Covalent binding design strategy: A prospective method for discovery of potent targeted anticancer agents

Accepted Manuscript Covalent binding design strategy: A prospective method for discovery of potent targeted anticancer agents Luhong Wang, Jingyuan Zhao, Yao Yao, Changyuan Wang, Jianbin Zhang, Xiaohong Shu, Xiuli Sun, Yanxia Li, Kexin Liu, Hong Yuan, Xiaodong Ma PII:

S0223-5234(17)30738-9

DOI:

10.1016/j.ejmech.2017.09.024

Reference:

EJMECH 9739

To appear in:

European Journal of Medicinal Chemistry

Received Date: 28 May 2017 Revised Date:

13 September 2017

Accepted Date: 14 September 2017

Please cite this article as: L. Wang, J. Zhao, Y. Yao, C. Wang, J. Zhang, X. Shu, X. Sun, Y. Li, K. Liu, H. Yuan, X. Ma, Covalent binding design strategy: A prospective method for discovery of potent targeted anticancer agents, European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.09.024. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Covalent binding design strategy: A prospective method for discovery of potent targeted anticancer agents Luhong Wanga, Jingyuan Zhaob, Yao Yaoc, Changyuan Wanga, Jianbin Zhanga, Xiaohong Shua,

a

College of Pharmacy, Dalian Medical University, Dalian 116044, PR China

b

Clinical Laboratory, Department of Respiratory Medicine, Hematology

Department, the First Affiliated

Hospital of Dalian Medical University, Dalian 116011, PR China ∗

College of laboratory medicine, Dalian Medical University, Dalian 116044, PR China Corresponding author

E-mail address: [email protected] (X. Ma)

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Abstract

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c

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Xiuli Sunb, Yanxia Lib, Kexin Liua, Hong Yuanb and Xiaodong Maa, ∗

Cancer remains the most serious disease that threatens human health. Molecularly targeted cancer therapies, specifically small-molecule protein kinase inhibitors, form an important part of cancer therapy. Targeted covalent modification represents a proven approach to drug discovery with the recent FDA approvals of afatanib,

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ibrutinib, and osimertinib agents, which were designed to undergo an irreversible hetero-Michael addition reaction with a unique cysteine residue of a specific protein. Covalent inhibitors possess numerous advantages, including increased biochemical

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efficacy, longer duration of action, the high potential for improved therapeutic index due to lower effective dose, and the potential to inhibit certain drug resistance

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mechanisms. In this regard, the novel targeted anticancer agents whose activity is presumably dependent upon a hetero-Michael addition reaction with thiols are summarized in this article. Keywords: Cancer; Kinase; Covalent; Design; Inhibitors.

1. Introduction Cancer is a major public health problem worldwide and in the United States, where an estimated 1,688,780 new cancer cases will be diagnosed and 600,920 cancer deaths death will occur in 2017 [1], it is the second leading cause of death. Abnormal 1

ACCEPTED MANUSCRIPT kinase regulation is responsible for more than 200 diseases, notably various cancers. Consequently, protein kinases have been very attractive drug targets for anticancer drug discovery [2-4]. Through July 2016, 30 kinase-targeted drugs had been approved by the U.S. Food and Drug Administration (FDA) (http://www.fda.gov/). Most of the

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protein kinase inhibitors (PKIs) are ATP competitive and are called type I inhibitors. Notably, covalently-bound kinase inhibitors, which target noncatalytic cysteines at the ATP binding site of protein kinases have been successfully designed as potential anticancer agents. To date, numerous covalent kinase inhibitors have been developed

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[5-7]. The most well-known covalent binding kinase inhibitors are afatinib (1) [8], ibrutinib (2) [9-13], and osimertinib (3) [14-16] (Figure 1). The FDA-approved drug

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afatinib is for patients with metastatic non-small-cell lung cancer (NSCLC), which is driven by the epidermal growth factor receptor (EGFR) exon 19 deletions or exon 21 (L858R) substitution mutations. Ibrutinib is the first-in-class Bruton's tyrosine kinase (BTK) inhibitor, for B-cell malignancies. The recently approved drug osimertinib (2015) is used to treat patients with EGFR T790M mutation-positive metastatic

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NSCLC. All three drugs were designed by combining a reversible-inhibitor scaffold possessing competence against their primary targets and an electrophilic functional group capable of covalent Michael addition to cysteine. Generally, acrylamides and

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other α,β-unsaturated groups, boronic acids, and α-halogen ketones are frequently used functional groups to design covalent targets that bind with the enzyme or

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receptor (Figure 2) [17-19].

N

O

Cl

O

O

N O

F N

HN

HN

N

N

O

N

HN

N

O

H2N

O N

N

N 2

NH N

N

1

N

3

Figure 1. Chemical structures of the well-known covalent binding kinase inhibitors afatinib (1), ibrutinib (2), and osimertinib (3). 2

ACCEPTED MANUSCRIPT Covalent inhibitors can possess several advantages over their reversible, noncovalent binding counterparts, including: (1) enhanced biochemical efficiency as competition with endogenous substrates, (2) less frequent dosing which could result in a lower overall patient burden, (3) improved pharmacokinetics property that may lead

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to a lower systemic drug exposure, (4) potential prevention of emergence of drug resistance produced by the continuous target suppression, (5) high efficiency in inhibiting targets with shallow binding sites, which made targets formerly thought of as “druggable” [20-23]. Accordingly, our considerable attention focusing on the

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discovery of potential covalent binding inhibitors against BTK [24-27], and EGFR T790M kinases [28-34], has resulted in a lot of new covalent-binding small molecules

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with improved anticancer activity. From this perspective, the novel biologically relevant compounds whose activity is presumably dependent upon a hetero-Michael addition reaction with thiols in cysteine are summarized in this review article. Significantly, this review suggests covalent binding drug design strategies that enable

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rational approaches to discover more effective anticancer drugs.

O

R

HS

Protein Kinase

Biological Thiols (Cysteine)

Protein Kinase

O

Michael molecule-thiol adducts

AC C

EP

Electrophile Small-molecule Inhibitors

S

R

Cysteine

S

O Small-molecule Inhibitors

Figure 2. Typical interaction mechanism of the covalent inhibitors with protein. 3

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2. EGFR and mutant EGFR inhibitors EGFR, a member of the ErbB family of receptor tyrosine kinases, plays an important role in the regulation of cell growth, differentiation and survival. NSCLC accounts for approximately 85 % of all lung cancers. The 5-year survival rate in

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advanced NSCLC patients is less than 5 % . Activating mutations in the EGFR have been identified in a subset of NSCLC [35,36]. Therefore, EGFR and its family

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EP

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members have emerged as attractive targets for NSCLC therapy.

4

ACCEPTED MANUSCRIPT Cl

Cl F

Cl

F

N O

HN

HN

O

HN

O

N

N

N

O

O

N

N

4

5 Cl

O

N

F

O

O

HN

HN

N N

N

7

R2 N

N N

HN

N

O

X

HN

N

R1

NH

N

N

N N

O

O

O

N

NH N

S

10

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8 R1 = Cl, R2 = Me, X = O 9 R1 = CF3, R2 = MeCO-, X= NH

N

HN

HN

O

N

O

M AN U O

N

HN

11 O N

NH2

O

NH

O

N

N

NH

F

HN

O N

N

O

AC C

N

N

EP

N

CN

O

6

O

HN

HN

N

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O

RI PT

Cl

Cl

12

N

N

N

N

HN

N

O

O N

N

HN

N

14

13

N

O

N HN

N

Cl

N

15

N

Figure 3. The novel covalently binding EGFR and EGFR T790M mutant kinases inhibitors

Gefitinib and erlotinib are the first class of EGFR-targeting therapeutic agents for the treatment of NSCLC. Both are effective inhibitors of wild-type (WT) EGFR, as well as some drug sensitive mutants associated with NSCLC, such as L858R and delE746_A750 [37-39]. However, acquired resistance to these inhibitors frequently develops after a median of 9 to 13 months. The common EGFR mutations with 5

ACCEPTED MANUSCRIPT clinical implications associated with acquired resistance are exon 19 deletions (del19), L858R mutation and the T790M mutation. The major mutation is EGFR T790M which is present in approximately 50 to 60 % of resistant cases [40,41]. The EGFR with the T790M mutation has a modest affinity for gefitinib or erlotinib binding,

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however, this mutation increases the binding affinity for ATP. This change in relative affinity is the primary mechanism whereby the T790M mutation confers drug resistance [42,43]. Second-generation EGFR inhibitors, such as Poziotinib (4) [44], Dacomitinib (5) [45,46], Allitinib (6) [47], and Neratinib (7) [48] have been

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developed to overcome resistance caused by the T790M mutation. These irreversible inhibitors contain an electrophilic Michael addition receptor moiety that can

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covalently alkylate the conserved cysteine residue (Cys797) close to the ATP binding site of EGFR. The formation of the covalent bond allows these irreversible inhibitors to achieve greater occupancy of the ATP site as well as higher selectivity for the EGFR-family tyrosine kinases relative to the reversible inhibitors. Unfortunately, all second-generation EGFR inhibitors possess equivalent binding affinity to wild-type

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and mutant EGFRs, which results in less safety and efficacy in clinic trials [49,50]. Recently, the third-generation EGFR inhibitors, osimertinib (3, AZD9291, mereletinib) [14-16], WZ4002 (8) [51], rociletinib (9, CO-1686) [52], AZ-5104 (10) [16],

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olmutinib (11, HM61713) [53], nazartinib (12) [54], PF-06747775 (13) [55], PF-06747778 (13) [56] and naquotinib (15) [57], have emerged as potential therapeutics to block the growth of EGFR T790M-positive tumors More importantly,

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the third-generation TKIs have a significantly increased potency against EGFR mutants than against the wild-type EGFR. Osimertinib is a novel selective EGFR T790M inhibitors approved by the US FDA in 2015 to treat locally advanced or metastatic NSCLC, when the cancer has the specific T790M mutation in the gene coding for EGFR (Figure 3, Table 1). The co-crystal structures of these exemplary inhibitors with the EGFR T790M enzyme clearly indicate that an important covalent bond between the acryl amide group and the amino acid Cys797 in the EGFR protein (Figure 4) was formed [15]. This irreversible contact may produce the strong inhibitory potency toward the drug resistance. 6

ACCEPTED MANUSCRIPT Table 1. Information of the novel covalently binding EGFR inhibitors. Compds.

Drug Name

Research Code

Status

Indication

BIBW-2992

Approved

NSCLC

AZD9291

Approved

NSCLC

Company

®

Gilotrif 1

(Afatinib dimaleate)

Boehringer Ingelheim

Tagrisso® (Osimertinib mesylate)

6

Poziotinib

phaseⅡ

NOV1201

Dacomitinib

Phase Ⅲ

AST-6;AST-1306

tosylate

Hanmi

NSCLC

PF-299804

Allitinib

cancer;

phaseⅡ

NSCLC

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5

Gastric

HM-78136;

Pfizer

Lung

Allist

cancer;

Pharmaceuticals,

Breast

Inc.

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4

AstraZeneca

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3

cancer

Nerlynx® 7

HKI-272;PF-0528767;

Neratinib

WAY-179272

maleate

WZ4002

8

13 14 15

Olita

Filing(US)

NSCLC

Pfizer Harvard university Clovis Oncology

phaseⅠ

NSCLC

AstraZeneca

Approved

NSCLC

Hanmi

EGF-816

phaseⅡ

NSCLC

Novartis

phaseⅠ

NSCLC

Pfizer

NSCLC

Pfizer

NSCLC

Astellas

®

(Olmutinib) Nazartinib

BI-1482694;

HM-61713;ZL-2303

PF-06747775 PF-06747778

Naquotinib

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12

NDA

AZ5104

10 11

CO-1686

cancer

NSCLC

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Rociletinib

Breast

EP

9

Approved

ASP-8273

Phase Ⅲ

7

ACCEPTED MANUSCRIPT A

B

Met790 Met790 Afatinib

Cys797 Cys797

D

C

Met790

Met790 Met790

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Val726 Leu718

Leu792

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Dacomitinib

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PF-06747775

WZ4002 Met793 Pro794

Cys797

Cys797 Cys797

Gly796

Figure 4. Crystal structure of the novel inhibitors bound to EGFR T790M, A: Afatinib (1) with

3. JAK inhibitors

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EGFR T790M (PDB code: 4G5P) [58]; B: Dacomitinib (5) with EGFR T790M (PDB code: 4I24) [59]; C: WZ4002 (8) with EGFR T790M (PDB code:3IKA) [51]; D: PF-06459988 (14) with EGFR T790M (PDB code: 5HG7) [56].

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Janus kinases (JAK) are non-receptor tyrosine kinases required for signaling through type I/II cytokine receptors. There are four JAK family members in mammals,

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JAK1, JAK2, JAK3, and tyrosine kinase 2 (TYK2), each of which can bind to distinct cytokines and/or growth factor receptors. The JAKs and their downstream effectors, signal transducer and activator of transcription proteins (STATs), form a critical immune cell signaling circuit. Among the JAK family members, JAK3 is unique in having a cysteine residue at the gatekeeper-plus-7 (GK+7) position [60,61]. This residue is Cys909 in human JAK3, and it is structurally equivalent to cysteine residues in EGFR and BTK that have been successfully targeted by covalent kinase inhibitors that are now approved drugs. In 2015, Gray et al. reported a medicinal chemistry campaign to derive potent disubstituted pyrimidine-based inhibitors that 8

ACCEPTED MANUSCRIPT exploit an acrylamide electrophile to form a covalent bond to Cys909 [62]. All their synthesized compounds exhibited effective and selective inhibition of JAK-dependent signaling within different contexts. Among them, 16, 17, and 18 stood out with overall favorable potency, selectivity, and mouse liver microsomes (MLM) stability

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(Table 2). In particular, compound 16 is the most selective JAK3 inhibitor, with at least 70-fold selectivity over other JAKs in the biochemical assays, at least 390-fold selectivity in JAK-transformed Ba/F3 cells, and decent selectivity in primary cells. In addition to the evidence provided by the JAK3−16 cocrystal structure, all three

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inhibitors were shown to covalently modify the Cys909 of JAK3 based on washout and pulldown experiments and mass spectrometry (MS) analysis (Figure 5).

Cl

N R

N

N H

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Table 2. Enzymatic IC50s of Key Compounds.

NH

H N O

Enzymatic activity (IC50, nM)

R

O

17

N

O

JAK

1

2

896

N

1050

JAK3 Cys909

4.8

N N

35

51

<0.5

Figure 5. X-ray cocrystal structure of JAK3 protein−compound 16 (PDB code: 4Z16)

N N

11

32

<0.5

[62].

AC C

18

JAK

EP

16

TE D

Compd

Compd 16

HO

9

ACCEPTED MANUSCRIPT Based on the pyrrolo[2,3-d]pyrimidine scaffold, Telliez et al. attempted to inhibit chemical reactivity by adding substituents to the acrylamide, as steric hindrance around the electrophile should reduce the ability of Glutathione S-transferases (GSTs) to catalyze glutathione (GSH) addition [63]. This approach provided a significant

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reduction in GST-mediated clearance, but unfortunately was not compatible with JAK3 affinity. A major effort by their research group has resulted in the identification of three active JAK3 specific inhibitors 19 (IC50 = 56 nM), 20 (IC50 = 29 nM) and 21 (IC50 = 47 nM), which achieve JAK isoform specificity through covalent interaction

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with a unique JAK3 residue Cys909 (Figure 6).

N

NH

O

N N

N H

19 IC50(JAK3)= 56 nM

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A

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Cys909

B

H N

EP

O

N

AC C

N

N H

20 IC50(JAK3)= 29 nM Cys909

10

ACCEPTED MANUSCRIPT C H N N

O

H

N

RI PT

N N H 21 IC50(JAK3)= 47 nM Cys909

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Figure 6. Crystal structures of pyrrolo[2,3-d]pyrimidine analogues bound to JAK3 with a covalent bond to Cys-909: (a) compound 19 (PDB code: 5TTS); (b) compound 20 (PDB code 5TTV); (c) compound 21 (PDB code 5TTU).

R N N N O O

N

H2N

O O

HN

O

O O S N

N

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4. BTK inhibitors

N

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2 Ibrutinib 22 23 IC50(Btk) < 0.5 nM IC50(Btk) = 0.58 nM IC50(Btk) = 0.72 nM

HN

N

NH

N F 26 Spebrutinib IC50(Btk) < 0.5 nM

O

O N

N

Cl

25 IC50(Btk) = 0.52 nM

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24 IC50(Btk) = 20 nM

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Figure 7. Chemical structures of the novel BTK inhibitors ibrutinib analogues (22-25) and spebrutinib (26).

11

ACCEPTED MANUSCRIPT CF3

R HN

O HN O

PLS-123

N N O

28 R= IC50(Btk) < 4.4 nM

O Cys481

N H O N H

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27 PLS-123 R = IC50(Btk) = 5 nM

RI PT

NH

HN

Figure 8. A: Chemical structures of the novel BTK inhibitor 27 and 28. B: Crystal structure of the

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human Btk in complex with compound 27 [68].

The B-cell receptor (BCR) signaling pathway plays an important role in B-cell development and differentiation. BTK, a TEC family kinase is a crucial component of the BCR pathway and is expressed only in hematopoietic cells except natural killer and T cells [64,65]. Therefore, BTK represents an attractive drug target for treating

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several B-cell lineage cancers. Ibrutinib (2) from Celera/Pharmacyclics/Janssen and spebrutinib (26, CC-292)16 from Celgene are the primary examples of covalent irreversible BTK inhibitors [9-13, 66]. In 2007, Pan et al. identified a series of

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pyrazolo[3,4-d]pyrimidine derivatives as potent BTK inhibitors. The representative compound 2 showed preference towards BTK over closely related kinases (Figure 7), and eventually was used to treat mantle cell lymphoma, chronic lymphocytic

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leukemia, and Waldenstrom's macroglobulinemia [13]. Spebrutinib is another highly selective BTK irreversible inhibitor (IC50 < 0.5 nM), being investigated for the treatment of CLL and other B-cell malignancies (Phase Ⅲ) [66].

12

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N N N QL47

O N

O

Cys481

29 QL47 IC50(Btk) = 7 nM

RI PT

N

Figure 9. Predicted binding mode of QL47 (29) to BTK based upon molecular modeling (PDB code: 3GEN) [69].

2014,

Pan

et

al

designed

and

synthesized

a

series

of

novel

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In

2,5-diaminopyrimidine covalent irreversible inhibitors of BTK. Compared with

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ibrutinib, these molecules exhibited a different selectivity profile for the analyzed kinases as well as a dual-action mode of inhibition of both BTK activation and catalytic activity, which counteracts a negative regulation loop for BTK [67,68]. Typically, compounds 27 and 28, showed potent antiproliferative activities toward multiple B-cell lymphoma cell lines, including germinal center B-cell-like diffuse

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large B cell lymphoma (GCB-DLBCL) cells (Figure 8). Moreover, PLS-123 significantly prevented tumor growth in a mouse xenograft model. Pan et al. also found that PLS-123 exhibited more potent anti-proliferative activity compared with

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ibrutinib in multiple cellular and in vivo models through effective apoptosis induction and the dual-action inhibitory mode of BTK activation. The crystal structure of BTK in complex with compound 27 indicated that it interacts with a cysteine residue

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(Cys481) located at the rim of the ATP-binding pocket in BTK. By using structure-based drug design in conjunction with kinome profiling and

cellular assays, Gray et al develop a potent, selective, and irreversible BTK inhibitor, namely QL47 (29), which covalently modifies Cys481 (Figure 9) [69]. QL47 inhibits BTK activity with an IC50 of 7 nM, inhibits autophosphorylation of BTK on Tyr223 in cells with an EC50 of 475 nM, and inhibits phosphorylation of the downstream effector PLCγ2 (Tyr759) with an EC50 of 318 nM. 5. BMX inhibitors 13

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O N N

NH OMe

O

Compd 30

OMe

O OMe

N H

Cys496

30 IC50(BMX)= 11nM

RI PT

HN

N H NH

Figure 10. Co-crystal structures of compound 30 with BMX kinase (PDB code: 3SXR) [72].

Bone marrow kinase in the X chromosome (BMX) is a nonreceptor tyrosine

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kinase belonging to the TEC family which also includes BTK, ITK, TEC, and TXK kinases. BMX is expressed primarily in bone marrow, but is also widely found in

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granulo/monocytic cells, epithelial and endothelial cells, as well as brain, prostate, heart, and lung cells. BMX participates in signal transduction in response to virtually all types of extracellular stimuli which are transmitted by growth factor receptors, G-protein coupled receptors, cytokine receptors, antigen receptors, and integrins [70,71]. Through a combination of irreversible inhibitor design and type II inhibitor

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design approaches, Liu et al. screened for and identified a highly selective and potent type II irreversible BMX kinase inhibitor 30 (IC50 = 11 nM), which forms a covalent bond with cysteine 496 residue in the DFG-out inactive conformation of BMX

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(Figure 10) [72]. This pyrimidine analogue, 30, displayed a high selectivity profile (S score(1) = 0.01) against the 468 kinases/mutants in the KINOME scan evaluation and

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achieved at least a 40-fold selectivity over BTK. The molecule docking results of 30 and BMX suggested a type II irreversible binding mode, which was demonstrated to be biologically relevant. Compound 30 would serve as a useful pharmacological tool to elucidate the detailed mechanism of BMX mediated signaling pathways. 6. FGFR inhibitors

14

ACCEPTED MANUSCRIPT NEt2 O N H Cl

O

MeO

N

N

NH N

N Cl OMe

RI PT

Cys486

31 FIIN-1 EC50 = 14 nM(Tel-FGFR1-transformed Ba/F3 cells)

Figure 11. Chemical structures of FGFR inhibitor 31.

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Fibroblast growth factor receptors (FGFR1, 2, 3, and 4) are members of a family of polypeptides synthesized by a variety of cell types during the processes of

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embryonic development and in adult tissues. FGFRs have a core structure containing an extracellular ligand-binding domain, a hydrophobic transmembrane domain, and an intracellular kinase domain. Fibroblast growth factors (FGFs) and their receptors perform key roles in multiple biological processes, including tissue repair, hematopoiesis, angiogenesis and embryonic development. Accordingly, FGFRs

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represent promising therapeutic targets in a number of cancers [72,73]. In 2010, Gray et al developed the first potent and selective irreversible inhibitor of FGFR, namely 31 (FIIN-1), which forms a covalent bond with cysteine 486 located in

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the P loop of the FGFR1 ATP binding site [74]. FIIN-1 exhibited nanomolar inhibition of FGFRs and, surprisingly, showed moderate or nearly no affinity to the c-FGR, LIMK1, c-SRC, TNK1, and YES proteins that bear an identical cysteine in P

loop

(Figure

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the

11).

Additionally,

FIIN-1

potently

inhibited

Tel-FGFR1-transformed Ba/F3 cells (EC50 = 14 nM) as well as numerous FGFR-dependent cancer cell lines. This remarkable selectivity towards FGFRs indicates that the selectivity and potency of FIIN-1 is determined primarily by noncovalent FGFR-drug interaction. This study showed that the irreversible FGFR inhibitor FIIN-1 should be considered a promising lead to target FGFR mutants that may emerge in the clinic. 7. NEK2 inhibitors 15

ACCEPTED MANUSCRIPT NEt2

N

O N H

N H O

R

N H

N H O N H 32 SU11652

H N

33 R=

H N

34 R=

35 R= O

N H H N OO

O OEt

O

RI PT

Cl

N H O

R

36 R=

Cl

Figure 12. Chemical structures of typical Nek 2 inhibitors 32-36.

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NEK2 (NIMA related kinase 2), is a homodimeric serine/threonine kinase that localizes to centrosomes, the microtubule organizing centers of the cell. Recent

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advances in the understanding of the biology of NEK2 suggest that its pharmacological inhibition has multifaceted therapeutic potential in bridging targeted and nontargeted routes of chemotherapy. NEK2 is involved in regulating four independent mechanisms of tumor biology: (1) cell-cycle regulation, (2) cell survival, (3) chemosensitization, and (4) metastasis [75-77]. The X-ray co-crystal structures

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reported by Taunton et al showed that compound SU11652 (32, Figure 12) reversibly binds to NEK2. Using structure-based design, oxindoles containing electrophilic groups poised to form a covalent bond with Cys22 of NEK2 were prepared in their

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further optimization [77]. The two acrylamide-containing derivatives, 33 and 34 were not effective, but the more reactive propiolamide 35 and chloromethylketone 36

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functioned as high nanomolar inhibitors of Nek2 were obtained (Figure 12). Although the chloromethylketone 36 was more active than the propiolamide 35 for inhibition of NEK2 in the enzymatic assays (IC50 = 130 nM vs 770 nM), 36 was less active in cells (50% of cells exiting mitosis vs 4% for 35, after treatment with 5 µM of inhibitor for 45 min). This is likely due to the increased reactivity of 36 with thiols (t1/2 = 3 min for 36 vs 60 min for 35 for the reaction with β-mercaptoethanol). Cys22 was confirmed as the target for propiolamide 35 and chloromethylketone 36 by MS analysis of tryptic peptides of the NEK2 kinase domain. Evidently, propiolamide 35 was the first selective NEK2 inhibitor with cellular activity. 16

ACCEPTED MANUSCRIPT 8. PI3Kα inhibitors

IC50 = 6.8 nM (PI3Kα ) = 166.0 nM ( PI3Kβ) = 240.3 nM (PI3Kγ) = 3020.0 nM (PI3Kδ) > 1 µM (PI3KC2A) O > 1 µM (PI3KC3 )

O

N

RI PT

O

N

N CNX-1351

S

N

N NH

N

37 CNX-1351

Cys862

SC

Figure 13. X-ray complex of compound 37 with PI3Kα [76].

Phosphatidylinositol-3-kinases (PI3Ks) are lipid kinases that phosphorylate

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phosphatidylinositol diphosphate (PIP2) to its corresponding phosphatidylinositol triphosphate (PIP3). It has recently been recognized that the PI3K/Akt signaling pathway is a key pathway in cell proliferation, growth, survival, protein synthesis, and glucose metabolism [78,79]. By application of a rational drug design, a representative targeted covalent inhibitor, namely 37 (CNX-1351), which potently and specifically

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inhibits PI3Kα (Figure 13) was created by Singh et al. [80]. They found that the selective inhibitor CNX-1351 covalently modifies PI3Kα on cysteine 862 (C862), an amino acid unique to the α isoform, and that PI3K-β, -γ, and -δ are not covalently

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modified. Compound CNX-1351can to potently (EC50 < 100 nM) and specifically inhibit signaling in PI3Kα-dependent cancer cell lines, and this leads to a potent

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antiproliferative effect (GI50 < 100 nM). Therefore, CNX-1351 is a powerful tool compound that will allow several important questions about PI3K biology to be addressed. Overall, their data demonstrate that the application of a targeted covalent inhibitor strategy to PI3Kα has a number of unique features related to the covalent mechanism of action. 9. Ras inhibitors

17

ACCEPTED MANUSCRIPT H N S O O

N

R

Cl R=

Cl N H

O O

O

Cl

OH

Cl

N H

38 10 µM (24h):

Cl

O

I

N H

39

50%

40

87%

100%

N N

R

R=

Cl

O

Cl

F3C

N H

Cl

O

41 14%

O

Cl

N H

I

42 28%

OH N H

43 100%

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SC

10 µM (24h):

RI PT

O

TE D

Compds 39, 40, 42

EP

Cys12

AC C

Figure 14. Electrophilic compounds 39, 40 and 42 bind to S-IIP of K-Ras (G12C) and disrupt switch-I and switch-II [84].

The RAS proteins are members of a large superfamily of low molecular-weight

GTP-binding proteins. Approximately 30% of all human cancers contain activating Ras mutations, making them one of the most common identifiable molecular drivers of cancer. H-, N-, and K-Ras are the founding members of the Ras superfamily of small GTPases, and are known to transduce signals from various cellular receptors, including receptor tyrosine kinases, G-protein-coupled receptors, and cytokine receptors, to participate in multiple intracellular signaling pathways leading to cell proliferation, differentiation, survival, and gene expression. Certain mutations in the 18

ACCEPTED MANUSCRIPT Ras genes contribute to the establishment and progression of cancers, with K-Ras being the most frequently mutated isoform (86%) among the three Ras genes, followed by N-Ras (11%) and H-Ras (3%). Efforts to target this oncogene directly have encouraged difficulties due to its picomolar affinity for GTP/GDP4 and the

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absence of known allosteric regulatory sites [81-83]. In 2013, Shokat et al reported the development of small molecules that irreversibly bind to a common oncogenic mutant, K-Ras (G12C) [84]. The representative compounds (39, 40, 42, Figure 14) featuring an acrylamide functional group rely on the mutant cysteine for binding and

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therefore do not affect the wild-type protein. Crystallographic studies reveal the formation of a new pocket that is not apparent in previous structures of Ras, beneath

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the effector bindingswitch-II region. Binding of these inhibitors to K-Ras (G12C) disrupts both switch-I and switch-II, subverting the native nucleotide preference to favor GDP over GTP and impairing binding to RAF kinase. 10. Hsp70 inhibitors

The heat shock protein 70 (Hsp70) family members are powerful proteins with

chemotherapy,

and

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major roles in malignancy, such as inhibition of apoptosis, induction of resistance to regulation of the stability of on coproteins. Unlike

the proteins mentioned above, Hsp70, which is not a kinase, but an ATPase, is also

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feasible to be used as cancer targets to design of covalent inhibitors [85,86]. In 2014, Chiosis et al first reported a series pyrimidine derivatives as potential Hsp70 inhibitor

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class rationally designed to bind to a novel allosteric pocket located in the N-terminal domain of the protein (Figure 15) [87]. These ligands contained an acrylamide to take advantage of an active cysteine267 embedded in the allosteric pocket and acted as covalent protein modifiers upon binding. The chemical modifications around the irreversible inhibitor scaffold served to demonstrate that covalent modification is not a requirement for activity within this class of compounds. Fortunately, their structure optimization led to the identification of compound 44, which mimics the biological effects of the irreversible inhibitors at comparable concentrations. In addition, their studies revealed that the acrylamide group could be eliminated altogether by 19

ACCEPTED MANUSCRIPT improving the enthalpy of the binding. This interaction weighs heavily toward the binding of these ligands to the allosteric pocket of Hsp70 (Figure 16). Phe68/Trp90

O N H

S

NH2+

O

O N

N H

N

S O

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H2N

Cys267-S

N

N

N

N

N

N

NH2 RO N N H

N

N

N

N

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O

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44

N

S

OR

45 R=Me 46 R=Et

Compd 45

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Figure 15. The reversible Hsp70 inhibitor 44-46 and its proposed mode of interaction with the Hsp70 pocket.

Compd Compd 44 45

Cys267

Figure 16. Binding interactions of Hsp70 with pyrimidine derivative 45 [88].

In their further structural optimizations on the pyrimidine template, two novel derivatives 45 and 46, which selectively bind to Hsp70 in cancer cells were discovered [88]. Addition of high nanomolar to low micromolar concentrations of these inhibitors to cancer cells leads to a reduction in the steady-state levels of 20

ACCEPTED MANUSCRIPT Hsp70-sheltered oncoproteins, an effect associated with inhibition of cancer cell growth and apoptosis. Their discovered pyrimidine scaffold represents a viable starting point for the development of druglike Hsp70 inhibitors as novel anticancer therapeutics

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11. Conclusion Protein kinases are key regulators that govern complex cellular processes, including cell growth, differentiation, proliferation and apoptosis. Tyrosine kinase inhibitors have achieved enormous success in molecular targeted therapies for cancer.

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Covalent kinase inhibitors, whose activity is presumably dependent upon a hetero-Michael addition reaction with thiols exhibited numerous advantages,

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including the particular ability to overcome drug resistance. A renewed interest in covalent drug design has been led by the recent approval of several covalent drugs for cancer treatment, and the success of targeted covalent inhibitors in enhancing the ligand binding selectivity for structurally-related proteins, increasing the binding affinity for proteins with shallow binding sites and the ability to overcome drug

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resistance. Accordingly, the purpose of this review was to provide an exhaustive coverage of biologically relevant and molecularly complex α,β-unsaturated carbonyls with demonstrated reactivity toward thiols. This review also highlighted the biological

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activity of α,β-unsaturated carbonyls through their cytotoxicity, inhibition of target proteins and sites of covalent modification. From the point of view of this review, it is

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important to understand the factors that govern their ability to form adducts with thiols, especially under biologically relevant conditions (pH = 6−8), when considering α,β-unsaturated carbonyls in the design of covalent inhibitors. Overall, the information presented in this perspective demonstrates the potential of Michael acceptors as modulators of biological activity through examples of the novel anticancer molecules.

Acknowledgements We are grateful to the National Natural Science Foundation of China (No. 81402788), the project of family noninvasive mechanical ventilation system 21

ACCEPTED MANUSCRIPT construction and promotion for AECOPD patients: Health and Family Planning Commission of Liaoning Province (No. 2015B004), and the Cultivation Project of Youth Techstars, Dalian, China (No. 2016RQ043) for the financial support of this research.

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upon a hetero-Michael addition reaction with thiols are summarized.