Epigenetics and Drug Discovery
Epigenetics refers to heritable differences in phenotype that are due to mechanisms other than differences in DNA sequence. Epigenetics involves a dynamic interplay between DNA methylation, posttranslational modification of histones and other proteins, and noncoding RNA networks that control gene expression programs in normal and diseased cells.
Mutations in chromatin regulatory genes and alterations of the cellular epigenome are prevalent in most cancers. Post-translational modifications (PTMs) on histone proteins serve as docking sites for chromatin-associated proteins, which in turn dictate dynamic conversion between transcriptionally active or silent chromatin states. The combinatorial nature of these modifications establishes a "histone code" which serves to expand the information present in the DNA-sequence of the genetic code. Lysine methylation is the most complex mark, and it plays a pivotal role in heterochromatin formation, transcriptional regulation and X-chromosome inactivation. Mutations or aberrant expression of proteins containing these domains often leads to deregulation of histone lysine methylation, leading to various diseases, most notably, cancer.
We work with the Structural Genomics Consortium (SGC) to develop structure-based potent, selective, cell-active small molecule inhibitors of individual epigenetic regulatory proteins. Such compounds – Chemical Probes - are extremely valuable for understanding epigenetic signaling mechanisms in cells. Chemical probes are highly complementary to genetic methods and more closely mimic strategies for therapeutic translation. We are providing our epigenetic chemical probes as an Open Access resource to the biological research community to facilitate understanding of epigenetic mechanisms and to more rapidly identify and validate therapeutic targets for cancer and other diseases.
p53, ubiquitin signaling, and cancer
p53 protein plays a major role in maintaining the integrity of the genome. One of its major roles in normal cells is the induction of cell cycle arrest or apoptosis in response DNA damage, by activating or repressing transcription of genes involved in these processes. Inactivation of the tumor suppressor p53 through deletion, mutation or interaction with proteins is a key step in over half of all human cancers. We are particularly interested in understanding, at the atomic levels, how various cellular proteins interact with and regulate the function of p53. Some of the proteins we have characterized include Replication Protein A (RPA), Ubiquitin Specific Protease 7 (USP7), Cullin 7 (Cul7) and Pirh2. Results from these studies have provided insight into the molecular mechanism of p53 regulation via protein-protein and protein-peptide interactions.
Our work on ubiquitin-mediated regulation of p53 has led us to wider consideration of ubuquitylation pathways. Again in collaboration with the SGC we have studied proteins involved in chromatin ubiquitylation (UHRF1, UHRF2, Ring1b) and the entire E2 ubiquitin ligase and ubiquitin-like domain families.
NMR spectroscopy and hybrid methods in structural biology
NMR spectroscopy and x-ray crystallography are the two common techniques used to determine the structure of proteins. We have developed NMR analysis resources such as ABACUS, is a protocol that analyses netoworks of J-correlated spectral peptide-linked peaks and NOE spectral peaks combined with a fragment monte carlo (FMC) procedure to sequence-specifically assign the backbone and side-chain resonances of proteins. To date, this method had been used to solve over 85 protein structures deposited in the PDB. The ABACUS protocol is available for download here.
We also employ x-ray crystallography, small angle X-ray scattering (SAXS) and chemical crosslinking as tools to characterize multidomain proteins and multiprotein complexes with hybrid computational strategies.
- Ryan Doherty
Selected References:Link to Pubmed Publications
Preclinical target validation using patient-derived cells. Edwards AM, Arrowsmith CH, Bountra C, Bunnage ME, Feldmann M, Knight JC, Patel DD, Prinos P, Taylor MD, Sundström M; SGC Open Source Target-Discovery Partnership. Nat Rev Drug Discov. 2015 Mar;14(3):149-50.
Cbx2 Targets PRC1 to Constitutive Heterochromatin in Mouse Zygotes in a Parent-of-Origin-Dependent Manner. Tardat M, Albert M, Kunzmann R, Liu Z, Kaustov L, Thierry R, Duan S, Brykczynska U, Arrowsmith CH, Peters AH. Mol Cell. 2015 Apr 2;58(1):157-71.
A global assessment of cancer genomic alterations in epigenetic mechanisms. Shah MA, Denton EL, Arrowsmith CH, Lupien M, Schapira M. Epigenetics Chromatin. 2014 Dec 4;7(1):29.
- Dalia Barsyte-Lovejoy, Fengling Li, Menno J. Oudhoff, John Tatlock, Aiping Dong, Hong Zheng, Hong Wu, Spencer A. Freeman, Matthieu Schapira, Guillermo Senisterra, Ekaterina Kuznetsova, Abdellah Allali-Hassani, Paul Fish, Brian Gerstenberger, Lee Roberts, Caroline Benn, Rachel Grimely, Gingras, A.-C., Fabio M.V. Rossi, Peter J. Brown, Mark Bunnage, Dafydd Owen, Colby Zaph, Masoud Vedadi, Cheryl H. Arrowsmith. PFI-2, a Potent and Selective Inhibitor of SETD7 Methyltransferase Activity, Proc Natl Acad Sci, 111, 12853-8 (2014).
- Kathy Ann Gelato, Maria Tauber, Michelle Ong, Stefan Winter, Kyoko Hiragami-Hamada, Julia Sindlinger, Alexander Lemak, Yvette Bultsma, Scott Houliston, Dirk Schwarzer, Nullin Divecha, Cheryl H. Arrowsmith, and Wolfgang Fischle. Accessibility of different histone H3-binding domains of UHRF1 is allosterically regulated by phosphatidylinositol 5-phosphate. Mol. Cell, 54:905-19 (2014)
- Alexander Lemak, Bin Wu, Adelinda Yee, Scott Houliston, Hsiau-Wei Lee, Aleksandras Gutmanas , Xianyang Fang , Maite Garcia, Anthony Semesi, Yun-Xing Wang, James H. Prestegard and Cheryl H. Arrowsmith. Structural characterization of flexible two-domain protein in solution using Small Angle X-Ray Scattering and NMR data. Structure, 22, 1862-74 (2014).
- Guillermo Senisterra, Hong Wu, Abdellah Allali Hassani, Gregory A Wasney, Dalia Barsyte-Lovejoy, Ludmila Dombrovsky, Aiping Dong, Kong T. Nguyen, David Smil, Yuri Bolshan, Taraneh Hajian, Hao He,Alma Seitova, Irene Chau, F Li, Gennadiy Poda, JF Couture, Peter J. Brown, Rima Al-awar, Matthieu Schapira, Cheryl H. Arrowsmith and Masoud Vedadi.Small molecule inhibition of MLL activity by disruption of its interaction with WDR5, Biochemical Journal 449:151-9 (2013)
- James, Lindsey I.; Barsyte-Lovejoy, Dalia; Zhong, Nan; Krichevsky, Liubov; Korboukh, Victoria K.; Herold, J. Martin; MacNevin, Christopher J.; Norris, Jacqueline L.; Sagum, Cari C.; Tempel, Wofram; Marcon, Edyta; Guo, Hongbo; Gao, Cen; Huang, Xi-Ping; Shili Duan; Kireev, Dmitri B.; Jin, Jian; Greenblatt, Jack; Roth, Bryan; Janzen, William P.; Brown, Peter J.; Bedford, Mark T.; Arrowsmith, Cheryl H.; Frye, Stephen V. Discovery of a chemical probe for a methyl-lysine reader domain: L3MBTL3, Nature Chem Biology 9, 184-91 (2013).
- Scott B. Rothbart, Krzysztof Krajewski, Nataliya Nady, Wolfram Tempel, Sheng Xue, Aimee N. Iberg, Dalia Barsyte-Lovejoy, Jorge Y. Martinez, Mark T. Bedford, Stephen M. Fuchs, Cheryl H. Arrowsmith, and Brian D. Strahl. Association of UHRF1 with H3K9 methylation directs the maintenance of DNA methylation, Nature Struct Mol Biol 19, 1155-60 (2012)
- Nataliya Nady, Liubov Krichevsky, Nan Zhang, Shili Duan, Maria F. Amaya, Wolfram Tempel, Mani Ravichandran, Cheryl H. Arrowsmith. Histone Substrate Recognition by Human Malignant Brain Tumor Domains, J Mol Biol, 423, 702-18 (2012)
- Wenyu Yu, Emma J. Chory, Amy K. Wernimont, Alex Scopton, Alexander Federation, Jason J. Marineau, Jun Qi, Dalia Barsyte-Lovejoy, Joanna Yi, Richard Marcellus, Roxana E. Iacob, John R. Engen, Erno Wienholds, Fengling Li, Javier Pineda, Guillermina Estiu, Tatiana Shatseva, Taraneh Hajian, Rima Al-awar, John E. Dick, Masoud Vedadi, Peter J. Brown, Cheryl H. Arrowsmith*, James E. Bradner*, Matthieu Schapira*. Catalytic site remodeling of the DOT1L methyltransferase by selective inhibitors, Nature Comm. 3, 1288 (2012)
- Lilia Kaustov, Hui Ouyang, Maria Amaya, Alexander Lemak, Nataliya Nady, Shili Duan, Greg Wasney, Masoud Vedadi, Matthieu Shapira, Jinrong Min & Cheryl Arrowsmith,Recognition and specificity determinants of the human Cbx chromodomains, J Biol Chem. 286, 521-9 (2011).
- Alexander Lemak, Aleksandras Gutmanas, Seth Chitayat, Murthy Karra, Christophe Farès, Maria Sunnerhagen, Cheryl H. Arrowsmith, A novel strategy for NMR resonance assignment and protein structure determination, J Biomol NMR, 49, 27-38 (2011).
- Natalie Nady, Alexander Lemak, John R. Walker, George V. Avvakumov, Michael S. Kareta, Mayada Achuor, Sheng Xue, Shili Duan, Abdellah Allali-Hassani, Frédéric Chédin, and Sirano Dhe-Paganon, Cheryl H.Arrowsmith. Recognition of Multivalent Histone States Associated with Heterochromatin by UHRF1 Protein J. Biol Chem 286, 24300-11 (2011).
- Masoud Vedadi, Dalia Barsyte-Lovejoy, Feng Liu, Sylvie Rival-Gervier, Abdellah Allali-Hassani, Tim J. Wigle, Peter A. DiMaggio, Gregory A. Wasney, Alena Siarheyeva, Aiping Dong, Wolfram Tempel, Xin Chen, Irene Chau, Thomas J. Mangano, Jon M. Evans, Catherine D. Simpson, Samantha G. Pattenden, Jacqueline L. Norris, Dmitri B. Kireev, Ashutosh Tripathy, Aled Edwards, Bryan L. Roth, William P. Janzen, Benjamin A. Garcia, James Ellis, Peter J. Brown, Stephen V. Frye, Cheryl H. Arrowsmith*, Jian Jin*, A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells, Nature Chem. Biol. 7(8):566 (2011).
- Schuetz A, Min J, Allali-Hassani A, Schapira M, Shuen M, Loppnau P, Mazitschek R, Kwiatkowski NP, Lewis TA, Maglathin RL, McLean TH, Bochkarev A, Plotnikov AN, Vedadi M, Arrowsmith CH. Human HDAC7 harbors a class IIa histone deacetylase-specific zinc binding motif and cryptic deacetylase activity. J. Biol. Chem. 283. 11355-63 (2008).
- Nady N, Min J, Kareta MS, Chédin F, Arrowsmith CH, A SPOT on the chromatin landscape? Histone peptide arrays as a tool for epigenetic research. Trends Biochem Sci, 33:305-13 (2008).
- Yi Sheng, R. Laister, A. Lemak, B. Wu, E. Tai, S. Duan, J. Lukin, M. Sunnerhagen, S. Srisailam, M. Karra, S. Benchimol, and CH Arrowsmith, Molecular Basis of Pirh2-mediated p53 ubiquitylation. Nat Struct Mol Biol, 15,1334-42 (2008).
- George V. Avvakumov, John R. Walker, Sheng Xue, Yanjun Li, Shili Duan, Christian Bronner, Cheryl H. Arrowsmith, and Sirano Dhe-Paganon, Structural basis for recognition of hemi-methylated DNA by the SRA domain of human UHRF1. Nature, 455; 822-5 (2008).
- Jinrong Min, Abdellah Allali-Hassani, Nataliya Nady, Chao Qi, Hui Ouyang, Yongsong Liu, Farrell MacKenzie, Masoud Vedadi and Cheryl H Arrowsmith. L3MBTL1 recognition of mono- and dimethylated histones. Nat Struct Mol Biol. 14, 1229-30 (2007).
- Sheng Y, Saridakis V. Sarkari F, Duan S, Wu T, Arrowsmith CH, and Frappier L. Molecular recognition of p53 and MDM2 by USP7/HAUSP, Nat Struct Mol Biol, 13, 285-91. (2006)Molecular recognition of p53 and MDM2 by USP7/HAUSP, Nat Struct Mol Biol, 13, 285-91. (2006).
- Schuetz A, Allali-Hassani A, Martin F, Loppnau P, Vedadi M, Bochkarev A, Plotnikov AN, Arrowsmith CH, Min J. Structural basis for molecular recognition and presentation of histone H3 By WDR5. EMBO J Aug 31 (2006).
- Saridakis V, Sheng Y, Sarkari F, Holowaty MN, Shire K, Nguyen T, Zhang RG, Liao J, Lee W, Edwards AM, Arrowsmith CH and Frappier L. Structure of the p53-binding domain of HAUSP/USP7 bound to Epstein-Barr Nuclear Antigen 1 Implications for EBV-mediated immortalization. Mol Cell. 18, 25-36. (2005).
- E. Botchareva, L. Kaustov, A. Ayed, G-S. Yi, Y. Lu, A. Pineda-Lucena, J.C.C. Liao, A. Okorokov, J. Milner, C.H. Arrowsmith and A. Botcharev, Single stranded DNA mimicry in the p53 transactivation domain interaction with RPA. Proc Natl Acad Sci. 102, 15412-7.(2005).
- Yee, A., Chang, X., Pineda-Lucena, A., Wu, B., Semesi, A., Le, B., Ramelot, T., Lee, G.M., Bhattacharyya, S., Gutierrez, P., Denisov, A., Lee, C.H., Cort, J.R., Kozlov, G., Liao, J., Finak, G., Chen, L., Wishart, D., Lee, W., McIntosh, L.P., Gehring, K., Kennedy, M.A., Edwards, A.M. and Arrowsmith, C.H. An NMR approach to structural proteomics. PNAS 99, (4), 1825-30 (2002)
- Christendat, D., Yee, A., Dharamsi, A., Kluger, Y., Savchenko, A., Cort, J.R., Booth, V., Mackereth, C.D., Saradakis, V., Ekiel, I., Kozlov, G., Maxwell, K.L., Wu, N., McIntosh, L.P., Gehring, K., Kennedy, M.A., Davison, A.R., Pai, E.F., Gerstein, M., Edwards, A.M. and Arrowsmith, C.H., Structural proteomics of an archeon, Nat. Struct. Biol. 7, (10), 903-909 (2000).