckley, D.L., Paulk, J., Brien, G.L., Sonnett, M., Seo, H.-S., Dastjerdi, S., Wu¨hr, M., Dhe-Paganon, S., Armstrong, S.A., and Bradner, J.E. (2017). Degradation of the BAF Complex Factor BRD9 by Heterobifunctional Ligands. Angew. Chem. Int. Ed. Engl. 56, 5738–5743.

Neklesa, T., Snyder, L.B., Willard, R.R., Vitale, N., Pizzano, J., Gordon, D.A., Bookbinder, M., Macaluso, J., Dong, H., Ferraro, C., et al. (2019). ARV-110: An oral androgen receptor PROTAC degrader for prostate cancer. J. Clin. Oncol. 37, 259.

Riching, K.M., Mahan, S., Corona, C.R., McDougall, M., Vasta, J.D., Robers, M.B., Urh, M., and Daniels, D.L. (2018). Quantitative Live-Cell Kinetic Degradation and Mechanistic Profiling of PROTAC Mode of Action. ACS Chem. Biol. 13, 2758–2770.

Nowak, R.P., DeAngelo, S.L., Buckley, D., He, Z., Donovan, K.A., An, J., Safaee, N., Jedrychowski, M.P., Ponthier, C.M., Ishoey, M., et al. (2018). Plasticity in binding confers selectivity in ligand-induced protein degradation. Nat. Chem. Biol. 14, 706–714.

Roy, M.J., Winkler, S., Hughes, S.J., Whitworth, C., Galant, M., Farnaby, W., Rumpel, K., and Ciulli, A. (2019). SPR-Measured Dissociation Kinetics of PROTAC Ternary Complexes Influence Target Degradation Rate. ACS Chem. Biol. 14, 361–368.

Nunes, J., McGonagle, G.A., Eden, J., Kiritharan, G., Touzet, M., Lewell, X., Emery, J., Eidam, H., Harling, J.D., and Anderson, N.A. (2019). Targeting IRAK4 for Degradation with PROTACs. ACS Med. Chem. Lett. 10, 1081–1085.

Saenz, D.T., Fiskus, W., Qian, Y., Manshouri, T., Rajapakshe, K., Raina, K., Coleman, K.G., Crew, A.P., Shen, A., Mill, C.P., et al. (2017). Novel BET protein proteolysis targeting chimera (BET-PROTAC) exerts superior lethal activity than bromodomain inhibitor (BETi) against post-myeloproliferative neoplasm (MPN) secondary (s) AML cells. Leukemia 31, 1951–1961.

Omenn, G.S., Lane, L., Overall, C.M., Corrales, F.J., Schwenk, J.M., Paik, Y.K., Van Eyk, J.E., Liu, S., Snyder, M., Baker, M.S., and Deutsch, E.W. (2018). Progress on Identifying and Characterizing the Human Proteome: 2018 Metrics from the HUPO Human Proteome Project. J. Proteome Res. 17, 4031–4041. Ottis, P., Toure, M., Cromm, P.M., Ko, E., Gustafson, J.L., and Crews, C.M. (2017). Assessing Different E3 Ligases for Small Molecule Induced Protein Ubiquitination and Degradation. ACS Chem. Biol. 12, 2570–2578. Ottis, P., Palladino, C., Thienger, P., Britschgi, A., Heichinger, C., Berrera, M., Julien-Laferriere, A., Roudnicky, F., Kam-Thong, T., Bischoff, J.R., et al. (2019). Cellular Resistance Mechanisms to Targeted Protein Degradation Converge Toward Impairment of the Engaged Ubiquitin Transfer Pathway. ACS Chem. Biol. 14, 2215–2223. Palmer, W.S., Poncet-Montange, G., Liu, G., Petrocchi, A., Reyna, N., Subramanian, G., Theroff, J., Yau, A., Kost-Alimova, M., Bardenhagen, J.P., et al. (2016). Structure-Guided Design of IACS-9571, a Selective High-Affinity Dual TRIM24-BRPF1 Bromodomain Inhibitor. J. Med. Chem. 59, 1440–1454.

Saenz, D.T., Fiskus, W., Manshouri, T., Mill, C.P., Qian, Y., Raina, K., Rajapakshe, K., Coarfa, C., Soldi, R., Bose, P., et al. (2019). Targeting nuclear b-catenin as therapy for post-myeloproliferative neoplasm secondary AML. Leukemia 33, 1373–1386. Sakamoto, K.M., Kim, K.B., Kumagai, A., Mercurio, F., Crews, C.M., and Deshaies, R.J. (2001). Protacs: chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination and degradation. Proc. Natl. Acad. Sci. USA 98, 8554–8559. Salami, J., Alabi, S., Willard, R.R., Vitale, N.J., Wang, J., Dong, H., Jin, M., McDonnell, D.P., Crew, A.P., Neklesa, T.K., and Crews, C.M. (2018). Androgen receptor degradation by the proteolysis-targeting chimera ARCC-4 outperforms enzalutamide in cellular models of prostate cancer drug resistance. Commun. Biol 1, 100.

Paramore, A., and Frantz, S. (2003). Bortezomib. Nat. Rev. Drug Discov. 2, 611–612.

Savitski, M.M., Zinn, N., Faelth-Savitski, M., Poeckel, D., Gade, S., Becher, I., Muelbaier, M., Wagner, A.J., Strohmer, K., Werner, T., et al. (2018). Multiplexed Proteome Dynamics Profiling Reveals Mechanisms Controlling Protein Homeostasis. Cell 173, 260–274.e25.

Parker, C.G., Galmozzi, A., Wang, Y., Correia, B.E., Sasaki, K., Joslyn, C.M., Kim, A.S., Cavallaro, C.L., Lawrence, R.M., Johnson, S.R., et al. (2017). Ligand and Target Discovery by Fragment-Based Screening in Human Cells. Cell 168, 527–541.e29.

Schneekloth, J.S., Jr., Fonseca, F.N., Koldobskiy, M., Mandal, A., Deshaies, R., Sakamoto, K., and Crews, C.M. (2004). Chemical genetic control of protein levels: selective in vivo targeted degradation. J. Am. Chem. Soc. 126, 3748–3754.

Patel, H.K., and Bihani, T. (2018). Selective estrogen receptor modulators (SERMs) and selective estrogen receptor degraders (SERDs) in cancer treatment. Pharmacol. Ther. 186, 1–24.

Schneekloth, A.R., Pucheault, M., Tae, H.S., and Crews, C.M. (2008). Targeted intracellular protein degradation induced by a small molecule: En route to chemical proteomics. Bioorg. Med. Chem. Lett. 18, 5904–5908.

Piya, S., Mu, H., Bhattacharya, S., Lorenzi, P.L., Davis, R.E., McQueen, T., Ruvolo, V., Baran, N., Wang, Z., Qian, Y., et al. (2019). BETP degradation simultaneously targets acute myelogenous leukemia stem cells and the microenvironment. J. Clin. Invest. 129, 1878–1894.

Sekine, K., Takubo, K., Kikuchi, R., Nishimoto, M., Kitagawa, M., Abe, F., Nishikawa, K., Tsuruo, T., and Naito, M. (2008). Small molecules destabilize cIAP1 by activating auto-ubiquitylation. J. Biol. Chem. 283, 8961–8968.

Popow, J., Arnhof, H., Bader, G., Berger, H., Ciulli, A., Covini, D., Dank, C., Gmaschitz, T., Greb, P., Karolyi-O¨zguer, J., et al. (2019). Highly Selective PTK2 Proteolysis Targeting Chimeras to Probe Focal Adhesion Kinase Scaffolding Functions. J. Med. Chem. 62, 2508–2520.

Serebrenik, Y.V., Hellerschmied, D., Toure, M., Lo´pez-Gira´ldez, F., Brookner, D., and Crews, C.M. (2018). Targeted protein unfolding uncovers a Golgi-specific transcriptional stress response. Mol. Biol. Cell 29, 1284–1298. Setten, R.L., Rossi, J.J., and Han, S.P. (2019). The current state and future directions of RNAi-based therapeutics. Nat. Rev. Drug Discov. 18, 421–446.

Pratz, K.W., Cortes, J., Roboz, G.J., Rao, N., Arowojolu, O., Stine, A., Shiotsu, Y., Shudo, A., Akinaga, S., Small, D., et al. (2009). A pharmacodynamic study of the FLT3 inhibitor KW-2449 yields insight into the basis for clinical response. Blood 113, 3938–3946.

Shibata, N., Miyamoto, N., Nagai, K., Shimokawa, K., Sameshima, T., Ohoka, N., Hattori, T., Imaeda, Y., Nara, H., Cho, N., and Naito, M. (2017). Development of protein degradation inducers of oncogenic BCR-ABL protein by conjugation of ABL kinase inhibitors and IAP ligands. Cancer Sci. 108, 1657–1666.

Raina, K., Noblin, D.J., Serebrenik, Y.V., Adams, A., Zhao, C., and Crews, C.M. (2014). Targeted protein destabilization reveals an estrogen-mediated ER stress response. Nat. Chem. Biol. 10, 957–962.

Shibata, N., Ohoka, N., Hattori, T., and Naito, M. (2019). Development of a Potent Protein Degrader against Oncogenic BCR-ABL Protein. Chem. Pharm. Bull. (Tokyo) 67, 165–172.

Raina, K., Lu, J., Qian, Y., Altieri, M., Gordon, D., Rossi, A.M., Wang, J., Chen, X., Dong, H., Siu, K., et al. (2016). PROTAC-induced BET protein degradation as a therapy for castration-resistant prostate cancer. Proc. Natl. Acad. Sci. USA 113, 7124–7129.

Silva, M.C., Ferguson, F.M., Cai, Q., Donovan, K.A., Nandi, G., Patnaik, D., Zhang, T., Huang, H.-T., Lucente, D.E., Dickerson, B.C., et al. (2019). Targeted degradation of aberrant tau in frontotemporal dementia patient-derived neuronal cell models. eLife 8, e45457.

12 Cell 181, April 2, 2020

Please cite this article in press as: Burslem and Crews, Proteolysis-Targeting Chimeras as Therapeutics and Tools for Biological Discovery, Cell (2020), https://doi.org/10.1016/j.cell.2019.11.031

Sima, J., Chakraborty, A., Dileep, V., Michalski, M., Klein, K.N., Holcomb, N.P., Turner, J.L., Paulsen, M.T., Rivera-Mulia, J.C., Trevilla-Garcia, C., et al. (2019). Identifying cis Elements for Spatiotemporal Control of Mammalian DNA Replication. Cell 176, 816–830.e18. Smagris, E., BasuRay, S., Li, J., Huang, Y., Lai, K.M., Gromada, J., Cohen, J.C., and Hobbs, H.H. (2015). Pnpla3I148M knockin mice accumulate PNPLA3 on lipid droplets and develop hepatic steatosis. Hepatology 61, 108–118.

van Steensel, B. (2015). A short guide to technology development in cell biology. J. Cell Biol. 208, 655–657. Visweshwaran, S.P., Thomason, P.A., Guerois, R., Vacher, S., Denisov, E.V., Tashireva, L.A., Lomakina, M.E., Lazennec-Schurdevin, C., Lakisic, G., Lilla, S., et al. (2018). The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes. EMBO J. 37, 97706.

Smith, M.H., Ploegh, H.L., and Weissman, J.S. (2011). Road to ruin: targeting proteins for degradation in the endoplasmic reticulum. Science 334, 1086–1090.

Ward, C.C., Kleinman, J.I., Brittain, S.M., Lee, P.S., Chung, C.Y.S., Kim, K., Petri, Y., Thomas, J.R., Tallarico, J.A., McKenna, J.M., et al. (2019). Covalent Ligand Screening Uncovers an RNF4 E3 Ligase Recruiter for Targeted Protein Degradation Applications. ACS Chem. Biol. 14, 2430–2440.

Smith, C.C., Wang, Q., Chin, C.-S., Salerno, S., Damon, L.E., Levis, M.J., Perl, A.E., Travers, K.J., Wang, S., Hunt, J.P., et al. (2012). Validation of ITD mutations in FLT3 as a therapeutic target in human acute myeloid leukaemia. Nature 485, 260–263.

Wardell, S.E., Marks, J.R., and McDonnell, D.P. (2011). The turnover of estrogen receptor a by the selective estrogen receptor degrader (SERD) fulvestrant is a saturable process that is not required for antagonist efficacy. Biochem. Pharmacol. 82, 122–130.

Smith, B.E., Wang, S.L., Jaime-Figueroa, S., Harbin, A., Wang, J., Hamman, B.D., and Crews, C.M. (2019). Differential PROTAC substrate specificity dictated by orientation of recruited E3 ligase. Nat. Commun. 10, 131.

Weissmiller, A.M., Wang, J., Lorey, S.L., Howard, G.C., Martinez, E., Liu, Q., and Tansey, W.P. (2019). Inhibition of MYC by the SMARCB1 tumor suppressor. Nat. Commun. 10, 2014.

Spradlin, J.N., Hu, X., Ward, C.C., Brittain, S.M., Jones, M.D., Ou, L., To, M., Proudfoot, A., Ornelas, E., Woldegiorgis, M., et al. (2019). Harnessing the anti-cancer natural product nimbolide for targeted protein degradation. Nat. Chem. Biol. 15, 747–755.

Winter, G.E., Buckley, D.L., Paulk, J., Roberts, J.M., Souza, A., Dhe-Paganon, S., and Bradner, J.E. (2015). DRUG DEVELOPMENT. Phthalimide conjugation as a strategy for in vivo target protein degradation. Science 348, 1376–1381.

Sun, B., Fiskus, W., Qian, Y., Rajapakshe, K., Raina, K., Coleman, K.G., Crew, A.P., Shen, A., Saenz, D.T., Mill, C.P., et al. (2018). BET protein proteolysis targeting chimera (PROTAC) exerts potent lethal activity against mantle cell lymphoma cells. Leukemia 32, 343–352. Sun, X., Wang, J., Yao, X., Zheng, W., Mao, Y., Lan, T., Wang, L., Sun, Y., Zhang, X., Zhao, Q., et al. (2019). A chemical approach for global protein knockdown from mice to non-human primates. Cell Discov. 5, 10. Tomoshige, S., Naito, M., Hashimoto, Y., and Ishikawa, M. (2015). Degradation of HaloTag-fused nuclear proteins using bestatin-HaloTag ligand hybrid molecules. Org. Biomol. Chem. 13, 9746–9750. Tomoshige, S., Hashimoto, Y., and Ishikawa, M. (2016). Efficient protein knockdown of HaloTag-fused proteins using hybrid molecules consisting of IAP antagonist and HaloTag ligand. Bioorg. Med. Chem. 24, 3144–3148. Toure´, B.B., Giraldes, J., Smith, T., Sprague, E.R., Wang, Y., Mathieu, S., Chen, Z., Mishina, Y., Feng, Y., Yan-Neale, Y., et al. (2016). Toward the Validation of Maternal Embryonic Leucine Zipper Kinase: Discovery, Optimization of Highly Potent and Selective Inhibitors, and Preliminary Biology Insight. J. Med. Chem. 59, 4711–4723. Tovell, H., Testa, A., Maniaci, C., Zhou, H., Prescott, A.R., Macartney, T., Ciulli, A., and Alessi, D.R. (2019). Rapid and Reversible Knockdown of Endogenously Tagged Endosomal Proteins via an Optimized HaloPROTAC Degrader. ACS Chem. Biol. 14, 882–892. Tsherniak, A., Vazquez, F., Montgomery, P.G., Weir, B.A., Kryukov, G., Cowley, G.S., Gill, S., Harrington, W.F., Pantel, S., Krill-Burger, J.M., et al. (2017). Defining a Cancer Dependency Map. Cell 170, 564–576.e16.

Yang, C.-Y., Qin, C., Bai, L., and Wang, S. (2019). Small-molecule PROTAC degraders of the Bromodomain and Extra Terminal (BET) proteins - A review. Drug Discov. Today. Technol. 31, 43–51. Yau, R., and Rape, M. (2016). The increasing complexity of the ubiquitin code. Nat. Cell Biol. 18, 579–586. Zarrinkar, P.P., Gunawardane, R.N., Cramer, M.D., Gardner, M.F., Brigham, D., Belli, B., Karaman, M.W., Pratz, K.W., Pallares, G., Chao, Q., et al. (2009). AC220 is a uniquely potent and selective inhibitor of FLT3 for the treatment of acute myeloid leukemia (AML). Blood 114, 2984–2992. Zhang, X., Lee, H.C., Shirazi, F., Baladandayuthapani, V., Lin, H., Kuiatse, I., Wang, H., Jones, R.J., Berkova, Z., Singh, R.K., et al. (2018). Protein targeting chimeric molecules specific for bromodomain and extra-terminal motif family proteins are active against pre-clinical models of multiple myeloma. Leukemia 32, 2224–2239. Zhang, L., Riley-Gillis, B., Vijay, P., and Shen, Y. (2019a). Acquired Resistance to BET-PROTACs (Proteolysis Targeting Chimeras) Caused by Genomic Alterations in Core Components of E3 ligase Complexes. Mol. Cancer Ther. 18, 1302–1311. Zhang, X., Crowley, V.M., Wucherpfennig, T.G., Dix, M.M., and Cravatt, B.F. (2019b). Electrophilic PROTACs that degrade nuclear proteins by engaging DCAF16. Nat. Chem. Biol. 15, 737–746. Zorba, A., Nguyen, C., Xu, Y., Starr, J., Borzilleri, K., Smith, J., Zhu, H., Farley, K.A., Ding, W., Schiemer, J., et al. (2018). Delineating the role of cooperativity in the design of potent PROTACs for BTK. Proc. Natl. Acad. Sci. USA 115, E7285–E7292.

Cell 181, April 2, 2020 13