Proteins of the bromodomain and extra-terminal (BET) family are epigenetics “readers” and promising therapeutic targets for cancer and other human diseases We describe herein a structure-guided design of [1,4]oxazepines as a new class of BET inhibitors and our subsequent design, synthesis, and evaluation of proteolysis-targeting chimeric (PROTAC) small-molecule BET degraders Our efforts have led to the discovery of extremely potent BET degraders, exemplified by QCA570, which effectively induces degradation of BET proteins and inhibits cell growth in human acute leukemia cell lines even at low picomolar concentrations QCA570 achieves complete and durable tumor regression in leukemia xenograft models in mice at well-tolerated dose-schedules QCA570 is the most potent and efficacious BET degrader reported to date

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Discovery of QCA570 as an Exceptionally Potent and Efficacious Proteolysis Targeting Chimera (PROTAC) Degrader of the Bromodomain and Extra-Terminal (BET) Proteins Capable of Inducing Complete and Durable Tumor Regression

Bromodomain-containing protein 4 (Brd4) plays an important role in mediating the expression of genes involved in cancers and non-cancer diseases such as inflammatory diseases and acute heart failure. Inactivating Brd4 or downregulating its expression inhibits cancer development, leading to the current interest in Brd4 as a promising anticancer drug target. Numerous Brd4 inhibitors have been studied in recent years and some of them are currently in various phases of clinical trials. Recently, selective degradation of target proteins by small bifunctional molecules (PROTACs) has emerged as an attractive drug discovery approach owing to the advantages it could offer over traditional small-molecule inhibitors. A number of Brd4 degraders have been reported and showed more efficient anticancer activities than just protein inhibition. In this review, we will discuss recent findings in the discovery and development of small-molecule inhibitors and degraders that target Brd4 as a potential anticancer agent.

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Targeting Brd4 for cancer therapy: inhibitors and degraders.

Epigenetic dysfunction has been widely implicated in several diseases especially cancers thus highlights the therapeutic potential for chemical interventions in this field. With rapid development of computational methodologies and high-performance computational resources, computer-aided drug design has emerged as a promising strategy to speed up epigenetic drug discovery. Herein, we make a brief overview of major computational methods reported in the literature including druggability prediction, virtual screening, homology modeling, scaffold hopping, pharmacophore modeling, molecular dynamics simulations, quantum chemistry calculation, and 3D quantitative structure activity relationship that have been successfully applied in the design and discovery of epi-drugs and epi-probes. Finally, we discuss about major limitations of current virtual drug design strategies in epigenetics drug discovery and future directions in this field.

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Computer-Aided Drug Design in Epigenetics

Inhibitor discovery for protein-protein interactions has proven difficult due to the large protein surface areas and dynamic interfaces involved. This is particularly the case when targeting transcription-factor-protein interactions. To address this challenge, structural biology approaches for ligand discovery using X-ray crystallography, mass spectrometry, and nuclear magnetic resonance (NMR) spectroscopy have had a significant impact on advancing small molecule inhibitors into the clinic, including the U.S. Food and Drug Administration approved drug, Venetoclax. Inspired by the protein-observed NMR approach using 1H-15N-HSQC NMR which detects chemical shift perturbations of 15N-labeled amides, we have applied a complementary protein-observed 19F NMR approach using 19F-labeled side-chains that are enriched at protein-protein-interaction interfaces. This protein-observed 19F NMR assay is abbreviated PrOF NMR to distinguish the experiment from the more commonly employed ligand-observed 19F NMR methods. In this Account, we describe our efforts using PrOF NMR as a ligand discovery tool, particularly for fragment-based ligand discovery (FBLD). We metabolically label the aromatic amino acids on proteins due to the enrichment of aromatic residues at protein interfaces. We choose the 19F nucleus due to its high signal sensitivity and the hyperresponsiveness of 19F to changes in chemical environment. Simultaneous labeling with two different types of fluorinated aromatic amino acids for PrOF NMR has also been achieved. We first describe the technical aspects of considering the application of PrOF NMR for characterizing native protein-protein interactions and for ligand screening. Several test cases are further described with a focus on a transcription factor coactivator interaction with the KIX domain of CBP/p300 and two epigenetic regulatory domains, the bromodomains of BRD4 and BPTF. Through these case studies, we highlight medicinal chemistry applications in FBLD, selectivity screens, structure-activity relationship (SAR) studies, and ligand deconstruction approaches. These studies have led to the discovery of some of the first inhibitors for BPTF and a novel inhibitor class for the N-terminal bromodomain of BRD4. The speed, ease of interpretation, and relatively low concentration of protein needed for NMR-based binding experiments affords a rapid, structural biology-based method to discover and characterize both native and new ligands for bromodomains, and it may find utility in the study of additional epigenetic proteins and transcription-factor-protein interactions.

SAR by (Protein-Observed) 19F NMR.

Epigenetics has become a focus of interest in drug discovery. In this sense, bromodomain-containing proteins have emerged as potential epigenetic targets in cancer research and other therapeutic areas. Several computational approaches have been applied to the prediction of bromodomain inhibitors. Nevertheless, such approaches have several drawbacks such as the fact that they predict activity against only one bromodomain-containing protein, using structurally related compounds. Also, there are no reports focused on meaningfully analyzing the physicochemical/structural features that are necessary for the design of a bromodomain inhibitor. This work describes the development of two different multi-target models based on quantitative structure-activity relationships (mt-QSAR) for the prediction and in silico design of multi-target bromodomain inhibitors against the proteins BRD2, BRD3, and BRD4. The first model relied on linear discriminant analysis (LDA) while the second focused on artificial neural networks. Both models exhibited accuracies higher than 85% in the dataset. Several molecular fragments were extracted, and their contributions to the inhibitory activity against the three BET proteins were calculated by the LDA model. Six molecules were designed by assembling the fragments with positive contributions, and they were predicted as multi-target BET bromodomain inhibitors by the two mt-QSAR models. Molecular docking calculations converged with the predictions performed by the mt-QSAR models, suggesting that the designed molecules can exhibit potent activity against the three BET proteins. These molecules complied with the Lipinski's rule of five.

BET bromodomain inhibitors: fragment-based in silico design using multi-target QSAR models

Chemical inhibition of epigenetic regulatory proteins BrdT and Brd4 is emerging as a promising therapeutic strategy in contraception, cancer, and heart disease We report an easily synthesized dihydropyridopyrimidine pan-BET inhibitor scaffold, which was uncovered via a virtual screen followed by testing in a fluorescence anisotropy assay Dihydropyridopyimidine 3 was subjected to further characterization and is highly selective for the BET family of bromodomains Structure–activity relationship data and ligand deconstruction highlight the importance of the substitution of the uracil moiety for potency and selectivity Compound 3 was also cocrystallized with Brd4 for determining the ligand binding pose and rationalizing subsequent structure–activity data An additional series of dihydropyridopyrimidines was synthesized to exploit the proximity of a channel near the ZA loop of Brd4, leading to compounds with submicromolar affinity and cellular target engagement Given these findings, novel and easily synth

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen

Diels-Alder reactions of fused 5-, 6- and 7-membered ring 2-vinylindoles: Synthesis of annulated tetrahydrocarbazoles

A Diels–Alder/Ene Cascade Leading to 5‐(Pyrrolidin‐3‐yl)thieno[3,2‐e]isoindoles from Ketone‐derived 2‐Vinylthiophenes and N‐Phenylmaleimide

The title crystals, C9H5Br2NO2, are the first reported 2,6-dihalophenyl cyanide–isocyanide pair that have neither three- nor two-dimensional isomorphism. Both crystals contain contacts between the carbonyl O atom and a Br atom. In the crystal of the cyanide, R22(10) inversion dimers form based on C≡N⋯Br contacts, a common packing feature in this series of crystals. In the isocyanide, the corresponding N≡C⋯Br contacts are not observed. Instead, the iso­cyano C atom forms contacts with the meth­oxy C atom. RNC was refined as a two-component pseudo-merohedral twin.

https://journals.iucr.org/e/issues/2018/03/00/lh5870/lh5870.pdf

Crystal structures of methyl 3,5-di-bromo-4-cyano-benzoate and methyl 3,5-di-bromo-4-iso-cyano-benzoate.

https://journals.iucr.org/x/issues/2021/04/00/hb4381/hb4381.pdf

Ryan J. Herzig

Papers

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen

Diels-Alder reactions of fused 5-, 6- and 7-membered ring 2-vinylindoles: Synthesis of annulated tetrahydrocarbazoles

A Diels–Alder/Ene Cascade Leading to 5‐(Pyrrolidin‐3‐yl)thieno[3,2‐e]isoindoles from Ketone‐derived 2‐Vinylthiophenes and N‐Phenylmaleimide

Crystal structures of methyl 3,5-di-bromo-4-cyano-benzoate and methyl 3,5-di-bromo-4-iso-cyano-benzoate.

3,5-Dibromo-4-carbamoylbenzoic acid 2-propanol monosolvate

Ryan J. Herzig

Papers

BET Bromodomain Inhibitors with One-Step Synthesis Discovered from Virtual Screen

Diels-Alder reactions of fused 5-, 6- and 7-membered ring 2-vinylindoles: Synthesis of annulated tetrahydrocarbazoles

A Diels–Alder/Ene Cascade Leading to 5‐(Pyrrolidin‐3‐yl)thieno[3,2‐e]isoindoles from Ketone‐derived 2‐Vinylthiophenes and N‐Phenylmaleimide

Crystal structures of methyl 3,5-di-bromo-4-cyano-benzoate and methyl 3,5-di-bromo-4-iso-cyano-benzoate.

3,5-Di­bromo-4-carbamoyl­benzoic acid 2-propanol monosolvate

3,5-Dibromo-4-carbamoylbenzoic acid 2-propanol monosolvate