Levi Blazer Ph.D.

Protein-Protein Interactions (PPIs) are notoriously difficult to inhibit with small molecules. However, pharmacological targeting of PPIs is quite attractive, due both to the sheer volume of PPIs that exist in biology and the physiological importance of these interactions. While there are examples of small molecule PPI inhibitors in the literature, few have progressed to clinical relevancy.^1,2 The inability to effectively prosecute this class of targets is predominantly due to the intrinsic properties of PPI interaction interfaces. Most PPI binding surfaces are large, shallow pockets or grooves with a series of diffuse, low-energy interactions, whereas small molecules generally bind to smaller, more distinct, sites using a few high-energy interactions.¹ In spite of the challenges associated with targeting PPIs, drugs that target these interactions successfully could be extremely beneficial to human health. Accordingly, the number of laboratories focused on targeting PPIs has been steadily increasing. Concomitant with this growth, the field has made substantial gains in understanding the fundamental nature of PPIs and in how to approach drug discovery for these challenging targets. This issue of Cayman Currents will introduce one of the most promising new areas of PPI inhibitor development and introduce some of the tools that Cayman has developed to help make chemical biology research in this area possible.

Epigenetics and the Histone Code

The regulation of gene transcription is dependent upon proper recruitment of transcription factors to the promoter regions of target genes. While the signals that regulate gene transcription are multivariate, one major mechanism is mediated by the posttranslational modifications of DNA and of the histones that package DNA into chromatin. These marks, and the repertoire of proteins that place and remove them, form a foundational cornerstone for transcriptional regulation. Recent work using mass spectrometry has identified hundreds of different posttranslational modifications (PTMs) that occur on histones.³ The majority of PTMs occur on the N-terminal tails of histone proteins, which protrude from the nucleosome into the nuclear milieu. While the physiological relevance of the majority of these marks is still unknown, there are several key posttranslational modifications that possess defined biological activities.

Arguably, the best-studied histone PTM to date is lysine acetylation. Through its ability to weaken the DNA-histone interaction and to recruit transcription factors, this common modification is a hallmark of genes undergoing active transcription.⁴ Acetyllysine is selectively recognized by a small protein motif called a bromodomain.⁵ There are at least 43 bromodomain-containing proteins (Figure 1) that have been identified in the human genome, the majority of which are important transcriptional regulators. Bromodomains are small (~120 amino acid) motifs formed by a left-handed bundle of 4 alpha helices linked by loop domains of varying length.⁵ The acetyllysine binding pocket is formed by a deep hydrophobic groove at the apex of the four helices.⁵ Often appearing in tandem, bromodomains selectively recognize acetyllysine through a hydrogen bond to an asparagine located inside the hydrophobic binding pocket.⁶ Mutations in bromodomain-containing proteins have been identified in human disease. For example, the translocation t(15:19) that fuses the N-terminal region of BRD4 with NUT (nuclear protein in testes) is known to play a major transformative role in NUT midline carcinomas, a particularly aggressive and lethal cancer.⁷ Through recruitment of P-TEFb, BRD4-NUT fusions function to repress expression of c-fos, an important mediator of epithelial differentiation. This blockade induces the carcinogenesis and rapid metastasis that is characteristic of undifferentiated NUT midline carcinomas.^8,9

Figure 1. Phylogenetic Analysis of Bromodomain-containing proteins. Cayman Chemical has purified single or tandem bromodomains from the proteins highlighted on the tree. In addition to purified bromodomains, Inhibitor Screening Assay Kits are also available for proteins highlighted in lime green.

While bromodomains have garnered a significant amount of attention from the biomedical community, aberrations in other types of reader domains can possess striking physiological effects. Genomic translocations leading to the fusion of methyllysine reader domains from JARID1A or PHF23 to Nucleoporin-98 can induce oncogenesis by causing the overexpression of several genes required to maintain pluripotency.¹⁰ The family of methyllysine reader domains contains at least 200 members in several different subfamilies. Members of this large class of protein domains vary widely from a structural perspective ( Figure 2) and in specificity for methylated lysine context and methylation status. The binding modality with which these domains recognize methylated lysine is generally formed through conserved interactions of the methylammonium residue with several aromatic residues in the binding pocket. ¹¹ In contrast to lysine acetylation, histone methylation happens in an exquisitely site-specific manner. Likewise, many methyllysine readers also interact with the residues surrounding the modified residue to provide sequence specificity to the domain.¹¹ This context-dependence is understandably important given the role of lysine methylation in the recruitment of transcription factors and other chromatin-modifying enzymes, and will likely provide chemical biologists a foothold from which they will develop selective chemical probes that target methyllysine reader domains.

Figure 2. Structural diversity of methyllysine reader domains. Unlike the consistent architecture of bromodomains, there is vast structural diversity in the methyllysine reader class of protein domains. Structures from PDB IDs: 1KNE, 2PQW, 2QIC, 2X4W, 3B95, 3DB3, 3IIW.

There are several other histone PTM recognition domains that have yet to be fully explored, including the phosphoserine-binding BRCT family, the methyl-DNA binding domains (e.g., MBD domains) and the methylarginine binding domains (e.g., ADD domains). Mutations in some of these reader domains are also linked to disease. For example, mutations in Methyl CpG binding protein 2 (MeCP2) that disrupt the DNA-binding motif lead to an X-linked mental and physical retardation known as Rett Syndrome.¹² BRCT domains, which recognize phosphorylated histone tails, are heavily involved in recruitment of DNA damage response elements,¹³ and mutations in the BRCT domain of the BRCA1 protein are associated with increased familial risk for breast and ovarian cancer.^12,14,15

Searching for Drugs in the Epigenetic Landscape

Due to the physiological importance of epigenetic reader domains, pharmacological manipulation of these PPIs may provide a novel approach to disease management. The most advanced class of inhibitors targeting epigenetic reader domains came from phenotypic screens designed to identify molecules that increased the production of Apolipoprotein A-1 by hepatocyte model cell lines.¹⁶ Mechanistic studies identified the lead compound from this screen, and the related compound (+)-JQ1 (Item No. 11187), as inhibitors of the BET-family of bromodomains.¹⁶ While the cardiovascular benefit of raising ApoA-1 levels is debatable,^17-19 BET bromodomain inhibition has proven to be particularly effective at slowing the growth of NUT midline carcinomas and a variety of other cancers.^20-24 This initial work led to the development of IBET-762, which is currently in Phase I clinical trials as a novel chemotherapeutic agent for midline carcinomas (NCT01587703). Another BET bromodomain inhibitor, RVX-208, has also entered Phase II trials on glucose metabolism in pre-diabetic patients (NCT01728467).

Encouraged by the serendipitous discovery of BET-family bromodomain inhibitors, a number of academic and industrial groups have undertaken the challenge of targeting other bromodomains. In a joint academic-industrial collaboration, the Structural Genomics Consortium has crystallized a large number of the bromodomains and has developed selective small molecule inhibitors for several different bromodomains (www.thesgc.org). These structures and related tool compounds many of which are available or in development at Cayman Chemical) will be invaluable in the development of next-generation of bromodomain inhibitors. Small molecule inhibitors of other reader domains have also been identified, including molecules targeting methyllysine binding domains of L3MBTL1 and L3MBTL3 (e.g., UNC1215, Item No. 13968), suggesting that bromodomains are not unique among this family of protein motifs in being amenable to small molecule inhibition.^25,26

Given the importance of epigenetic reader domains in gene transcription, this broad family of protein motifs represents a promising new area for drug discovery against a broad array of disease states. Indeed, BET bromodomain inhibitors are currently being pursued beyond their anti-proliferative effects, including anti-viral and anti-inflammatory indications.^27-30 To help make reader domain research possible, Cayman Chemical offers a broad panel of purified reader domains, inhibitor screening assay kits, and chemical probes. As this field progresses, expect to see novel inhibitors of epigenetic reader domains driving early stage drug discovery across a broad scope of disease states. Cayman Chemical will be there every step of the way to provide high-quality research reagents to make discovery possible.

References

1. Blazer, L.L. and Neubig, R.R. Neuropsychopharmacology 34(1), 126-141 (2009).

2. Arkin, M.R. and Whitty, A. Curr. Opin. Chem. Biol. 13(3), 284-290 (2009).

3. Arnaudo, A.M. and Garcia, B.A. Epigenetics Chromatin 6(1), 24 (2013).

4. Bannister, A.J. and Kouzarides, T. Cell Res. 21, 381-395 (2011).

5. Sanchez, R. and Zhou, M.M. Curr. Opin. Drug Discov. Devel. 12(5), 659-665 (2009).

6. Filippakopoulos, P., Picaud, S., Mangos, M., et al. Cell 149(1), 214-231 (2012).

7. French, C.A., Miyoshi, I., Kubonishi, I., et al. Cancer Res. 63(2), 304-307 (2003).

8. French, C.A. Cancer Genet. Cytogenet. 203(1), 16-20 (2010).

9. Jang, M.K., Mochizuki, K., Zhou, M., et al. Mol. Cell 19(4), 523-534 (2005).

10. Wang, G.G., Song, J., Wang, Z., et al. Nature 459(7248), 847-851 (2013).

11. Yun, M., Wu, J., Workman, J.L., et al. Cell Res. 21(4), 564-578 (2013).

12. Amir, R.E., Van den Veyver, I.B., Wan, I.M., et al. Nat. Genet. 23(2), 185-188 (1999).

13. Woods, N.T., Mesquita, R.D., Sweet, M., et al. Sci. Signal. 5(242), rs6 (2012).

14. Glover, J.N. Fam. Cancer 5(1), 89-93 (2006).

15. Clapperton, J.A., Manke, I.A., Lowery, D.M., et al. Nat. Struct. Mol. Biol. 11(6), 512-518 (2004).

16. Mirguet, O., Gosmini, R., Toum, J., et al. J. Med. Chem. 56(19), 7501-7515 (2013).

17. Landmesser, U. Curr. Vasc. Pharmacol. 10(6), 718-719 (2012).

18. Nicholls, S.J., Gordon, A., Johannson, J., et al. Cardiovasc. Drugs Ther. 26(2), 181-187 (2012).

19. Joy, T.R. Pharmacol. Ther. 135(1), 18-30 (2012).

20. Filippakopoulos, P., Qi, J., Picaud, S., et al. Nature 468(7327), 1067-1073 (2010).

21. Roos, G. and Messens, J. Free Radic. Biol. Med. 51(2), 314-326 (2011).

22. Hawkins, C.L., Morgan, P.E., and Davies, M.J. Free Radic. Biol. Med. 46(8), 965-988 (2009).

23. Lockwood, W.W., Zejnullahu, K., Bradner, J.E., et al. Proc. Natl. Acad. Sci. USA 109(47), 19408- 19413 (2013).

24. Cheng, Z., Gong, Y., Ma, Y., et al. Clin. Cancer Res. 19(7), 1748-2759 (2013).

25. Herold, J.M., Wigle, T.J., Norris, J.L., et al. J. Med. Chem 54, 2504-11 (2011).

26. James, L.I., Barsyte-Lovejoy, D., Zhong,N., et al. Nat. Chem. Biol. 9(3), 184-191 (2013).

27. Belkina, A.C., Nikolajczyk, B.S., and Denis, G.V. J. Immunol. 190(7), 3670-3678 (2013).

28. Boehm, D., Calvanese, V., Dar, R.D., et al. Cell Cycle 12(3), 452-462 (2013).

29. Wang, X., Li, J., Schowalter, R.M., et al. PLoS Pathog. 8(11), e1003021

30. Zhu, J., Gaiha, G.D., John, S.P., et al. Cell Rep. 2(4), 807-816 (2012).