Epigenetic Reader Proteins Research Articles

Mechanism and Kinetics of Acetyl-Lysine Binding to Bromodomains.

Bromodomains are four-helix bundle proteins that specifically recognize acetylation of lysine side chains on histones. The available X-ray structures of bromodomain/histone tail complexes show that the conserved Asn residue in the loop between helices B and C is involved in a hydrogen bond with the acetyl-lysine side chain. Here we analyze the spontaneous binding of acetyl-lysine to the bromodomain TAF1(2) by the first molecular dynamics simulations of histone mark binding to an epigenetic reader protein. Multiple events of reversible association sampled along the unbiased simulations allow us to determine the pathway and kinetics of binding. The simulations show that acetyl-lysine has two major binding modes in TAF1(2) one of which corresponds to the available crystal structures and is stabilized by a hydrogen bond to the conserved Asn side chain. The other major binding mode is more buried than in the crystal structures and is stabilized by two hydrogen bonds with conserved residues of the loop between helices Z and A. In the more buried binding conformation, three of the six structured water molecules at the bottom of the binding pocket are displaced by the acetyl-lysine side chain. The kinetic analysis shows that the two binding modes interconvert on a faster time scale with respect to the association/dissociation process. The atomic-level description of the binding pathway and binding modes is useful for the design of small molecule modulators of histone binding to bromodomains.

Structured Water Molecules in the Binding Site of Bromodomains Can Be Displaced by Cosolvent

Bromodomains are α-helical bundles of approximately 110 residues that recognize acetylated lysine side chains mainly on histone tails. Bromodomains are known to play an important role in cancer and inflammation, and as such, significant efforts are being made to identify small-molecule inhibitors of these epigenetic reader proteins. Here, explicit solvent molecular dynamics (MD) simulations of two bromodomains (BAZ2B and CREBBP) are used to analyze the water molecules that seem to be conserved at the bottom of the acetyl-lysine binding site in most crystal structures of bromodomains. The MD runs suggest that the occupancy of the structured water molecules is influenced by conformational transitions of the loop that connects helices Z and A. Additional simulations in the presence of 50 molecules of cosolvent (i.e., 440 mM of dimethylsulfoxide, methanol, or ethanol) indicate that some of the structured water molecules can be displaced transiently. The residence time in the acetyl-lysine binding site is calculated to be about 1 ns, 2-5 ns, and 10-30 ns for methanol, ethanol, and dimethylsulfoxide, respectively, while the affinity of the three cosolvents for BAZ2B and CREBBP is in the range of 50-500 mM. The results described have implications for ligand design, suggesting that only structured water molecules that do not exchange with cosolvent should be maintained in crystal structures used for docking campaigns, and that hydroxy substituents should be incorporated in the ligand so as to map the structured water molecules replaced by (m)ethanol.

Abstract LB-2: Open innovation drug discovery: A study in JQ1.

Abstract Collaborative research from our laboratory, in 2010, produced a first-in-class inhibitor of BET bromodomain proteins (JQ1). As an effort to promote a more open-innovation model of drug discovery, we endeavored to provide JQ1 without restriction of cost or delay to the scientific community. Toward this objective, we have optimized and completed a facile scaling synthesis capable of producing 100 g quantities of the active (+)-JQ1 stereoisomer. In the past 30 months, JQ1 has been provided to over 350 laboratories in more than a dozen countries. Through this research, BET bromodomain proteins have emerged as pharmacologically validated targets in multiple model systems of cancer including squamous carcinoma of the lung, head and neck, acute myeloid leukemia, multiple myeloma, Burkitt lymphoma, B-cell acute lymphoblastic leukemia, neuroblastoma and non-small cell lung cancer. More than 800 cancer cell lines and 25 murine models of cancer have been systematically screened with JQ1 for sensitivity to bromodomain inhibition, identifying additional therapeutic opportunities. Open availability of JQ1 has also lead to unexpected innovation outside of cancer research through studies identifying opportunities for BET bromodomain inhibition in such diverse fields as male contraception and HIV therapeutics. Since publication of the chemical structure and synthetic route to JQ1, seven new BET bromodomain inhibitors have appeared in the literature each derivatized from and/or benchmarked to the JQ1 molecule. Importantly, BET bromodomain inhibition has already accomplished clinical translation. To more objectively assess the consequence of open-innovation as a model of drug discovery, we have systematically analyzed metadata collected from published research and our ongoing efforts in compound distribution. Here, we present an interim analysis which relates the effect of open-innovation in drug discovery to the scope, productivity, reproducibility and importantly the pace of scientific research in epigenetic reader proteins. These results identify open-innovation in drug discovery as a catalyst for scientific discovery, bridging a gap between fields of research and the basic and clinical sciences. Citation Format: Daniel L. Shaw, Charles Y. Lin, Richard P. Oakley, Nicholas P. Semenkovich, Jun Qi, James E. Bradner. Open innovation drug discovery: A study in JQ1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-2. doi:10.1158/1538-7445.AM2013-LB-2

An update on the potential for male contraception: emerging options

An update on the potential for male contraception: emerging options Deborah A Garside,1 Ayman Gebril,2 Manal Alsaadi,2 Natalie Nimmo,2 Alexander B Mullen,2 Valerie A Ferro2 1Imperial College London, Faculty of Medicine, London, 2University of Strathclyde, Strathclyde Institute of Pharmacy and Biomedical Sciences, Glasgow, UK Abstract: The human population continues to grow and is estimated to rise to 10.1 billion by the end of the century. Therefore, there is still an unmet need for safe and highly effective contraceptive options for both men and women. Current options available to men include withdrawal, condoms, and vasectomy. Methods in development fall into two categories: hormonal and nonhormonal. This review will provide an overview of the testosterone combinations and immunocontraception of hormonal targets. Nonhormonal immunocontraception of sperm proteins will also be examined, together with the use of agents to disrupt other sperm-associated targets and pathways. The categories focused on include epididymal proteins, testicular kinases, epigenetic reader proteins, opioids, lonidamine derivatives, retinoic acid, microRNAs associated with spermatogenesis, and plant extracts. Considering these developments, the number of options available to men is likely to increase in the near future. Keywords: hormonal, immunocontraception, nonhormonal contraceptives

Targeting Bromodomains in Myeloma

Abstract SCI-6In cancer, epigenetic proteins are among the most promising and intently pursued targets in drug discovery. This relates to the growing appreciation that cancer growth pathways are centrally influenced by proteins that modify chromatin. Already, inhibitors of DNA methyltransferases and histone deacetylases have demonstrated substantial clinical efficacy, leading to regulatory approval for use in hematologic malignancies. These events have triggered intense competition to develop inhibitors of the chromatin-modifying enzymes known as epigenetic “writers” and “erasers.” Perhaps owing to perceptions regarding the feasibility of targeting protein-protein interactions, small-molecule inhibitors of chromatin-binding modules or epigenetic “readers” have received considerably less attention. Recently, we reported the first potent small-molecule inhibitor of human bromodomains, JQ1, which exhibits selectivity for the BET family of bromodomain-containing transcriptional coactivators. Of broad relevance to cancer, we demonstrated that c-Myc expression is critically dependent on BRD4 function and localization to discrete upstream regulatory regions. Exposure of cancer cells to JQ1 prompts immediate downregulation of c-Myc expression, leading to suppression of a transcriptional program associated with proliferation, survival, and metabolic adaptation. In translational models of MYC-dependent hematologic malignancies, in particular multiple myeloma, the efficacy of JQ1 treatment establishes a mechanistic rationale for the leveraged clinical development of drug-like JQ1 derivatives. Toward this objective, we have completed chemical optimization of novel, drug-like inhibitors of BET bromodomains as reagents capable of supporting human clinical investigation. This research has established the feasibility of inhibiting epigenetic reader proteins with efficient, cell-permeable small molecules. In addition, we have developed a high-throughput platform for modeling mechanism-based, drug-drug combinations to achieve synergy in translational model systems of multiple myeloma. In support of an open-innovation model of chemical biology, we have created a chemical probe allowing the broad study of chromatin biology, which has already been provided to more than 350 academic, governmental, and industrial laboratories worldwide. Together, we have rapidly identified mechanism-based opportunities for clinical translation in cancer. Disclosures:Bradner:SHAPE Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Acetylon Pharmaceuticals: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Tensha Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees.

Mouse study shows spermatogenesis inhibitor is effective as a reversible male contraceptive

A novel small-molecule inhibitor of BRDT, a testis-specific member of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins, has the potential to be used as a potent and reversible male contraceptive, according to a study recently published in Cell.

Small-Molecule Inhibition of BRDT for Male Contraception

SummaryA pharmacologic approach to male contraception remains a longstanding challenge in medicine. Toward this objective, we explored the spermatogenic effects of a selective small-molecule inhibitor (JQ1) of the bromodomain and extraterminal (BET) subfamily of epigenetic reader proteins. Here, we report potent inhibition of the testis-specific member BRDT, which is essential for chromatin remodeling during spermatogenesis. Biochemical and crystallographic studies confirm that occupancy of the BRDT acetyl-lysine binding pocket by JQ1 prevents recognition of acetylated histone H4. Treatment of mice with JQ1 reduced seminiferous tubule area, testis size, and spermatozoa number and motility without affecting hormone levels. Although JQ1-treated males mate normally, inhibitory effects of JQ1 evident at the spermatocyte and round spermatid stages cause a complete and reversible contraceptive effect. These data establish a new contraceptive that can cross the blood:testis boundary and inhibit bromodomain activity during spermatogenesis, providing a lead compound targeting the male germ cell for contraception.PaperClip

Abstract IA16: Chemical inhibition of bromodomains

Abstract In cancer, epigenetic proteins are among the most promising and intently pursued targets in drug discovery. This relates to the growing appreciation that cancer growth pathways are centrally influenced by proteins which modify chromatin. Already, inhibitors of DNA methyltransferases and histone deacetylases have demonstrated substantial clinical efficacy leading to regulatory approval for use in hematologic malignancies. These events have triggered intense competition to develop inhibitors of chromatin modifying enzymes, or so-called epigenetic “writers” and “erasers.” Perhaps owing to perceptions regarding the feasibility of targeting protein:protein interactions, small-molecule inhibitors of chromatin-binding modules or epigenetic “readers” have not been described. Recently, we reported the first potent, small-molecule inhibitor of human bromodomains, JQ1, which exhibits selectivity for the BET family of bromodomain-containing transcriptional co-activators. Of broad relevance to cancer, we will present research demonstrating that c-Myc expression is critically dependent on BRD4 function and localization to discrete upstream regulatory regions. Exposure of cancer cells to JQ1 prompts immediate downregulation of c-Myc expression leading to suppression of a transcriptional program associated with proliferation, survival and metabolic adaptation. In translational models of Myc-dependent hematologic malignancies, the efficacy of JQ1 treatment establishes a mechanistic rationale for the leveraged clinical development of drug-like JQ1 derivatives. Toward this objective, we have completed chemical optimization of novel, drug-like inhibitors of BET bromodomains as reagents capable of supporting human clinical investigation. This research has established the feasibility of inhibiting epigenetic reader proteins with efficient, cell-permeable, small molecules. In support of an open-innovation model of chemical biology, we have created a chemical probe allowing the broad study of chromatin biology, which has already been provided to more than 250 academic, governmental and industrial laboratories worldwide. Together, we have rapidly identified mechanism-based opportunities for clinical translation in cancer, inflammation, and metabolic diseases. Citation Format: James E. Bradner. Chemical inhibition of bromodomains [abstract]. In: Proceedings of the AACR Special Conference on Chemical Systems Biology: Assembling and Interrogating Computational Models of the Cancer Cell by Chemical Perturbations; 2012 Jun 27-30; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2012;72(13 Suppl):Abstract nr IA16.

Abstract SY02-03: Bromodomain inhibition in cancer

Abstract Cellular states are maintained by coordinated gene expression programs, attributable to master regulatory transcription factors. Toward the pharmacologic control of cellular states we have undertaken a chemical biological approach to inhibit chromatin-dependent transcriptional signaling. Transcriptional activation by master regulatory proteins is facilitated by chromatin remodeling at promoter/enhancer regions, in particular side-chain acetylation of lysine residues on nearby histone tails. Context-specific lysine acetylation prompts molecular recognition by transcriptional co-activator proteins possessing acetyl-lysine recognition modules, or bromodomains. Perhaps owing to perceptions regarding the feasibility of abrogating protein-protein interactions, research toward the direct inhibition of human bromodomains (or epigenetic reader proteins, in general) has received comparatively little attention. Based on the above rationale, we have developed chemical and biochemical platforms for the development and characterization of novel bromodomain inhibitors. Recently, we reported the first potent, small-molecule inhibitor of human bromodomains, JQ1, which exhibits selectivity for the BET family of bromodomain-containing transcriptional co-activators. Of broad relevance to cancer, we will present research demonstrating that c-Myc expression is critically dependent on BRD4 function and localization to discrete upstream regulatory regions. Exposure of cancer cells to JQ1 prompts immediate down-regulation of c-Myc expression leading to suppression of a transcriptional program associated with proliferation, survival and metabolic adaptation. In translational models of Myc-dependent hematologic malignancies, the efficacy of JQ1 treatment establishes a mechanistic rationale for the leveraged clinical development of drug-like JQ1 derivatives. Toward this objective, we have completed chemical optimization of novel, drug-like inhibitors of BET bromodomains as reagents capable of supporting human clinical investigation. This research has established the feasibility of inhibiting epigenetic reader proteins with efficient, cell-permeable, small molecules. In support of an open-innovation model of chemical biology, we have created a chemical probe allowing the broad study of chromatin biology, which has already been provided to more than 150 academic, governmental and industrial laboratories worldwide. Together, we have rapidly identified mechanism-based opportunities for clinical translation in cancer, inflammation and metabolic diseases. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr SY02-03. doi:1538-7445.AM2012-SY02-03

Supramolecular hosts that recognize methyllysines and disrupt the interaction between a modified histone tail and its epigenetic reader protein

Post-translational modifications of proteins (including phosphorylation, acetylation and methylation, among others) frequently carry out their biological functions by serving as ‘on’ switches for protein–protein interactions. As highly localized and perfectly defined hot-spots for protein–protein binding, they are a diverse set of motifs that collectively offer great promise as targets for therapeutic intervention and fundamental studies of chemical biology. Recent years have seen the discovery of a very large number of such modification sites on the unstructured tails of proteins, including histones and the tumor suppressor p53. These unstructured protein elements do not present concave binding pockets, and as such cannot be targeted by the conventional small-molecule agents of chemical biology and medicinal chemistry. We report here a family of calixarene-based supramolecular hosts that bind selectively and with high affinity to histone trimethyllysine motifs that are relevant to gene regulation and oncogenesis. We show that these compounds constitute a novel class of protein–protein interaction disruptors and that they can operate selectively against their targeted trimethyllysine sites even in highly complex protein substrates bearing a background of many unmethylated lysines and arginines.