Small molecule inhibitors targeting the bromodomain and extra-terminal domain (BET) family proteins have emerged as a promising class of anti-cancer drugs. Nevertheless, the clinical advancement of these agents has been significantly hampered by challenges related to their potency, oral bioavailability, or toxicity. In this study, virtual screening approaches were employed to discover novel inhibitors of the bromodomain-containing protein 4 (BRD4) by analyzing their comparable chemical structural features to established BRD4 inhibitors. Several of these compounds exhibited inhibitory effects on BRD4 activity ranging from 60 % to 70 % at 100 µM concentrations, while one compound also exhibited an 84 % inhibition of Sirtuin 2 (SIRT2) activity. Furthermore, a subset of structurally diverse compounds from the BRD4 inhibitors was selected to investigate their anti-cancer properties in both 2D and 3D cell cultures. These compounds exhibited varying effects on cell numbers depending on the specific cell line, and some of them induced cell cycle arrest in the G0/G1 phase in breast cancer (MDA-MB-231) cells. Moreover, all the compounds studied reduced the sizes of spheroids, and the most potent compound exhibited a 90 % decrease in growth at a concentration of 10 µM in T47D cells. These compounds hold potential as epigenetic regulators for future studies.

UNASSIGNED: Bromodomain and ExtraTerminal (BET) domain proteins are transcriptional cofactors that, recognizing acetylated lysines of histone and non-histone proteins, can modulate gene expression. The BET family consists of four members, each of which contains two bromodomains (BD1 and BD2) able to recognize the acetylated mark. Pan-BET inhibitors (BETi) have shown a promising anticancer potential in many clinical trials; however, their further development has been in part hampered by the side effects due to their lack of selectivity. Mounting evidence suggests that BD1 is primarily involved in cancer and that its selective inhibition can phenocopy the anticancer effects of pan-BETi with increased tolerability. Therefore, the development of BD1 selective inhibitors is highly pursed in both academia and industry.
UNASSIGNED: This review aims at giving an overview of the patent literature of BD1-selective BETi between 2014 and 2023. WIPO, USPTO, EPO, and SciFinder® databases were used for the search of patents.
UNASSIGNED: The development of BD1-selective BETi, despite challenging, is highly desirable as it could have a great impact on the development of new safer anticancer therapeutics. Several strategies could be applied to discover potent and selective compounds with limited side effects.

The polybromo, brahma-related gene 1-associated factors (PBAF) chromatin remodeling complex subunit polybromo-1 (PBRM1) contains six bromodomains that recognize and bind acetylated lysine residues on histone tails and other nuclear proteins. PBRM1 bromodomains thus provide a link between epigenetic posttranslational modifications and PBAF modulation of chromatin accessibility and transcription. As a putative tumor suppressor in several cancers, PBRM1 protein expression is often abrogated by truncations and deletions. However, ∼33% of PBRM1 mutations in cancer are missense and cluster within its bromodomains. Such mutations may generate full-length PBRM1 variant proteins with undetermined structural and functional characteristics. Here, we employed computational, biophysical, and cellular assays to interrogate the effects of PBRM1 bromodomain missense variants on bromodomain stability and function. Since mutations in the fourth bromodomain of PBRM1 (PBRM1-BD4) comprise nearly 20% of all cancer-associated PBRM1 missense mutations, we focused our analysis on PBRM1-BD4 missense protein variants. Selecting 16 potentially deleterious PBRM1-BD4 missense protein variants for further study based on high residue mutational frequency and/or conservation, we show that cancer-associated PBRM1-BD4 missense variants exhibit varied bromodomain stability and ability to bind acetylated histones. Our results demonstrate the effectiveness of identifying the unique impacts of individual PBRM1-BD4 missense variants on protein structure and function, based on affected residue location within the bromodomain. This knowledge provides a foundation for drawing correlations between specific cancer-associated PBRM1 missense variants and distinct alterations in PBRM1 function, informing future cancer personalized medicine approaches.

Feline leukemia virus (FeLV) is a cosmopolitan gammaretrovirus that causes lifelong infections and fatal diseases, including leukemias, lymphomas, immunodeficiencies, and anemias, in domestic and wild felids. There is currently no definitive treatment for FeLV, and while existing vaccines reduce the prevalence of progressive infections, they neither provide sterilizing immunity nor prevent regressive infections that result in viral reservoirs with the potential for reactivation, transmission, and the development of associated clinical diseases. Previous studies of murine leukemia virus (MuLV) established that host cell epigenetic reader bromodomain and extra-terminal domain (BET) proteins facilitate MuLV replication by promoting proviral integration. Here, we provide evidence that this facilitatory effect of BET proteins extends to FeLV. Treatment with the archetypal BET protein bromodomain inhibitor (+)-JQ1 and FeLV challenge of two phenotypically disparate feline cell lines, 81C fibroblasts and 3201 lymphoma cells, significantly reduced FeLV proviral load, total FeLV DNA load, and p27 capsid protein expression at nonlethal concentrations. Moreover, significant decreases in FeLV proviral integration were documented in 81C and 3201 cells. These findings elucidate the importance of BET proteins for efficient FeLV replication, including proviral integration, and provide a potential target for treating FeLV infections.

The BET family protein BRD4, which forms the CDK9-containing BRD4-PTEFb complex, is considered to be a master regulator of RNA polymerase II (Pol II) pause release. Because its tandem bromodomains interact with acetylated histone lysine residues, it has long been thought that BRD4 requires these bromodomains for its recruitment to chromatin and transcriptional regulatory function. Here, using rapid depletion and genetic complementation with domain deletion mutants, we demonstrate that BRD4 bromodomains are dispensable for Pol II pause release. A minimal, bromodomain-less C-terminal BRD4 fragment containing the PTEFb-interacting C-terminal motif (CTM) is instead both necessary and sufficient to mediate Pol II pause release in the absence of full-length BRD4. Although BRD4-PTEFb can associate with chromatin through acetyl recognition, our results indicate that a distinct, active BRD4-PTEFb population functions to regulate transcription independently of bromodomain-mediated chromatin association. These findings may enable more effective pharmaceutical modulation of BRD4-PTEFb activity.

The development and optimisation of a photoaffinity labelling (PAL) displacement assay is presented, where a highly efficient PAL probe was used to report on the relative binding affinities of compounds to specific binding sites in multiple recombinant protein domains in tandem. The N- and C-terminal bromodomains of BRD4 were used as example target proteins. A test set of 264 compounds annotated with activity against the bromodomain and extra-terminal domain (BET) family in ChEMBL were used to benchmark the assay. The pIC50 values obtained from the assay correlated well with orthogonal TR-FRET data, highlighting the potential of this highly accessible PAL biochemical screening platform.

Here we report a detailed structure-activity relationship (SAR) study related to [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the reader module of bromodomain containing-protein 9 (BRD9). 3D structure-based pharmacophore models, previously introduced by us, were here employed to evaluate a second generation of compounds, exploring different substitution patterns on the heterocyclic core. Starting from the promising data obtained from our previously identified [1,2,4]triazolo[4,3-a]quinoxaline-based compounds 1-4, the combination of in silico studies, chemical synthesis, biophysical and in vitro assays led to the identification of a new set of derivatives, selected for thoroughly exploring the chemical space of the bromodomain binding site. In more details, the investigation of different linkers at C-4 position highlighted the amine spacer as mandatory for the binding with the protein counterpart and the crucial role of the alkyl substituents at C-1 for increasing the selectivity toward BRD9. Additionally, the importance of a hydrogen bond donor group, critical to anchor the ZA region and required for the interaction with Ile53 residue, was inferred from the analysis of our collected results. Herein we also propose an optimization and an update of our previously reported \"pharm-druglike2\" 3D structure-based pharmacophore model, introducing it as \"pharm-druglike2.1\". Compounds 24-26, 32, 34 and 36 were identified as new valuable BRD9 binders featuring IC50 values in the low micromolar range. Among them, 24 and 36 displayed an excellent selectivity towards BRD9 and a good antiproliferative effect on a panel of leukemia models, especially toward CCRF-CEM cell line, with no cytotoxicity on healthy cells. Notably, the interaction of 24 and 36 with the bromodomain and PHD finger-containing protein 1 (BRPF1) also emerged, disclosing them as new and unexplored dual inhibitors for these two proteins highly involved in leukemia. These findings highlight the potential for the identification of new attractive dual epidrugs as well as a promising starting point for the development of chemical degraders endowed with anticancer activities.

DNA-encoded cyclic peptide libraries can yield high-potency, high-specificity ligands against target proteins. We used such a library to seek ligands that could distinguish between paralogous bromodomains from the closely related bromodomain and extra-terminal domain family of epigenetic regulators. Several peptides isolated from a screen against the C-terminal bromodomain of BRD2, together with new peptides discovered in previous screens against the corresponding domain from BRD3 and BRD4, bound their targets with nanomolar and sub-nanomolar affinities. X-ray crystal structures of several of these bromodomain-peptide complexes reveal diverse structures and binding modes, which nevertheless display several conserved features. Some peptides demonstrate significant paralog-level specificity, although the physicochemical explanations for this specificity are often not clear. Our data demonstrate the power of cyclic peptides to discriminate between very similar proteins with high potency and hint that differences in conformational dynamics might modulate the affinity of these domains for particular ligands.

Acetylation signaling pathways in trypanosomatids, a group of early branching organisms, are poorly understood due to highly divergent protein sequences. To overcome this challenge, we used interactomic datasets and AlphaFold2 (AF2)-multimer to predict direct interactions and validated them using yeast two and three-hybrid assays. We focused on MORF4 related gene (MRG) domain-containing proteins and their interactions, typically found in histone acetyltransferase/deacetylase complexes. The results identified a structurally conserved complex, TcTINTIN, which is orthologous to human and yeast trimer independent of NuA4 for transcription interaction (TINTIN) complexes; and another trimeric complex involving an MRG domain, only seen in trypanosomatids. The identification of a key component of TcTINTIN, TcMRGBP, would not have been possible through traditional homology-based methods. We also conducted molecular dynamics simulations, revealing a conformational change that potentially affects its affinity for TcBDF6. The study also revealed a novel way in which an MRG domain participates in simultaneous interactions with two MRG binding proteins binding two different surfaces, a phenomenon not previously reported. Overall, this study demonstrates the potential of using AF2-processed interactomic datasets to identify protein complexes in deeply branched eukaryotes, which can be challenging to study based on sequence similarity. The findings provide new insights into the acetylation signaling pathways in trypanosomatids, specifically highlighting the importance of MRG domain-containing proteins in forming complexes, which may have important implications for understanding the biology of these organisms and developing new therapeutics. On the other hand, our validation of AF2 models for the determination of multiprotein complexes illuminates the power of using such artificial intelligence-derived tools in the future development of biology.

Cancer cells display pervasive changes in DNA methylation, disrupted patterns of histone posttranslational modification, chromatin composition or organization and regulatory element activities that alter normal programs of gene expression. It is becoming increasingly clear that disturbances in the epigenome are hallmarks of cancer, which are targetable and represent attractive starting points for drug creation. Remarkable progress has been made in the past decades in discovering and developing epigenetic-based small molecule inhibitors. Recently, epigenetic-targeted agents in hematologic malignancies and solid tumors have been identified and these agents are either in current clinical trials or approved for treatment. However, epigenetic drug applications face many challenges, including low selectivity, poor bioavailability, instability and acquired drug resistance. New multidisciplinary approaches are being designed to overcome these limitations, e.g., applications of machine learning, drug repurposing, high throughput virtual screening technologies, to identify selective compounds with improved stability and better bioavailability. We provide an overview of the key proteins that mediate epigenetic regulation that encompass histone and DNA modifications and discuss effector proteins that affect the organization of chromatin structure and function as well as presently available inhibitors as potential drugs. Current anticancer small-molecule inhibitors targeting epigenetic modified enzymes that have been approved by therapeutic regulatory authorities across the world are highlighted. Many of these are in different stages of clinical evaluation. We also assess emerging strategies for combinatorial approaches of epigenetic drugs with immunotherapy, standard chemotherapy or other classes of agents and advances in the design of novel epigenetic therapies.