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Y08060: A Selective BET Inhibitor for Treatment of Prostate Cancer Qiuping Xiang, Yan Zhang, Jiaguo Li, Xiaoqian Xue, Chao Wang, Ming Song, Cheng Zhang, Rui Wang, Chenchang Li, Chun Wu, Yulai Zhou, Xiaohong Yang, Guohui Li, Ke Ding, and Yong Xu ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00003 • Publication Date (Web): 13 Feb 2018 Downloaded from http://pubs.acs.org on February 15, 2018

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ACS Medicinal Chemistry Letters

Y08060: A Selective BET Inhibitor for Treatment of Prostate Cancer Qiuping Xiang,†,‡,# Yan Zhang,†,‡,# Jiaguo Li,§,# Xiaoqian Xue,†,‡ Chao Wang,† Ming Song,† Cheng Zhang,†,§ Rui Wang,† Chenchang Li,†,§ Chun Wu,† Yulai Zhou,§ Xiaohong Yang,§Guohui Li,⊥ Ke Ding, ¶ and Yong Xu†* †

Guangdong Provincial Key Laboratory of Biocomputing, Joint School of Life Sciences, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences; Guangzhou Medical University, Guangzhou, China. ‡

University of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China.

§

College of Pharmacy, Jilin University, Changchun, China, No. 1266 Fujin Road, Chaoyang District, Changchun, Jilin 130021, China. ⊥

Laboratory of Molecular Modeling and Design, State Key Laboratory of Molecular Reaction Dynamics, Dalian Institute of Chemical Physics, The Chinese Academy of Sciences, Dalian, Liaoning 116023, China. ¶

School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou 510632, China.

KEYWORDS: bromodomain, BRD4, prostate cancer, epigenetics

ABSTRACT: Prostate cancer is a commonly diagnosed cancer and a leading cause of cancer-related deaths. The bromodomain and extra terminal domain (BET) family proteins have emerged as potential therapeutic targets for the treatment of castration-resistant prostate cancer. A series of 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives were designed and synthesized as selective bromodomain containing protein 4 (BRD4) inhibitors. The compounds potently inhibit BRD4(1) with nanomolar IC50 values, and exhibit high selectivity over most non-BET subfamily members. One of the representative compounds 36 (Y08060) effectively suppresses cell growth, colony formation, and expression of androgen receptor (AR), AR regulated genes, and MYC in prostate cancer cell lines. In in vivo studies, 36 demonstrates a good PK profile with high oral bioavailability (61.54%), and is a promising lead compound for further prostate cancer drug development.

Prostate cancer (PC) is the most common cancer in men worldwide.1-3 The activation of androgen receptor (AR) signaling has a central role in the initiation and progression of prostate cancer, even after castration.4, 5 Endocrine therapy for prostate cancer, which is based on the classic work of Huggins and Hodges,6 has been used to reduce androgen synthesis or block AR activation. Initially, prostate cancer is sensitive to androgen deprivation therapy (ADT), which can induce marked tumor regression and normalize prostate specific antigen (PSA) in serum. Most patients, however, acquire resistance to treatment, thus leading to castrate-resistant prostate cancer (CRPC). Men who develop CRPC always succumb to the disease. 7 Although some success has been achieved with therapies such as Abiraterone and the second generation anti-androgen agent Enzalutamide, which target AR signaling, durable responses to these therapies are limited, presumably due to acquired resistance. 8-12 Clinically, there is an urgent need for alternative therapeutic strategies for CRPC. Asangani and colleagues reported that BRD4 (bromodomaincontaining protein 4) can physically interact with the Nterminal domain of AR and this interaction can be disrupted by (+)-JQ1 (1).13, 14 Bromodomain and extra terminal domain (BET) inhibitors may have a promising therapeutic potential for the treatment of CRPC.7 Several classes of BET bromodomain inhibitors have been discovered, including 1 ((+)-JQ1), 2 (I-BET-762), 3 (PFI-1), and 4 (Y02224) (Figure 1A).14-19

Notwithstanding the discovery of these BET inhibitors, chemicals that have entered clinical trials for CRPC are still limited. Among these, 2, OTX-015, ZEN003694 and GS-5829, are currently being evaluated either as a single agent or in combination with AR-antagonists (Enzalutamide or ARN-509)20-23 in clinical trials in patients with CRPC, but potent and specific BET inhibitors with different chemotypes are still in high demand to assist in our understanding of the therapeutic potential of BET inhibition. Herein, we describe the discovery and optimization of 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives and the efforts which yielded 5-bromo-2-methoxyN-(6-methoxy-2,2-dimethyl-3-oxo-3,4-dihydro-2Hbenzo[b][1,4]oxazin-7-yl)benzenesulfonamide (Y08060, 36) as a specific BRD4 inhibitor with promising therapeutic activity in vivo. X-ray crystallographic analysis reveals that 3 binds to the BRD4(1) bromodomain with three key interactions, which are shown in Figure 1C. The lactam of the quinazolinone forms two hydrogen bonds with the conserved residue Asn140. Oxygen interacts with the conserved residue Tyr97 through a watermediated hydrogen bond. The sulfonamide forms two additional hydrogen bonds with two water molecules. In this paper, we described further optimization of 3 with the aim of improving its oral absorption property (compound 3, after oral dosing in the rat, showed a low oral bioavailability of

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32%). Based on this analysis, we rationally designed two heterocyclic structures, 2,2-dimethyl-2H-benzo[b][1,4]oxazin3(4H)-ones (I) and 2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin3(4H)-ones (II), as BRD4 bromodomain inhibitors by scaffold hopping (Figure 1B). In both series, the 2,2dimethylmorpholin-3-one scaffold was utilized as the acetylated lysine (KAc) mimics and forms important hydrogen bonds with the BRD4(1) protein. The dimethyl group mimics the terminal methyl group of the acetyl lysine and occupies the small pocket surrounded by residues Pro82, Phe83, Val87, and Ile146 (Figure 1C). The sulfonamide moiety was maintained to form hydrogen bond networks with water. To develop compounds with improved affinity for BRD4(1), we performed an extensive structure-activity relationship (SAR) study to analyze the protein-ligand interactions from three different areas, the WPF shelf (which is composed of Trp81, Pro82, Phe83), the acetylated lysine binding region and the BC loop.

nitrophenols (5a−5b) through subsequent procedures including standard acylation and cyclization.24 Compounds 7a−7d were obtained after an alkylation or hydrocarboxylation reaction of 6a−6b with amine or sodium alcoholate, respectively. Reduction of the nitro group was achieved with Pd/C in a hydrogen atmosphere or with iron and ammonium chloride. Direct sulfonylation of 8a−8f with different sulfonyl chlorides yielded the final sulfonamides (13−26 and 32−37) with good to moderate yields.

The 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one derivatives were designed and synthesized as shown in Scheme 1. The amides (6a−6b) were prepared from 2-amino-5-

To optimize the interactions with the WPF shelf, various substituents at the R1 position of 2,2-dimethyl-2Hbenzo[b][1,4]oxazin-3(4H)-one were designed to explore the chemical space with a view to improving the affinity (Table 1).

The synthesis of 2,2-dimethyl-2H-pyrido[3,2-b][1,4]oxazin3(4H)-one derivatives (27−31) is shown in Scheme 2. Compound 10 was synthesized using procedures similar to those used with amides 6a−6b, then reacted with nitric acid and concentrated sulfuric acid to produce the nitro-substituted product (11). Compounds 27−31 could be synthesized similarly using the procedures outlined in Scheme 1.

A

B

C

D

E

Figure 1. (A) Structures of representative BRD4(1) bromodomain inhibitors. IC 50 values for the inhibitors are shown. (B) Design of new BRD4 bromodomain inhibitors. (C) Superimposition of the binding modes of compounds I, II and 3. The crystal structures of BRD4(1) protein and compound 3 were taken from PDB ID 4E96. (D) Co-crystal structure of compound 25 in a complex with BRD4(1) protein (PDB ID: 5Z1R). (E) Co-crystal structure of compound 36 in a complex with the BRD4(1) protein (PDB ID: 5Z1S). The binding site residues are shown as sticks. Hydrogen bond interactions are indicated by yellow dashed lines.

To ensure the compounds reach the WPF shelf and become involved in hydrophobic interactions with residues Trp81, Pro82, and Phe83 of the WPF shelf, alkyl, cycloalkyl or aromatic groups were introduced and compounds 13−17 were synthesized (Table 1). Compounds 13 and 14 show moderate

activity with temperature shifts of 3.9 and 4.5 °C in the thermal shift assay (TSA) and IC50 values of 12.01 and 11.87 μM in the AlphaScreen assay, respectively. Compound 15 in which R1 is a cyclohexyl group, exhibits 3-fold increase of activity with an IC50 value of 3.87 μM. Replacement of the cyclohexyl group in


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ACS Medicinal Chemistry Letters 15 with a thienyl group led to 16, which in the TSA, exhibits potency similar to that of 15 (5.1 °C vs 5.5 °C). Replacement of the cyclohexyl group in 15 with a phenyl group resulted in 17, which shows activity (IC50 = 3.34 μM) similar to that of 15. The results suggest that 17 may serve as a lead compound for discovery of new BRD4(1) inhibitors. An extensive SAR investigation was then conducted, revealing that various substitutions at different positions of the phenyl ring in 17 could have diverse impacts on the BRD4(1) bromodomain inhibitory activity. For instance, the ortho-methyl ester substituted analogue (18) shows moderate activity (IC50 = 9.32 μM and ΔTm = 4.0 °C), but when a Br atom is introduced at the ortho-position, the resulting compound 19 has no activity. Further study showed that compounds bearing methyl or F atom substitutents at the para-position of the phenyl ring exhibit inhibitory activity similar to 17 with IC50 values of 4.63 and 4.79 μM, respectively. Scheme 1. Synthesis of 2,2-dimethyl-2Hbenzo[b][1,4]oxazin-3(4H)-one Derivatives*

*

Reagents and conditions: (a) (i) 2-bromo-2-methylpropanoyl bromide, Et3N, DCM, 0 °C, 4 h. (ii) K2CO3, DMF, 80 °C, 4 h, 50%; (b) amine, K2CO3, DMSO 100 °C, 4 h, 40−70% or CH3ONa, CuI, DMF, 100 °C, 18 h, 60%; (c) Pd/C, H2, CH3OH, rt, 5 h, 70−80%. or Fe, NH4Cl, AcOH, EtOH, H2O, 80 °C, 30 min, 90%; (d) sulfonyl chloride, pyridine, 80 °C, 2 h, 50−90%; (e) BBr3, DCM, 0 °C to rt, 5 h, 90%.

*

Reagents and conditions：(a) (i) 2-bromo-2-methylpropanoyl bromide, DCM, Et3N, 0 °C, 4 h; (ii) K2CO3, DMF, 80 °C, 4 h, 50%; (b) HNO3, H2SO4, rt, 2 h, 17%; (c) Pd/C, CH3OH, H2, rt, 5 h, 90%; (e) sulfonyl chloride, pyridine, 80 °C, 2 h, 50−70%. Next, we further investigated another head group, 2,2dimethyl-2H-pyrido[3,2-b][1,4]oxazin-3(4H)-one, which is similar to 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one except that a nitrogen atom has been introduced at 10-C position (Figure 1B). We combined the 2,2-dimethyl-2Hpyrido[3,2-b][1,4]oxazin-3(4H)-one with the optimal alkyl and aromatic motifs obtained above, leading to compounds 27−31 (Table 1). These compounds show significant loss of inhibition. Compound 28 binds to BRD4(1) with an IC50 value of 25.43 μM, and is approximately 4-fold weaker than 16. Compound 31 is 14-fold less potent than the corresponding compound (22) (40.1 μM vs 2.73 μM ). Compounds 27, 29 and 30 have maximum inhibitions of less than 40% at 20 μM. These results indicate that adding the nitrogen atom at the 10-C position on the head group is detrimental to potency. Therefore, we focused on the scaffold of 2,2-dimethyl-2H-benzo[b][1,4]oxazin-3(4H)one as described below. To understand the structural basis for the activity, we determined the co-crystal structure of 25 bound to BRD4(1). The crystal structure of 25 shows that the binding mode of the head group is similar to that of 3. As shown in Figure 1D, the 2,2dimethyl-2H-benzo[b][1,4]oxazin-3(4H)-one scaffold binds to and forms extensive hydrophobic interactions with the deep and narrow acetyl lysine binding pocket formed by residues Val87, Leu92, Leu94, Tyr97 and Pro82, Phe83, Ile146. The key hydrogen bond interactions with Asn140 and Tyr97 are maintained. The sulfonamide linker provides a suitable angle turn for the 1-bromo-4-methylbenzene group to reach the hydrophobic WPF shelf, as is also seen in the crystal structure of 4 in a complex with BRD4(1). The NH of the sulfonamide linker participates in a hydrogen bond interaction with external water. The water molecule presents a suitable geometry enabling good contact with the methoxyl group on the phenyl ring. All the structure information demonstrates that 2,2-dimethyl2H-benzo[b][1,4]oxazin-3(4H)-one is a good starting point for bromodomain inhibitor development.

When two halogen atoms were added to the phenyl ring at different positions, improved potency was obtained. The 2,4Table 1. Structure-activity relationship of compounds with difluorophenyl compound (22) exhibits improved activity, with modifications of the WPF shelf, acetylated lysine binding rean IC50 of 2.73 μM. The TSA assay also demonstrated stabilizagion and the BC loop. tion of the BRD4(1) protein with a temperature shift of 6.3 °C. The activity of 22 was better than that of other dihalogen substituents. Compound 23, with a 2-chloro-4-fluorophenyl group, has an IC50 value of 1.44 μM and is 2 times more potent than compound 17. The 2-fluoro-3-chlorophenyl compound (24) is essentially inactive, its maximum inhibition being less than 20% TSA AlphaScreen at 20 μM. This result indicates that substitution at this position Compd R1 R2 X is unfavorable. Compounds 25 and 26 were also synthesized, △Tm Inhibition rate IC50 at 20 μM (%) (μM)c (°C)a and bind to BRD4(1) with IC50 values of 1.99 and 2.96 μM, respectively, and are equipotent with 22 and 23, respectively. d --

--

12.0

98.2

0.12

13

H

C

3.9

61.1

12.01

14

H

C

4.5

73.4

11.87

1

Scheme 2. Synthesis of 2,2-dimethyl-2H-pyrido[3,2b][1,4]oxazin-3(4H)-one Derivatives*

--



15

H

C

6.5

97.1

3.87

16

H

C

5.1

81.7

6.54

17

H

C

5.5

80.0

3.34

18

H

C

4.0

76.0

9.32

19

H

C

2.7

26.2

>50

20

H

C

2.9

70.6

4.63

21

H

C

4.5

60.3

4.79

22

H

C

6.3

98.2

2.73

23

H

C

5.7

97.7

1.44

24

H

C

2.4

50

25

H

C

4.5

46.0

1.99

26

H

C

4.5

42.0

2.96

27

H

N

1.4

19.7

>50

28

H

N

2.3

37.9

25.43

29

H

N

1.1

15.2

>50

30

H

N

2.6

33.7

>50

31

H

N

2.3

42.8

40.1

32

Cl

C

4.2

99.0

3.59

33

(CH3)2N-

C

3.6

97.0

4.77

34

C

1.8

79.0

4.73

35

C

0

0

NA

a hydroxyl group (37) without any obvious effect on the BRD4(1) inhibitory potency. The crystal structures of 36 and 37 (Figure 1E and Figure S1) bound to BRD4(1) indicates that a size restriction exists in this area. These results suggest that 5bromo-2-methoxybenzene substitution was most favorable in the WPF region. In fact, this substituent had been validated for compound 4 in our previous study.19 Our modifications at this site have therefore yielded compounds 36 and 37 which have much improved binding affinities over that of our initial compound (25). Bromodomain proteins have a conserved module, and share a common 3D structure pattern of three long helices (helix A−C) and one short helix (helix Z). The acetylated lysine can recognize the bromodomains by forming several hydrogen bonds with the conserved residue Asn140. This selectivity is critical for the success of drug discovery. To investigate the selectivity profile, representative compounds were tested against 8 bromodomain-containing proteins by thermal stability shift assay. As shown in Figure 2A, the results illustrate that all the compounds displayed excellent selectivity for BET subfamily over other non-BET family with the exception of their moderate inhibition of CBP/EP300. The data from the above assays show that 36 and 37 have good profiles for further evaluation. The binding abilities of 36, 37 and reference compound 1 to BRD4(1) were then determined by an isothermal titration calorimetry (ITC) experiment (Figure 2B, Figure S2). Compound 1 showed a Kd value of 92 nM. The Kd of 36 and 37 were determined as 302 and 498 nM, respectively, which is consistent with the data from the AlphaScreen and TSA assays (Table 1).

A

36

CH3O-

C

6.6

100.0

0.69

37

HO-

C

7.2

100.0

0.63

The BC loop is also an active site. To find more potent analogues based on the experimentally determined co-crystal structure of 25 and BRD4(1), we designed various substituents on the R2 group to explore the chemical space of the BC loop for affinity improvement (Table 1). When R2 in compound 25 is replaced by Cl, analogue 32 shows slightly weaker activity than 25 in the TSA and AlphaScreen assays (ΔTm = 4.2 °C and IC50 = 3.59 μM). Compounds 33 and 34, bearing a larger group, dimethylamine or pyrrolidine respectively, show moderate activity. However compound 35, which bears a morpholine group, is inactive. The biochemical activity results showed that a larger group may have steric conflict with residues around this pocket. Then we synthesized compound 36 (Table 1), in which the H atom at the R2 position was replaced by a methoxyl group. Compound 36 has an IC50 value of 0.69 μM and temperature shift of 6.6 °C in the TSA assay. It was also shown that the methoxyl group at R2 position in 36 can be replaced by

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B

Figure 2. Compounds 36 and 37 are potent and selective BRD4(1) inhibitors. (A) The bromodomain selectivity profiles were determined by a TSA assay. Compound concentration, 200 μM; protein concentration, 10 μM. The heat map shows the relative ΔTm values. Red indicates large ΔTm , and green indicates small ΔTm . (B) ITC assay.

Recent studies demonstrated that small molecule inhibitors of BET proteins exhibit in vitro efficacy in models of CRPC and are currently being evaluated in several clinical trials. Potent and specific BET inhibitors such as 1 and 2 effectively inhibit cell growth in prostate cancer cell lines such as C4-2B, LNCaP, and 22Rv1. Our representative compounds were evaluated for their cellular antiproliferative activity and specificity in the C42B, LNCaP, 22Rv1, PC3 and DU145 cell lines (Table 2). They exhibit reasonable potency against C4-2B, LNCaP and 22Rv1 cell lines but could not be well correlated with the other cell


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ACS Medicinal Chemistry Letters lines, indicating that these analogues are more active against AR-dependent prostate cancers. For example, 36 has IC50 values of 3.23 and 4.41 μM in inhibition of cell viability in the C42B and LNCaP cell lines, respectively. Compound 37 has similar potencies in the same cell lines when compared to 36. The results illustrated that our BET inhibitors, similar to compound 1, display excellent cellular specificity. To further investigate the growth inhibitory effects, colony formation assays were performed for the representative compound 36. As in the cell viability assay, 36 reduced colony formation in a dose-dependent manner (Figure 3A and Figure S3). Colony formations were reduced to less than 10% in 22Rv1 and C4-2B cells at 5 μM. Thus, 36 significantly inhibits growth of prostate cancer cells.

To assess the potential of this series of BRD4(1) inhibitors in vivo, compound 36 was selected for preliminary pharmacokinetic (PK) analysis (Table 3). The plasma level of 36 was determined after an i.v. dose of 2 mg/kg or a single oral dose of 10 mg/kg. Encouragingly, compound 36 exhibits reasonable PK properties with an oral AUC value of 3765.71 μg/L·h, a T1/2 value of 1.96 h, and an oral bioavailability of 61.5%. The results indicate that 36 could be advanced for in vivo pharmacological studies. Table 3. Intravenous (iv) and oral (po) pharmacokinetic profiles of compound 36 in ratsa Route Cmax (µg/L) Tmax (h) AUC(0-t) (µg/L·h) AUC(0-∞) (µg/L·h) T1/2 (h) MRT(0-t) (h) Cl (L/h/kg) Vz (L/kg) F（%）

Table 2. Antiproliferative effects against prostate cell lines C42B, LNCaP, 22Rv1, PC3 and Du145 Compd

Cell growth inhibition (IC50, μM)a LNCaP 22Rv1 PC-3 0.16 0.071 3.01

1

C4-2B 0.19

Du145 2.52

15

7.04

8.40

8.02

30.40

35.93

17

13.26

7.52

13.08

40.13

92.56

23

7.16

7.80

7.05

26.95

54.50

25

6.56

6.28

4.85

21.01

30.93

36

3.23

4.41

3.38

17.22

23.19

37

7.75

7.70

7.59

22.85

33.24

a

The IC50 was calculated from cell viability assay by Cell-Titer GLO (Promega).

To investigate whether BRD4(1) inhibitors suppress the ARmediated gene and other oncogene expression in prostate cancer-related cell lines, qRT-PCR was performed in LNCaP cells. As shown in Figure 3B, compound 36 strongly inhibits mRNA expression of full length AR, AR-regulated genes PSA, KLK2 and TMPRSS2. MYC is a known oncogenic driver in prostate cancers. c-MYC mRNA level is also significantly downregulated by 36. The results demonstrated that the BRD4(1) inhibitor (36), by functioning downstream of AR, may have a profound effect on CRPC as it suppress AR-, PSA-, and cMyc- mediated pathways.

ivb 1000.37 0.083 1199.17 1223.82 1.65 1.17 1.64 4.01 --

poc 926.40 1.67 3520.79 3765.71 1.96 2.60 --61.54

a

Compounds were formulated in 5% DMSO, 40% PEG400 and 55% (saline). bA dose of 2 mg/kg. cA dose of 10 mg/kg. Values are given as mean of 3 independent experiments.

In summary, a series of 2,2-dimethyl-2Hbenzo[b][1,4]oxazin-3(4H)-one derivatives have been designed and synthesized as new selective BRD4(1) inhibitors. These compounds potently inhibit BRD4(1) with nanomolar IC50 values and selectively inhibit non-BET bromodomain-containing proteins. One of the most promising compounds (36) has nanomolar IC50 value against BRD4(1) and potently inhibits the proliferation of a panel of prostate cancer cell lines, C4-2B, LNCaP and 22Rv1. This compound also suppresses the gene expression in LNCaP cells and demonstrates favorable pharmacokinetic properties. Our study provides a new research probe and lays a basic foundation for further drug discovery efforts targeting prostate cancer and other cancers. Further optimization of the lead compound (36) based on these SAR results and in vivo tumor xenograft data will be reported in due course. ASSOCIATED CONTENT Supporting Information

A

Experimental procedures for the synthesis, 1H NMR and 13C NMR for final compounds, and details of in vitro assays.

B

This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *Tel: +86-20-32093612; Fax: 86-20-32093612; Email: [email protected] (Y. X.) Author Contributions # These authors contributed equally to this work Figure 3. Growth inhibitory effects of CBP inhibitors in different prostate cancer cell lines. (A) Compound 36 inhibits 22Rv1 cancer cell colony formation. (B) qRT–PCR analysis of full length AR, PSA, KLK2, TRPRSS2 and c-MYC expression in LNCaP cell treated with vehicle, 1 (1 μM or 5 μM), or 36 (5 μM) for 48 h. Data represent mean ± s.d. (n = 3) from one of three independent experiments. NS, not significant.* P ≥ 0.05, ** P ≤ 0.005 by two-tailed Student’s t test.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS We gratefully acknowledge financial support from the National Natural Science Foundation of China (grant 21602222 and 81673357), the “Personalized Medicines − Molecular Signature-



based Drug Discovery and Development”, Strategic Priority Research Program of the Chinese Academy of Sciences (grant No. XDA12020363), the Chinese National Programs for Key Research and Development (grant 2016YFB0201701), the Natural Science Foundation of Guangdong Province (2015A030312014). The authors thank the staffs from BL17U1, BL18U1, BL19U1 beamlines of National Facility for Protein Science Shanghai (NFPS) at Shanghai Synchrotron Radiation Facility, for assistance during data collection. The authors gratefully acknowledge support from the Guangzhou Branch of the Supercomputing Center of Chinese Academy of Sciences.

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