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Identification of Selective, Cell Active Inhibitors of Protein Arginine Methyltransferase 5 (PRMT5) through Structure-Based Virtual Screening and Biological Assays Fei Ye, Weiyao Zhang, Xiaoqing Ye, Jia Jin, Zhengbing Lv, and Cheng Luo J. Chem. Inf. Model., Just Accepted Manuscript • DOI: 10.1021/acs.jcim.8b00050 • Publication Date (Web): 19 Apr 2018 Downloaded from http://pubs.acs.org on April 21, 2018

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Journal of Chemical Information and Modeling

Identification of Selective, Cell Active Inhibitors of Protein Arginine

Methyltransferase

5

(PRMT5)

through

Structure-Based Virtual Screening and Biological Assays Fei Ye,†,* Weiyao Zhang,† Xiaoqing Ye,† Jia Jin,† Zhengbing Lv,† Cheng Luo‡,* †

College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China

‡

Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai

Institute of Materia Medica, Chinese Academy, Shanghai 201203, China

ABSTRACT Protein arginine methyltransferase 5 (PRMT5), a type II PRMT enzyme, is reported as an important therapeutic target in leukemia and lymphoma. In the present study, based on the combination of virtual screening and biochemical validations, we discovered a series of small-molecule inhibitors targeting PRMT5. Among those, DC_Y134 exhibited the most potent activity with IC50 value of 1.7 µM and displayed good selectivity against other methyltransferases. Further treatment with DC_Y134 inhibited the proliferation of several hematological malignancy cell lines by causing cell cycle arrest and apoptosis. Western blot assays indicated that DC_Y134 reduced the cellular symmetrically dimethylated levels. In addition, we analyzed the binding mode of DC_Y134 through molecular docking, which revealed that DC_Y134 occupies the binding site of substrate arginine and explained the selectivity of this inhibitor. Taken together, compound DC_Y134 could be used to elucidate the biological roles of PRMT5 and serve as a lead compound for treatment of hematologic malignancies.

INTRODUCTION Arginine methylation, an important epigenetic modification, is catalyzed by protein arginine methyltransferases (PRMTs). During the catalytic process, PRMTs ACS Paragon Plus Environment

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transfer one or two methyl groups from Sadenosyl-L-methionine (SAM) to the target arginine residue.1 PRMTs are very important as they participate in diverse biological processes, including cell growth, differentiation, proliferation, and development.2-4 So far, nine mammalian PRMTs have been discovered, which could be classified into three groups according to the products of enzymatic reactions.5 Type I PRMTs (PRMT1, 2, 3, 4, 6 and 8) catalyze the formation of ω-NG monomethylarginine (ω-MMA) and asymmetric ω-NG, NG-dimethylarginine (ω-aDMA); Type II PRMTs (PRMT5, 9) produce ω-MMA and symmetric ω-NG, NG-dimethylarginine (ω-sDMA); Type III PRMTs (PRMT7) generate ω-MMA only.6 PRMT5, the predominant Type II PRMT, could methylate a variety of substrates, including histones H2A arginine 3 (H2AR3), H4 arginine 3 (H4R3), H3 arginine 8 (H3R8)7 and many other non-histone substrates such as p53,8 HOXA9.9 Accordingly, PRMT5 participates in various biological processes, such as transcriptional regulation,3,

8, 10

ribosome biogenesis,11 RNA metabolism2,

12

and cell cycle

regulation.8 Overexpression or dysregulation of PRMT5 has been implicated in diverse cancers, including lung cancer,13 prostate cancer,14 ovarian cancer15 and breast cancer.16 Recently, PRMT5 has been demonstrated to play essential roles in both normal and dysfunctional hematopoiesis,17-19 making PRMT5 a promising therapeutic target for hematologic malignancies, such as chronic myelocytic leukemia (CML),20 acute myeloid leukemia (AML)21, 22 and mantle cell lymphoma (MCL).23 For decades, many efforts have been put in developing inhibitors targeting PRMTs.24-27 However, up to now, very few selective PRMT5 inhibitors have been discovered. In 2015, CMP5 was reported as a first-in-class, highly selective, small-molecule inhibitor of PRMT5, showing an IC50 value lower than 50 µM.28 PRMT5 inhibition mediated by CMP5 led to the disruption the B-cell immortalization, providing a promising therapeutic agent for B-cell lymphomas.28 Meanwhile, EPZ015666 was identified as another selective PRMT5 inhibitor with potent activity (IC50 = 0.022 µM), and this molecule showed antitumor activity in MCL xenograft models.23 Our previous work identified two types of PRMT5 inhibitors with good selectivity, showing IC50 values of 0.33 µM and 2.8 µM respectively.29, 30 Recently, ACS Paragon Plus Environment

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several PRMT inhibitors were discovered with IC50 values in the micromolar range.31, 32

Considering the potential therapeutic value of PRMT5 inhibitors in cancer therapy,

there is an urgent need to discover more novel PRMT5 inhibitors. Here, by using structure-based virtual screening and biological experiments, we identified a hit compound toward PRMT5. Subsequent similarity-based analog searching led to the discovery of DC_Y134, a novel and selective PRMT5 inhibitor with increased activity (IC50 = 1.7 µM). Molecular docking study was carried out to investigate the binding mode of DC_Y134, suggesting that this compound occupies the substrate-binding pocket. Further cellular experiments revealed that DC_Y134 induced growth inhibition, cell cycle arrest, cell apoptosis and reduction of the cellular symmetrically dimethylated levels in leukemia cells. Therefore, DC_Y134 could be further optimized and used as a lead compound for treatment of PRMT5-related hematologic malignancies.

METHODS Virtual-Screening Protocol Protein Preparation. The crystal structure of human PRMT5 in complex with SAM and the inhibitor EPZ015666 (PDB ID 4X61) 23 was selected for docking-based virtual screening. After removing the water molecules, ions and cofactors, the receptor structure was prepared by the Protein Preparation Wizard Workflow integrated in the Maestro 9.0.33 Ligand Database Preparation. The ligand database is prepared by the same methods described previously.25 The SPECS database which contains ~200,000 compounds was chosen for the virtual screening. Considering the drug-like properties of the candidates, we filtered the SPECS database using Lipinski’s rule of five34 and removed “pan-assay interference compounds (PAINS)”,35, 36 the remaining 139,176 molecules were then prepared with LigPrep37 provided in the Maestro 9.0, the protonation states were generated with Epik38 with a pH of 7.0 ± 2.0. Virtual Screening protocol. The program Glide39 embedded in Maestro 9.0 was

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employed to perform the molecular docking studies. To validate the applicability of this docking program, the compound EPZ015666 was re-docked into the prepared protein structure with a root-mean-square deviation (RMSD) value of 0.4971 Å (Glide SP mode) and 0.1965 Å (Glide XP mode), respectively (Figure S1), indicating program Glide was able to reproduce EPZ015666’s active conformation. The grid box was set to 50 Å × 50 Å × 50 Å around the ligand EPZ015666, which is large enough to cover the two potential binding sites: the SAM-binding pocket and the substrate arginine-binding pocket. Subsequently, Glide SP mode and Glide XP mode were utilized to dock the prepared ligand database into the defined docking grid. The top-ranked 1000 compounds were clustered to 60 groups by SciTegic functional class fingerprints (FCFP_4) in Pipeline Pilot,40 1-3 compounds were selected from each group based on visual inspection. Finally, 120 compounds were chosen for biological assays.

Similarity-Based Analog Searching Similarity Searching was carried out by the same methods described previously,41 which run a two-dimensonal similarity search through the prepared SPECS database using Pipeline Pilot.40 Finally, 21 compounds were selected for biological validations.

PRMT5 Inhibition Assays The inhibition activities against PRMT5 were performed by Shanghai Chempartner Co., Ltd, using 3H-labeled radioactive methylation assay. Biotinylated H4 derived peptide and [3H]-SAM (cat#2146246, PerkinElmer) were utilized as substrate, SAH (cat#A9384, Sigma) was utilized as positive control, the radioactive methylation assays were carried out by the same method described previously.30 In simple terms, firstly, the compounds were diluted to customized concentrations and transferred into the assay plate. Then the PRMT5 (cat#51045, BPS) solution was diluted and incubated with the compounds in succession. After 15 minutes incubation, ACS Paragon Plus Environment

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the cofactor and substrate solutions were added into the plate in proper order, which means the beginning of the enzymatic reaction. Finally, cold SAH was used to stop the reaction and the solution was transferred into Flashplate to measure. All the data was processed by GraphPad Prism 5.0 software.

Selectivity Assay The

inhibition

activities

of

compounds

against

PRMT1

and

DNA

methyltransferase 1 (DNMT1) were carried out by Shanghai Chempartner Co., Ltd, using 3H-labeled radioactive methylation assays according to the method described previously.29, 42 In brief, DNMT1 emzyme solution (DNMT1: cat#51101, BPS) were pre-incubated with the diluted compounds and SAH (SAH: cat#A9384, Sigma) as positive control. After 15 minutes, the substrate (Poly (dI-dC) . poly (dI-dC): cat# P4929, sigma) and cofactor (3H-SAM: cat#2146246, PerkinElmer Inc) were sequentially added into the plate and started the reaction. Finally, cold SAM solution (SAM: cat#A7007, Sigma) was used to stop the reaction and the signals were detected with MicroBeta. The inhibitory activities of compounds against coactivator-associated arginine methyltransferase 1 (CARM1, PRMT4), euchromatic histone lysine methyltransferase (G9a) and enhancer of zeste homolog 2 (EZH2) were performed by Shanghai Chempartner Co., Ltd, also using AlphaLISA assays. In the experiment, SAH (SAH: cat#A9384, Sigma) was used as the positive control for CARM1, G9a and GSK-126 (GSK-126: cat#M60071, Xcessbi) was used for EZH2. The compounds were pre-incubated with corresponding emzyme solution (CARM1: cat#51047, BPS; G9a: cat#51001, BPS; EZH2: cat#51004, BPS) for 15 minutes at RT. Next, the corresponding substrate mixture (SAM: cat#A7007, Sigma; corresponding designed peptides ) was added to start the reaction. After 1 hour reaction, the beads mixture was added and incubate at RT, subdued light for second 1 hour. Finally, the signals were detected with EnSpire.



Cell Viability Assay Cellular experiments was measured by the method described previously.29 Briefly, Cells were plated into 96-well plates in an appropriate density and then incubated with DC_Y134 at different concentrations or DMSO control for 1~3 days respectively. The long-term viability assays were performed according to the method described previously.29 Cell Titer-Glo luminescent assays (cat#G7572, Promega) were carried out to measure cell viability following the manufacturer's protocol in corresponding time point. The data was calculated and normalized in GraphPad Prism 5.0.

Flow Cytometric Analysis MV4-11 cell line was seeded in 6-well plates and then incubated with DC_Y134 at different concentrations or DMSO. For apoptotic assays, cells harvested after 72 hours treatment were washed with pre-cooling PBS twice, then measured by Annexin V-FITC Apoptosis Detection Kit (Vazyme Biotech). For cell-cycle assays, cells were incubated with DC_Y134 for 96 hours. After fixing with 70% (v/v) ethanol overnight, cells were then stained in the dark using Cell Cycle and Apoptosis Analysis Kit (Yeasen Biotech) following the manufacturer's protocol. All samples were observed by FACSCalibur (BD Pharmingen) and results were analyzed by ModFit, LT and FlowJo V7.6.1 software respectively.

Western Blot MV4-11 cell line was seeded in 6 -well plates in an appropriate density and then then incubated with DC_Y134 at different concentrations or DMSO control. After 4 days treatment, the numbers of cells were quantified using the Cell Titer-Glo Luminescent Cell Viability assays (cat#G7572, Promega) and then cells were harvested. Following PBS washes twice, cells were lysed in 1× SDS sample buffer in equal ratio and boiled for about 10 min. 4%-12% SDS−PAGE were run and


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transferred to nitrocellulose membranes (cat#66485, Pall Life Sciences). Blots were blocked with 5% skimmed milk for 30 minutes at RT, then incubated with primary antibody (SDMA: cat#13222S, Cell Signaling Technology; GAPDH: cat# 60004-1-lg, Proteintech) at 4 °C overnight. Next day, after being incubated with HRP-conjugated Goat Anti-Rabbit IgG (cat#D110058-0100, Sangon Biotech), the blots were visualized by Amersham Imager 600.

RESULTS AND DISCUSSION Structure-Based Virtual Screening Currently, several X-ray crystal structures of PRMT5 have been reported,23, 43-46 and they show that there are two potential ligand binding sites in PRMT5: a SAM-binding site and a substrate arginine-binding site (Figure S2A). Here, the crystal structure which contains the cofactor SAM and a substrate-competitive inhibitor EPZ015666 (PDB ID 4X61) 23 was selected for structure-based virtual screening. To enrich the hit discovery rate, we defined a ligand box including both of the two binding sites. As shown in Figure 1A, the prepared ligands were docked into the defined docking grids of PRMT5 utilizing the Glide39 SP and XP modes. The top-ranked 1000 compounds were chosen and then clustered into 60 groups for visual inspection. Consequently, 120 compounds with diverse scaffolds were chosen for subsequent biological evaluation at enzymatic level.

PRMT5 Enzyme Inhibition Assays The 120 candidate compounds mentioned above were measured for inhibitory activity against PRMT5 by 3H-labeled radioactive methylation assay. Among them, five compounds displayed pronounced inhibition against PRMT5, inhibiting PRMT5 activity by >50% at 100 µM (blue bars in Figure 1B). Next, the IC50 values against PRMT5 were determined (Table S1). Among the five compounds, one hit compound DC_Y53 displayed the best inhibitory activity (IC50 = 5.7 µM, Figure 2A).



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Similarity-Based Analog Searching To validate the scaffold of DC_Y53 as well as to obtain analogues with higher activities, a similarity-based analog search was carried out.40 A total of 21 analogues of DC_Y53 were chosen for biochemical evaluation using the same radioactive methylation inhibition assay mentioned above. Three molecules showed higher than 70% inhibition of PRMT5 activity at the concentration of 100 µM (Figure S3), the IC50 values of them were determined as shown in Figure 2B-D. DC_Y134, which exhibited the best inhibitory effect (IC50 = 1.7 µM), was chosen for our subsequent research.

Binding-Mode Analysis As shown in Figure S2A, there are two potential ligand binding pocket in PRMT5: a substrate-binding site and a SAM-binding site. To determine the binding pocket of DC_Y134, blind molecular docking study was performed, which revealed that DC_Y134 preferentially occupy the substrate-binding site (Table S2 and Figure S2B-C). Subsequently, we obtained a more accurate picture of the interaction mode from the focused docking at the substrate-binding site. The putative binding mode of DC_Y134, which is similar to that of EPZ015666, suggested that this compound interacts directly with many of the residues that participate in substrate interactions or enzyme catalysis of methyl transfer (Figure 3A). The hexahydrocarbazole ring of DC_Y134 is situated at the hydrophobic pocket which is composed of residues Phe327,

Glu435,

Glu444,

Trp579

(Figure

3B-C).

In

particular,

the

hexahydrocarbazole ring establishes π-π stacking interaction with the side chain of Phe327, which was supposed to play an essential role in arginine symmetric dimethylation by PRMT5.43 The fluorobenzene ring of DC_Y134 fits into the small hydrophobic cavity surrounded by Tyr304, Phe577, Phe580. Furthermore, two hydrogen bonds are established between the NH groups of DC_Y134 and the carbonyl oxygen of Ser578 (Figure 3B-C). Inspired by comparison of DC_Y134 with ACS Paragon Plus Environment

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EPZ015666 (Figure S4), hydrogen bond donor could be introduced to the aliphatic chain and fluorobenzene ring to form polar interactions with residues Leu437, Ser439, Glu444, Phe577, Phe580, thus leading to increased inhibitory activity. Taken together, we concluded that DC_Y134 can efficiently occupy the substrate-binding pocket of PRMT5.

Enzymatic Selectivity To study the selectivity of DC_Y134 for PRMT5, we measured inhibitory activities of the compound against several methyltransferases including PRMT1, CARM1, G9a, EZH2 and DNMT1. As shown in Table1, DC_Y134 exhibited much lower inhibition against these methyltransferases, indicating that this compound could be demonstrated as a potent and selective inhibitor targeting PRMT5. Next, we conducted multiple sequence alignment and structure superimposition analysis to explain the selectivity of DC_Y134 for PRMT5. As shown in Figure S5A, the substrate-binding sites of these methyltransferases are not conserved, especially for DNMT1, G9a and EZH2. The structure alignment indicated that there are clear differences between the substrate-binding sites of PRMT5 and CARM1, a typical type I PRMT (Figure S5B). Several residues critical for inhibitor binding in PRMT5 are substituted by other residues in CARM1, e.g., the residue Phe327 of PRMT5 that is important for catalyzing symmetric dimethylation of arginine and form π-π stacking interaction with DC_Y134 is replaced by methionine in CARM1 (Figure S5A-B), which would create steric clashes with the inhibitor. The residues Phe577 and Phe580 that located in the hydrophobic pocket which interact with fluorobenzene ring of DC_Y134 are substituted as threonine and tyrosine in CARM1, leading to weaker hydrophobic interactions. In addition, the replacement of Ser578 in PRMT5 with histidine in CARM1 may result in destruction of hydrogen bonds between the protein and compound. Taken together, the putative binding mode provides structural basis for explaining selectivity of DC_Y134 against PRMT5, and give clues on development of the selective PRMT5 inhibitors.



Cell Viability and Western Blot Analysis Since PRMT5 plays important roles in leukemia and lymphoma,20-23 we evaluated the anti-proliferation capacity of DC_Y134 in multiple hematological cancer cell lines. MCL cell lines (JeKo-1, MAVER-1), CML cell line (K562), AML cell lines (Kasumi-1, THP-1), and biphenotypic B myelomonocytic leukemia cell line (MV4-11) were selected and treated with DC_Y134 to study the anti-proliferative effects. As shown in Figure 4A-B and Figure S6, DC_Y134 inhibited cell proliferation in a time- and dose-dependent manner, and displayed relative better inhibitory activities against leukemia cell lines than MCL cell lines. Among all of the cells, MV4-11 cells were the most sensitive towards DC_Y134 (Figure 4B). Therefore, MV4-11 cell line was chosen for the subsequent western blot analysis, which indicated that DC_Y134 could inhibited PRMT5 activity in cells and reduce the global SDMA level especially at 25 KDa in a dose-dependent pattern (Figure 4C).

Induction of Cell Cycle Arrest and Apoptosis To explore the anti-proliferation mechanism, flow cytometric analysis was carried out to detect the effect of DC_Y134 towards cell cycle and apoptosis. As show in Figure 5A, DC_Y134 could induce cell cycle arrest at G0/G1 phase in a concentration-dependent pattern. Additionally, DC_Y134 accelerated apoptosis effect in MV4-11 cell line after 96h treatment (Figure 5B). The results indicated that DC_Y134 was both a cell-cycle arrestor and an apoptosis inducer.

CONCLUSION Due to the important roles of PRMT5 in both normal and dysfunctional hematopoiesis, PRMT5 has became a potential therapeutic target for drug discovery of hematologic malignancies. However, up to now, there are very few of PRMT5 inhibitors have been reported. In this work, by using molecular docking-based virtual


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screening and biological validations, DC_Y134 has been discovered as a novel PRMT5 inhibitor. Further selectivity assays showed DC_Y134 possessed great selectivity against several methyltransferases. The binding-mode prediction revealed DC_Y134 occupied the substrate arginine pocket, which also explained the selectivity of this inhibitor. In addition, DC_Y134 showed anti-proliferative effects in leukemia cells by causing G0/G1 cell-cycle arrest and cell apoptosis synergistically. Taken together, this study provided a novel chemical template for further hit-to-lead optimization and a good hit compound for development of PRMT5-targeting therapeutic agents.

ASSOCIATED CONTENT Supporting Information Available: The re-docking validation results of compound EPZ015666; Docking poses of DC_Y134 binding to PRMT5; Inhibitory activity of the 21 analogues of DC_Y53; A comparison of DC_Y134 with PRMT5 inhibitor EPZ015666; Selectivity of DC_Y134 toward different methyltransferases; Cell activity of DC_Y134; IC50 Values of Active Compounds Identified in Virtual Screening; List of poses in blind docking of py21 targeting PRMT5.

AUTHOR INFORMATION Corresponding Authors *E-mail: [email protected] *E-mail: [email protected].

ACKNOWLEDGMENTS We thank Drs. Gang Chen and Qinghe Zhou for guide of the cellualr experiments and critical reading of the manuscript. We gratefully acknowledge financial support from Zhejiang Province Natural Science Foundation (LY18H300008), Public Projects of Zhejiang Province (2016C31017), Zhejiang Provincial Top Key Discipline of Biology, ACS Paragon Plus Environment


Science Foundation of Zhejiang Sci-Tech University (13042163-Y, 13042159-Y) and the 521 Talent Cultivation Plan of Zhejiang Sci-Tech University.


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Figure 1. Virtual screening procedures and activity assays for PRMT5 in vitro. (A) Schematic representation of the virtual screening procedure. (B) Inhibitory activity of the 120 candidate molecules based on virtual screening at 100 µM. The red columnar bar represents the activity of the reference compound SAH.



Figure 2. Chemical structures and inhibitory data against PRMT5 of DC_Y53 and its analogues selected by similarity-based searching. (A-D) The chemical structures and IC50 values of DC_Y53, DC_Y125, DC_Y126, DC_Y134 respectively.


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Figure 3. Predicted binding mode of DC_Y134. (A) Superimposition of the binding modes of DC_Y134 and EPZ015666 (PDB ID 4X61). Compound DC_Y134 is shown as magenta sticks, and EPZ015666 is shown as cyan sticks, with the surface of PRMT5 depicted in vacuum electrostatics. (B) Binding mode of DC_Y134. It is shown as magenta sticks, and key residues are shown as gray sticks. Hydrogen bonds are shown as a red dashed line. (C) Schematic diagram showing putative interactions between PRMT5 and DC_Y134. Residues involved in the hydrophobic interactions are shown as starbursts, and hydrogen-bonding interactions are denoted by dotted green lines.



Figure 4. Cell Viability and Western Blot Analysis. (A-B) Relative proliferation rates of Kasumi1 and MV4-11 after treatment with DC_Y134 over 12 days.(C) Treatment with DC_Y134 for 96 h decreased the cellular symmetrically dimethylated levels in MV4-11 cells.


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Figure 5. Cell cycle arrest, apoptosis induction in MV4-11 cells after treatment with DC_Y134. (A) Treatment of DC_Y134 for 72 h induced cell cycle arrest at G0/G1 phase. (B) Treatment of DC_Y134 for 96 h promoted apoptosis.



Table 1. Selectivity of DC_134 over other methyltransferases

Target PRMT5 PRMT1 CARM1 G9a DNMT1 EZH2

Inhibition ratio at 50 and 100µM, % 50 µM 100 µM 90 94 8 17 28 39 19 21 6 -4 15 19


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