- Email: [email protected]
Discovery of novel DNA methyltransferase 3A inhibitors via structure-based virtual screening and biological assays
Accepted Manuscript Discovery of Novel DNA Methyltransferase 3A Inhibitors via Structure-based Virtual Screening and Biological Assays Zhiyuan Shao, Pan Xu, Wen Xu, Linjuan Li, Shien Liu, Rukang Zhang, YuChih Liu, Chenhua Zhang, Shijie Chen, Cheng Luo PII: DOI: Reference:
S0960-894X(16)31163-5 http://dx.doi.org/10.1016/j.bmcl.2016.11.023 BMCL 24418
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
5 September 2016 8 November 2016 10 November 2016
Please cite this article as: Shao, Z., Xu, P., Xu, W., Li, L., Liu, S., Zhang, R., Liu, Y-C., Zhang, C., Chen, S., Luo, C., Discovery of Novel DNA Methyltransferase 3A Inhibitors via Structure-based Virtual Screening and Biological Assays, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.023
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.
Discovery of Novel DNA Methyltransferase 3A Inhibitors via Structure-based Virtual Screening and Biological Assays Zhiyuan Shaoa,†, Pan Xu b,†, Wen Xuc,†, Linjuan Lib,d, Shien Liub, Rukang Zhangb,d, Yu-Chih Liue, Chenhua Zhange, Shijie Chenb,*, and Cheng Luo b,* a
Nano Science and Technology Institute, University of Science and Technology of China,
Suzhou 215123, China. b
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai
Institute of MateriaMedica, Chinese Academy of Sciences, Shanghai 201203, China. c
Obstetrics & Gynecology Hospital of Fudan University, No.419 Fangxie Road. Shanghai
200011, China. d
School of Life Science and Technology, Shanghai Tech University, Shanghai 200031,
China. e
Shanghai ChemPartner Co., LTD, Building 5, 998, Halei Road, Zhangjiang Hi-Tech
Park, Pudong New Area, Shanghai, P.R. China 201203. †
These authors contributed equally to this work.
* To whom correspondence should be addressed. E-mail: [email protected], [email protected].
Abstract: DNA methyltransferases are involved in diverse biological processes and abnomal methylation patterns play essential roles in cancer initiation and progression. DNA methyltransferase 3A (DNMT3A) acting as a de novo DNA methyltransferase, has gained widespread attention especially in hematological diseases. To date, large numbers of DNMTs inhibitors have been discovered, however, the small molecular inhibitors targeting DNMT3A are still in its infancy. In this study, structure-based virtual screening in combination with biological assays was performed to discovery potent novel DNMT3A inhibitors. Compound 40 and 40_3 displayed comparable in vitro inhibitory activity against DNMT3A with IC50 values of 46.5µM and 41µM, respectively. Further binding mode analysis suggested these molecules inhibit DNMT3A activity through binding the S-adenosyl-L-methionine (SAM) pocket. Overall, 40 and 40_3 may serve as novel scaffolds for further optimization and small molecular probes for investigating DNMT3A function.
Keywords: epigenetics; DNMT3A inhibitors; virtual screening; molecular docking
DNA methylation, together with histone modification, is an important epigenetic event involved in diverse biological processes, especially playing pivotal roles in development, stem cell regulation and several diseases.1 In mammals, three kinds of DNA methyltransferases are identified for ensuring DNA methylation at C5 position of cytosine mainly in the context of CpG dinucleotides, namely DNMT1, DNMT3A and DNMT3B. 2 To be more precise, DNMT3A and DNMT3B function as de novo DNA methyltransferases during embryonic development.2 Besides, the non-active DNMT3-like (DNMT3L) is also indispensable for the methylation although lacking of a catalytic domain shared by other DNMTs.3 DNMT1, the most abundant one, primarily carries out maintaining DNA methylation pattern during DNA replication.4 During the past decades, it has been suggested that abnormal DNA methylation in gene promoter CpG islands can contribute to cancer initiation and progression, such as the disruption of p53 pathway due to DNA hypermethylation of specific
genes.5 Thus, inactivation of DNMTs could serve as an effective way of reversing abnormal DNA methylation. 6 To date, different DNMTs inhibitors are identified which could be classified into two types: nucleoside analogs and non-nucleoside analogs.6 Among the nucleoside analogs, azacitidine and decitabine have been approved by FDA to treat myelodysplastic syndrome (MDS) and leukemia.7 However, these drugs could exert significant toxic side effects as well as poor stability, which also need to incorporate into DNA to take effect as covalent inhibitors.8 The second type of DNMTs inhibitors has emerged in multitude during recent years,9-11 whereas the selectivity and activity can still hinder further optimization. Moreover, most of the DNMTs inhibitors target for DNMT1. Since DNMT3A plays essential roles in haematological malignancies, such as frequent DNMT3A mutations associated with haematological disorders,1 the discovery of specific small molecules targeted for DNMT3A is of high importance as chemical regulators for investigating DNMT3A function. Additionally, the high resolutional autoinhibitory structure of DNMT3A brings new opportunity to discover novel chemotype as DNMT3A inhibitors.12 In this study, aiming at discovering novel DNMT3A inhibitors with potent activity, a hierarchical virtual screening combining pharmacophore-based mapping and docking-based approach was performed against the SPECS database (http://www.specs.net) of about 200,000 commercially available compounds (Fig. 1).13 The crystal structure of DNMT3A in complex with its substrate S-adenosyl-L-homocysteine (SAH) was selected for receptor-ligand pharmacophore generation using Accelrys Discovery Studio 3.0.14 Considering that the accuracy and rationality of receptor-ligand pharmacophore largely relies on the initial structure of complex, both the determined crystal structures12
(PDB15:
4U7P,
4U7T)
were
utilized
for
pharmacophore
construction. By carefully inspection of the generated pharmacophore features, two pharmacophore models were selected for subsequent library screening (Fig. S1). Finally, after removing the inorganic molecules, duplicates and pan-assay
interference compounds through Pipeline Pilot 7.5,16, 17 77,205 molecules were obtained for further screening via docking-based methods. In light of the bias introduced by single docking software, docking-based virtual screening was performed through different docking software including Glide 5.5 18 and DOCK 6.7. 19 The crystal structure of DNMT3A with SAH obtained from the Protein Data Bank 15 (PDB: 4U7T) was adopted for docking studies. Both docking programs were validated by re-docking studies (Fig. S2). By calculating the root mean square deviation (RMSD) values, it indicated that both docking procedures were reliable. Then the initial 77,205 molecules were separated into two parts and both parts were docked into SAH binding site of DNMT3A using Glide SP mode and DOCK 6.7 respectively.20 After scoring with grid-based score provided by DOCK, AMBER score was employed to re-dock the top-ranked 5000 molecules to further evaluate the accuracy. In the end, top-ranked 1000 molecules of each docking procedure were selected and merged into one file for cluster analysis with defined cluster number of 100 and fingerprint FCFP_4. As a result of carefully visual inspection, 107 small molecules with diverse scaffolds were selected and purchased for further biological evaluation. To confirm inhibitory activity of hit compounds from virtual screening, we performed in vitro DNMT3A inhibition assays based on radioactive methylation method, in which sinefungin was used as the reference compound. Sinefungin acts as SAM competitor and has been widely used as reference inhibitor in SAM-competition studies. 21 The 107 compounds were tested at the concentration of 100µM, with compound 40 almost totally inhibiting DNMT3A/3L enzymatic activity (Fig. 2). We then tested the IC50 value of compound 40 using the same assay (Fig. 2). It displayed potential inhibitory activity against DNMT3A with IC50 of 46.5µM. On account of the moderate inhibitory activity of 40 against DNMT3A, similarity-based analog searching was performed using 40 as the query molecule to find out more potent potential compounds. Moreover, scaffold
rationality could be validated via evaluating the biological activity of similar molecules. Tanimoto similarity coefficient was deployed to assess the similarities based on fingerprint FCFP_6 and ECFP_6 using Pipeline Pilot 7.5,17 finally 15 compounds with similar scaffolds were purchased from SPECS Corp.13 and the inhibitory activity was tested against DNMT3A via radioactive methylation assay. Table 1 displays the chemical structures and corresponding inhibitory activity. Among the tested compounds, 40_3 and 40_8 showed effective inhibition at the concentration of 100 µM. To compare them with 40, the IC50 values of the most potent compounds were tested (Fig S3). 40_3 (IC50 = 41µM) displayed equivalent inhibition as 40 (IC50 = 46.5µM), whereas 40_8 (IC50 = 64µM) was less potent. In conclusion, these molecules with similar scaffolds could validate the inhibitory activity of parental compound 40 to some extent and provide useful information for further structure-activity relationship (SAR) analysis. The structure-activity relationships of compound 40 and its analogs were investigated based on the inhibitory activity of derivatives and docking studies. To obtain more reliable docking results, induced-fit docking was adopted using Induced Fit Docking procedure implemented in Schrödinger.22, 23 As shown in Fig. 3, compound 40 was partly located in SAH binding pocket. Contrary to SAH, compound 40 binds to DNMT3A in a shallower pocket due to the rigid benzoic acid moiety while SAH contains a more flexible aliphatic side chain. Besides, the carboxyl group of 40 could form several hydrogen bonds with neighbouring residues, including Arg887 and Asn711, this hydrogen bond interacting network may contribute largely to the inhibitory activity against DNMT3A. More detailed interaction between 40 or SAH and DNMT3A were illustrated in Fig. S4. For further validating the pivotal role of carboxyl group, the binding modes of 40_3 and 40_8 were also analysed. By superimposing docking conformation of 40, 40_3 and 40_8, all these compounds occupy the similar binding pocket with almost consistent tendency. Moreover, the carboxyl group of 40_3 could establish extra hydrogen bonds with Arg891 while that of
40_8 could only form two hydrogen bonds with Arg792 and Arg790, respectively. These results could reasonably elucidate the difference of inhibitory activity between 40 and the analogs, and indicate the importance of carboxyl group which is in accordance with the in vitro inhibition assay. More interestingly, these three compounds could interact with Phe640 by aromatic stacking in spite of the specific manner. 40 and 40_8 form edge-to-face π-π stacking with Phe640 and 40_3 forms face-to-face aromatic stacking. These interactions may serve as important clues for interpreting the importance of phenyl group. As indicated in Table 1, when the phenyl group of 40 was substituted by aliphatic side chains, such as methyl and ethyl, 40_1 and 40_2 displayed no inhibition against DNMT3A. In view of the in vitro inhibitory activity of 40 and its analogs together with good structure-activity relationship, we then determined whether 40 and 40_3 could effectively influence cell viability. Taking into consideration of the significant role of DNMT3A in AML cell lines, we tested 40 and 40_3 in Kasumi-1, KG-1, MV4-11 and THP-1 AML cells. Additionally, we also tested the inhibitory effect of 40 and 40_3 in cervical cancer cells (Hela, Fig. S5). The results indicated that 40 and 40_3 were more potent in AML cell lines comparing with other cell lines especially in MV4-11 (Fig. 4). Of note, both 40 and 40_3 showed comparable inhibition against almost all the tested cell lines. Taken together, DNA methylation, one of the most important epigenetic modifications as catalysed by DNMTs family, has gained widespread attention in recent years. Aberrant methylation status always connects to diverse diseases including cancer. Increasing number of DNMT inhibitors were reported in literatures, but most of them are targeted for DNMT1. Herein, we performed structure-based virtual screening against DNMT3A and in vitro enzymatic assays combining with cell-based assays. Compound 40 and 40_3 displayed good inhibitory activity against DNMT3A with IC50 values of 46.5µM and 41µM, subsequent binding mode analysis indicated both compounds could occupy part of the SAM binding pocket. The phenyl moiety of 40 and 40_3
could interact with Phe640 via aromatic stacking, while the carboxyl group could form hydrogen bond interacting network with surrounding residues by SAR analysis. This result may provide useful clues for further structure optimization to discovery more potent DNMT3A inhibitors. In conclusion, this study provided a reasonable strategy combining hierarchical structured-based virtual screening with enzymatic assays for discovering potent DNMT3A inhibitors. Compound 40 and 40_3 may serve as small molecular probes for investigating the biological function of DNMT3A, and as starting scaffold for further optimization as well.
Acknowledgements The computation resources were supported by Computer Network Information Center, Chinese Academy of Sciences and Tianjin Supercomputing Center. We gratefully acknowledge financial support from the Ministry of Science and Technology of China [2015CB910304 to H.J.]; the National Natural Science Foundation of China (21472208 to C.L.), CAS Strategic Priority Research Program (XDA12020304) and Fund of State Key Laboratory of Toxicology and Medical Countermeasures, Academy of Military Medical Science [TMC201505 to C.L.].
Notes and references 1. Yang, L.; Rau, R.;Goodell, M.A. Nat Rev Cancer 2015, 15, 152. 2. Du, J.; Johnson, L.M.; Jacobsen, S.E.;Patel, D.J. Nat Rev Mol Cell Biol 2015, 16, 519. 3. Jia, D.; Jurkowska, R.Z.; Zhang, X.; Jeltsch, A.;Cheng, X. Nature 2007, 449, 248. 4. Goll, M.G.;Bestor, T.H. Annu Rev Biochem 2005, 74, 481. 5. Baylin, S.B.;Jones, P.A. Nat Rev Cancer 2011, 11, 726. 6. Yoo, C.B.;Jones, P.A. Nat Rev Drug Discov 2006, 5, 37. 7. Esteller, M. N Engl J Med 2008, 358, 1148. 8. Stresemann, C.;Lyko, F. Int J Cancer 2008, 123, 8. 9. Chen, S.; Wang, Y.; Zhou, W.; Li, S.; Peng, J.; Shi, Z.; Hu, J.; Liu, Y.C.; Ding, H.; Lin, Y.; Li, L.; Cheng, S.; Liu, J.; Lu, T.; Jiang, H.; Liu, B.; Zheng, M.;Luo, C. J Med Chem 2014, 57, 9028. 10. Erdmann, A.; Halby, L.; Fahy, J.;Arimondo, P.B. J Med Chem 2015, 58, 2569. 11. Medina-Franco, J.L.; Mendez-Lucio, O.; Duenas-Gonzalez, A.;Yoo, J. Drug Discov Today 2015, 20, 569.
12. Guo, X.; Wang, L.; Li, J.; Ding, Z.; Xiao, J.; Yin, X.; He, S.; Shi, P.; Dong, L.; Li, G.; Tian, C.; Wang, J.; Cong, Y.;Xu, Y. Nature 2015, 517, 640. 13. Specs, http://www.specs.net/, (accessed April, 2015). 14. Accelrys Discovery Studio (version 3.0), Accelrys, San Diego, CA, 2010. 15. Berman, H.M.; Westbrook, J.; Feng, Z.; Gilliland, G.; Bhat, T.N.; Weissig, H.; Shindyalov, I.N.;Bourne, P.E. Nucleic Acids Res 2000, 28, 235. 16. Baell, J.B.;Holloway, G.A. J Med Chem 2010, 53, 2719. 17. Pipeline Pilot (version 7.5), Accelrys Software Inc, San Diego, CA, 2008. 18. Glide (version 5.5), Schrödinger, LLC, New York, 2009. 19. Allen, W.J.; Balius, T.E.; Mukherjee, S.; Brozell, S.R.; Moustakas, D.T.; Lang, P.T.; Case, D.A.; Kuntz, I.D.;Rizzo, R.C. J Comput Chem 2015, 36, 1132. 20. Friesner, R.A.; Banks, J.L.; Murphy, R.B.; Halgren, T.A.; Klicic, J.J.; Mainz, D.T.; Repasky, M.P.; Knoll, E.H.; Shelley, M.; Perry, J.K.; Shaw, D.E.; Francis, P.;Shenkin, P.S. J Med Chem 2004, 47, 1739. 21. Gros, C.; Chauvigne, L.; Poulet, A.; Menon, Y.; Ausseil, F.; Dufau, I.;Arimondo, P.B. Nucleic Acids Res 2013, 41, e185. 22. Glide (version 6.7), Schrödinger, LLC, New York, NY, 2015. 23. Prime (version 4.0), Schrödinger, LLC, New York, NY, 2015.
Figure legends
Fig. 1 The structure-based virtual screening procedure.
Fig 2. In vitro Inhibitory activity of compounds against DNMT3A using radioactive methylation assay. (A) The inhibitory activity of 107 compounds from virtual screening at the tested concentration of 100µM, compound 40 and the reference inhibitor, sinefungin were highlighted using red columns. (B) The chemical structure and IC50 value of compound 40.
Fig. 3 Putative binding modes of compound 40 and its analogs in the binding pocket of DNMT3A structure (PDB: 4U7T). (A) Binding modes of compound 40 (green) and SAH (yellow) with interacting residues depicted. (B) The superimposition of compound 40 (green) and its analogs 40_3 (salmon) as well as 40_8 (cyan) in the binding pocket, where the catalytic pocket of DNMT3A is displayed as gray surface. (C) The interacting modes of 40_3 with surrounding residues in the binding pocket. (D) The binding mode of 40_8. The DNMT3A structure is displayed as gray cartoons, while the interacting residues are depicted as gray sticks and the corresponding residue names are showed in three-letter abbreviations. Dashed lines represent the hydrogen bonds, specifically, red ones indicated hydrogen bonds formed with 40 or its analogs and yellow ones are those formed with SAH. Aromatic stacking are indicated with yellow transparent circles. Of note, bromines are in dark red and chlorines are in green.
Fig. 4 Cellular activity of inhibitors. (A) Kasumi-1 cells were treated with 12.5, 25, 50, 100 and 200µM compound 40 and 40_3 for 48 hours. (B) MV4-11 cells were treated with 12.5, 25, 50, 100 and 200µM compound 40 and 40_3 for 48 hours. (C) THP-1 cells were treated with 12.5, 25, 50, 100 and 200µM compound 40 and 40_3 for 48 hours. (D) KG-1 cells were treated with 12.5, 25, 50, 100 and 200µM compound 40 and 40_3 for 72 hours.
Table 1 Chemical structures and inhibitory activity of analogs against DNMT3A using radioactive methylation assay. Br O
O
N H HO
N
O O
R1 Compound Structure
R2
R3 % Inhibition
IC50 (µM)
40_1
R3 = methyl
-15
NDa
40_2
R3 = ethyl
-13
NDa
40_3
67
41
-8
NDa
11
NDa
56
NDa
8
NDa
92
64
1
NDa
19
NDa
11
NDa
4
NDa
6
NDa
13
NDa
3
NDa
R1 = 40_4
3
R = 40_5
1
R = 40_6
3
;R =
3
R = 40_7
1
R = 40_8
R1 =
3
;R = ; R3 =
40_9 1
R = 40_10
;R =
1
3
1
R = 40_13
1
R = 40_14
40_15 a
3
N
R = 40_12
;R =
1
R = 40_11
3
;R = 3
;R = 3
;R =
1
R =
;R =
1
3
R =
3
;R =
ND represents not determined.
Graphical Abstract Novel DNMT3A inhibitor 40 was discovered via combining pharmacophore mapping and docking-based virtual screening, with in vitro IC50 of 46.5µM.
S0960-894X(16)31163-5 http://dx.doi.org/10.1016/j.bmcl.2016.11.023 BMCL 24418
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date: Revised Date: Accepted Date:
5 September 2016 8 November 2016 10 November 2016
Please cite this article as: Shao, Z., Xu, P., Xu, W., Li, L., Liu, S., Zhang, R., Liu, Y-C., Zhang, C., Chen, S., Luo, C., Discovery of Novel DNA Methyltransferase 3A Inhibitors via Structure-based Virtual Screening and Biological Assays, Bioorganic & Medicinal Chemistry Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.11.023
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.
Discovery of Novel DNA Methyltransferase 3A Inhibitors via Structure-based Virtual Screening and Biological Assays Zhiyuan Shaoa,†, Pan Xu b,†, Wen Xuc,†, Linjuan Lib,d, Shien Liub, Rukang Zhangb,d, Yu-Chih Liue, Chenhua Zhange, Shijie Chenb,*, and Cheng Luo b,* a
Nano Science and Technology Institute, University of Science and Technology of China,
Suzhou 215123, China. b
Drug Discovery and Design Center, State Key Laboratory of Drug Research, Shanghai
Institute of MateriaMedica, Chinese Academy of Sciences, Shanghai 201203, China. c
Obstetrics & Gynecology Hospital of Fudan University, No.419 Fangxie Road. Shanghai
200011, China. d
School of Life Science and Technology, Shanghai Tech University, Shanghai 200031,
China. e
Shanghai ChemPartner Co., LTD, Building 5, 998, Halei Road, Zhangjiang Hi-Tech
Park, Pudong New Area, Shanghai, P.R. China 201203. †
These authors contributed equally to this work.
* To whom correspondence should be addressed. E-mail: [email protected], [email protected].
Abstract: DNA methyltransferases are involved in diverse biological processes and abnomal methylation patterns play essential roles in cancer initiation and progression. DNA methyltransferase 3A (DNMT3A) acting as a de novo DNA methyltransferase, has gained widespread attention especially in hematological diseases. To date, large numbers of DNMTs inhibitors have been discovered, however, the small molecular inhibitors targeting DNMT3A are still in its infancy. In this study, structure-based virtual screening in combination with biological assays was performed to discovery potent novel DNMT3A inhibitors. Compound 40 and 40_3 displayed comparable in vitro inhibitory activity against DNMT3A with IC50 values of 46.5µM and 41µM, respectively. Further binding mode analysis suggested these molecules inhibit DNMT3A activity through binding the S-adenosyl-L-methionine (SAM) pocket. Overall, 40 and 40_3 may serve as novel scaffolds for further optimization and small molecular probes for investigating DNMT3A function.
Keywords: epigenetics; DNMT3A inhibitors; virtual screening; molecular docking
DNA methylation, together with histone modification, is an important epigenetic event involved in diverse biological processes, especially playing pivotal roles in development, stem cell regulation and several diseases.1 In mammals, three kinds of DNA methyltransferases are identified for ensuring DNA methylation at C5 position of cytosine mainly in the context of CpG dinucleotides, namely DNMT1, DNMT3A and DNMT3B. 2 To be more precise, DNMT3A and DNMT3B function as de novo DNA methyltransferases during embryonic development.2 Besides, the non-active DNMT3-like (DNMT3L) is also indispensable for the methylation although lacking of a catalytic domain shared by other DNMTs.3 DNMT1, the most abundant one, primarily carries out maintaining DNA methylation pattern during DNA replication.4 During the past decades, it has been suggested that abnormal DNA methylation in gene promoter CpG islands can contribute to cancer initiation and progression, such as the disruption of p53 pathway due to DNA hypermethylation of specific
genes.5 Thus, inactivation of DNMTs could serve as an effective way of reversing abnormal DNA methylation. 6 To date, different DNMTs inhibitors are identified which could be classified into two types: nucleoside analogs and non-nucleoside analogs.6 Among the nucleoside analogs, azacitidine and decitabine have been approved by FDA to treat myelodysplastic syndrome (MDS) and leukemia.7 However, these drugs could exert significant toxic side effects as well as poor stability, which also need to incorporate into DNA to take effect as covalent inhibitors.8 The second type of DNMTs inhibitors has emerged in multitude during recent years,9-11 whereas the selectivity and activity can still hinder further optimization. Moreover, most of the DNMTs inhibitors target for DNMT1. Since DNMT3A plays essential roles in haematological malignancies, such as frequent DNMT3A mutations associated with haematological disorders,1 the discovery of specific small molecules targeted for DNMT3A is of high importance as chemical regulators for investigating DNMT3A function. Additionally, the high resolutional autoinhibitory structure of DNMT3A brings new opportunity to discover novel chemotype as DNMT3A inhibitors.12 In this study, aiming at discovering novel DNMT3A inhibitors with potent activity, a hierarchical virtual screening combining pharmacophore-based mapping and docking-based approach was performed against the SPECS database (http://www.specs.net) of about 200,000 commercially available compounds (Fig. 1).13 The crystal structure of DNMT3A in complex with its substrate S-adenosyl-L-homocysteine (SAH) was selected for receptor-ligand pharmacophore generation using Accelrys Discovery Studio 3.0.14 Considering that the accuracy and rationality of receptor-ligand pharmacophore largely relies on the initial structure of complex, both the determined crystal structures12
(PDB15:
4U7P,
4U7T)
were
utilized
for
pharmacophore
construction. By carefully inspection of the generated pharmacophore features, two pharmacophore models were selected for subsequent library screening (Fig. S1). Finally, after removing the inorganic molecules, duplicates and pan-assay
interference compounds through Pipeline Pilot 7.5,16, 17 77,205 molecules were obtained for further screening via docking-based methods. In light of the bias introduced by single docking software, docking-based virtual screening was performed through different docking software including Glide 5.5 18 and DOCK 6.7. 19 The crystal structure of DNMT3A with SAH obtained from the Protein Data Bank 15 (PDB: 4U7T) was adopted for docking studies. Both docking programs were validated by re-docking studies (Fig. S2). By calculating the root mean square deviation (RMSD) values, it indicated that both docking procedures were reliable. Then the initial 77,205 molecules were separated into two parts and both parts were docked into SAH binding site of DNMT3A using Glide SP mode and DOCK 6.7 respectively.20 After scoring with grid-based score provided by DOCK, AMBER score was employed to re-dock the top-ranked 5000 molecules to further evaluate the accuracy. In the end, top-ranked 1000 molecules of each docking procedure were selected and merged into one file for cluster analysis with defined cluster number of 100 and fingerprint FCFP_4. As a result of carefully visual inspection, 107 small molecules with diverse scaffolds were selected and purchased for further biological evaluation. To confirm inhibitory activity of hit compounds from virtual screening, we performed in vitro DNMT3A inhibition assays based on radioactive methylation method, in which sinefungin was used as the reference compound. Sinefungin acts as SAM competitor and has been widely used as reference inhibitor in SAM-competition studies. 21 The 107 compounds were tested at the concentration of 100µM, with compound 40 almost totally inhibiting DNMT3A/3L enzymatic activity (Fig. 2). We then tested the IC50 value of compound 40 using the same assay (Fig. 2). It displayed potential inhibitory activity against DNMT3A with IC50 of 46.5µM. On account of the moderate inhibitory activity of 40 against DNMT3A, similarity-based analog searching was performed using 40 as the query molecule to find out more potent potential compounds. Moreover, scaffold
rationality could be validated via evaluating the biological activity of similar molecules. Tanimoto similarity coefficient was deployed to assess the similarities based on fingerprint FCFP_6 and ECFP_6 using Pipeline Pilot 7.5,17 finally 15 compounds with similar scaffolds were purchased from SPECS Corp.13 and the inhibitory activity was tested against DNMT3A via radioactive methylation assay. Table 1 displays the chemical structures and corresponding inhibitory activity. Among the tested compounds, 40_3 and 40_8 showed effective inhibition at the concentration of 100 µM. To compare them with 40, the IC50 values of the most potent compounds were tested (Fig S3). 40_3 (IC50 = 41µM) displayed equivalent inhibition as 40 (IC50 = 46.5µM), whereas 40_8 (IC50 = 64µM) was less potent. In conclusion, these molecules with similar scaffolds could validate the inhibitory activity of parental compound 40 to some extent and provide useful information for further structure-activity relationship (SAR) analysis. The structure-activity relationships of compound 40 and its analogs were investigated based on the inhibitory activity of derivatives and docking studies. To obtain more reliable docking results, induced-fit docking was adopted using Induced Fit Docking procedure implemented in Schrödinger.22, 23 As shown in Fig. 3, compound 40 was partly located in SAH binding pocket. Contrary to SAH, compound 40 binds to DNMT3A in a shallower pocket due to the rigid benzoic acid moiety while SAH contains a more flexible aliphatic side chain. Besides, the carboxyl group of 40 could form several hydrogen bonds with neighbouring residues, including Arg887 and Asn711, this hydrogen bond interacting network may contribute largely to the inhibitory activity against DNMT3A. More detailed interaction between 40 or SAH and DNMT3A were illustrated in Fig. S4. For further validating the pivotal role of carboxyl group, the binding modes of 40_3 and 40_8 were also analysed. By superimposing docking conformation of 40, 40_3 and 40_8, all these compounds occupy the similar binding pocket with almost consistent tendency. Moreover, the carboxyl group of 40_3 could establish extra hydrogen bonds with Arg891 while that of
40_8 could only form two hydrogen bonds with Arg792 and Arg790, respectively. These results could reasonably elucidate the difference of inhibitory activity between 40 and the analogs, and indicate the importance of carboxyl group which is in accordance with the in vitro inhibition assay. More interestingly, these three compounds could interact with Phe640 by aromatic stacking in spite of the specific manner. 40 and 40_8 form edge-to-face π-π stacking with Phe640 and 40_3 forms face-to-face aromatic stacking. These interactions may serve as important clues for interpreting the importance of phenyl group. As indicated in Table 1, when the phenyl group of 40 was substituted by aliphatic side chains, such as methyl and ethyl, 40_1 and 40_2 displayed no inhibition against DNMT3A. In view of the in vitro inhibitory activity of 40 and its analogs together with good structure-activity relationship, we then determined whether 40 and 40_3 could effectively influence cell viability. Taking into consideration of the significant role of DNMT3A in AML cell lines, we tested 40 and 40_3 in Kasumi-1, KG-1, MV4-11 and THP-1 AML cells. Additionally, we also tested the inhibitory effect of 40 and 40_3 in cervical cancer cells (Hela, Fig. S5). The results indicated that 40 and 40_3 were more potent in AML cell lines comparing with other cell lines especially in MV4-11 (Fig. 4). Of note, both 40 and 40_3 showed comparable inhibition against almost all the tested cell lines. Taken together, DNA methylation, one of the most important epigenetic modifications as catalysed by DNMTs family, has gained widespread attention in recent years. Aberrant methylation status always connects to diverse diseases including cancer. Increasing number of DNMT inhibitors were reported in literatures, but most of them are targeted for DNMT1. Herein, we performed structure-based virtual screening against DNMT3A and in vitro enzymatic assays combining with cell-based assays. Compound 40 and 40_3 displayed good inhibitory activity against DNMT3A with IC50 values of 46.5µM and 41µM, subsequent binding mode analysis indicated both compounds could occupy part of the SAM binding pocket. The phenyl moiety of 40 and 40_3
could interact with Phe640 via aromatic stacking, while the carboxyl group could form hydrogen bond interacting network with surrounding residues by SAR analysis. This result may provide useful clues for further structure optimization to discovery more potent DNMT3A inhibitors. In conclusion, this study provided a reasonable strategy combining hierarchical structured-based virtual screening with enzymatic assays for discovering potent DNMT3A inhibitors. Compound 40 and 40_3 may serve as small molecular probes for investigating the biological function of DNMT3A, and as starting scaffold for further optimization as well.
Acknowledgements The computation resources were supported by Computer Network Information Center, Chinese Academy of Sciences and Tianjin Supercomputing Center. We gratefully acknowledge financial support from the Ministry of Science and Technology of China [2015CB910304 to H.J.]; the National Natural Science Foundation of China (21472208 to C.L.), CAS Strategic Priority Research Program (XDA12020304) and Fund of State Key Laboratory of Toxicology and Medical Countermeasures, Academy of Military Medical Science [TMC201505 to C.L.].
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Figure legends
Fig. 1 The structure-based virtual screening procedure.
Fig 2. In vitro Inhibitory activity of compounds against DNMT3A using radioactive methylation assay. (A) The inhibitory activity of 107 compounds from virtual screening at the tested concentration of 100µM, compound 40 and the reference inhibitor, sinefungin were highlighted using red columns. (B) The chemical structure and IC50 value of compound 40.
Fig. 3 Putative binding modes of compound 40 and its analogs in the binding pocket of DNMT3A structure (PDB: 4U7T). (A) Binding modes of compound 40 (green) and SAH (yellow) with interacting residues depicted. (B) The superimposition of compound 40 (green) and its analogs 40_3 (salmon) as well as 40_8 (cyan) in the binding pocket, where the catalytic pocket of DNMT3A is displayed as gray surface. (C) The interacting modes of 40_3 with surrounding residues in the binding pocket. (D) The binding mode of 40_8. The DNMT3A structure is displayed as gray cartoons, while the interacting residues are depicted as gray sticks and the corresponding residue names are showed in three-letter abbreviations. Dashed lines represent the hydrogen bonds, specifically, red ones indicated hydrogen bonds formed with 40 or its analogs and yellow ones are those formed with SAH. Aromatic stacking are indicated with yellow transparent circles. Of note, bromines are in dark red and chlorines are in green.
Fig. 4 Cellular activity of inhibitors. (A) Kasumi-1 cells were treated with 12.5, 25, 50, 100 and 200µM compound 40 and 40_3 for 48 hours. (B) MV4-11 cells were treated with 12.5, 25, 50, 100 and 200µM compound 40 and 40_3 for 48 hours. (C) THP-1 cells were treated with 12.5, 25, 50, 100 and 200µM compound 40 and 40_3 for 48 hours. (D) KG-1 cells were treated with 12.5, 25, 50, 100 and 200µM compound 40 and 40_3 for 72 hours.
Table 1 Chemical structures and inhibitory activity of analogs against DNMT3A using radioactive methylation assay. Br O
O
N H HO
N
O O
R1 Compound Structure
R2
R3 % Inhibition
IC50 (µM)
40_1
R3 = methyl
-15
NDa
40_2
R3 = ethyl
-13
NDa
40_3
67
41
-8
NDa
11
NDa
56
NDa
8
NDa
92
64
1
NDa
19
NDa
11
NDa
4
NDa
6
NDa
13
NDa
3
NDa
R1 = 40_4
3
R = 40_5
1
R = 40_6
3
;R =
3
R = 40_7
1
R = 40_8
R1 =
3
;R = ; R3 =
40_9 1
R = 40_10
;R =
1
3
1
R = 40_13
1
R = 40_14
40_15 a
3
N
R = 40_12
;R =
1
R = 40_11
3
;R = 3
;R = 3
;R =
1
R =
;R =
1
3
R =
3
;R =
ND represents not determined.
Graphical Abstract Novel DNMT3A inhibitor 40 was discovered via combining pharmacophore mapping and docking-based virtual screening, with in vitro IC50 of 46.5µM.