IDENTIFICATION OF POTENT BROMODOMAIN4 (BRD4) INHIBITORS BY ENERGY-PHARMACOPHORE BASED VIRTUAL SCREENING TO TARGET BRD4-NUT MIDLINE CARCINOMA (original) (raw)
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Computational screening is a method to prioritize small-molecule compounds based on the structural and biochemical attributes built from ligand and target information. Previously, we have developed a scalable virtual screening workflow to identify novel multitarget kinase/ bromodomain inhibitors. In the current study, we identified several novel N-[3-(2-oxo-pyrrolidinyl)phenyl]-benzenesulfonamide derivatives that scored highly in our ensemble docking protocol. We quantified the binding affinity of these compounds for BRD4(BD1) biochemically and generated cocrystal structures, which were deposited in the Protein Data Bank. As the docking poses obtained in the virtual screening pipeline did not align with the experimental cocrystal structures, we evaluated the predictions of their precise binding modes by performing molecular dynamics (MD) simulations. The MD simulations closely reproduced the experimentally observed protein−ligand cocrystal binding conformations and interactions for all compounds. These results suggest a computational workflow to generate experimental-quality protein−ligand binding models, overcoming limitations of docking results due to receptor flexibility and incomplete sampling, as a useful starting point for the structure-based lead optimization of novel BRD4(BD1) inhibitors.
Structural variation of protein-ligand complexes of the first bromodomain of BRD4
The bromodomain-containing protein 4 (BRD4), a member of the bromodomain and extra-terminal domain(BET) family, plays a key role in several diseases, especially cancers. With increased interest in BRD4 as atherapeutic target, over 300 X-ray crystal structures of the protein in complex with small molecule inhibitorshave become publicly available over the recent decade. In this study, we use this structural information toinvestigate the conformations of the first bromodomain (BD1) of BRD4. Structural alignment shows a highlevel of similarity between the structures of BRD4-BD1, regardless of the bound ligand. We employ WONKA,a tool for detailed analyses of protein binding sites, to compare the active site of over 100 crystal structures,with a focus on the highly conserved network of water molecules, which line the binding pocket of BRD4-BD1.The analysis presented in this work helps guide the selection of the best structure of BRD4-BD1 to use instructure-based drug design, an important ...
Bromodomains are evolutionarily conserved structural motifs that recognize acetylated lysine residues on histone tails. They play a crucial role in shaping chromatin architecture and regulating gene expression in various biological processes. Mutations in bromodomains containing proteins leads to multiple human diseases, which makes them attractive target for therapeutic intervention. Extensive studies have been done on BRD4 as a target for several cancers, such as Acute Myeloid Leukemia (AML) and Burkitt Lymphoma. Several potential inhibitors have been identified against the BRD4 bromodomain. However, most of these inhibitors have drawbacks such as nonspecificity and toxicity, decreasing their appeal and necessitating the search for novel non-toxic inhibitors. This study aims to address this need by virtually screening natural compounds from the NPASS database against the Kac binding site of BRD4-BD1 using high throughput molecular docking followed by similarity clustering, pharmac...
This manuscript focuses on the structure-based design of selective inhibitors of the first bromodomain of BRD4. This manuscript uses describes organic synthesis to make inhibitors, and biophysical analysis to evaluate their inhibitor potency in competive inhibition assays (fluorescence anisotropy assays and AlphaScreen). Binding mode is evaluate from protein co-crystal structures. Cell activity is evaluated in cell viability assays, target engagement CETSA assays analyzed via western blot, and inhibition of Myc via western blot analysis.
A Structure-based Design Approach for Generating High Affinity BRD4 D1-Selective Chemical Probes
Journal of Medicinal Chemistry, 2022
Chemical probes for epigenetic proteins are essential tools for dissecting the molecular mechanisms for gene regulation and therapeutic development. The bromodomain and extraterminal (BET) proteins are master transcriptional regulators. Despite promising therapeutic targets, selective small molecule inhibitors for a single bromodomain remain an unmet goal due to their high sequence similarity. Here, we address this challenge via a structure-activity relationship study using 1,4,5-trisubstituted imidazoles against the BRD4 N-terminal bromodomain (D1). Leading compounds 26 and 30 have 15 and 18 nM affinity against BRD4 D1 and over 500-fold selectivity against BRD2 D1 and BRD4 D2 via ITC. Broader BET selectivity was confirmed by fluorescence anisotropy, thermal shift, and CETSA. Despite BRD4 engagement, BRD4 D1 inhibition was unable to reduce c-Myc expression at low concentration in multiple myeloma cells. Conversely, for inflammation, IL-8 and chemokine downregulation were observed. These results provide new design rules for selective inhibitors of an individual BET bromodomain.
Proteins: Structure, Function, and Bioinformatics, 2019
Bromodomains (BrDs), a conserved structural module in chromatin-associated proteins, are well known for recognizing ε-N-acetyl lysine residues on histones. One of the most relevant BrDs is BRD4, a tandem bromodomain containing protein (BrD1 and BrD2) that plays a critical role in numerous diseases including cancer. Growing evidence shows that the two BrDs of BRD4 have different biological functions; hence selective ligands that can be used to study their functions are of great interest. Here, as a follow-up of our previous work (Gacias et al., Chem Biol. 2014; 21:841-854), we first provide a detailed characterization study of the in silico rational design of Olinone as part of a series of five tetrahydro-pyrido indole-based compounds as BRD4 BrD1 inhibitors. Additionally, we investigated the molecular basis for Olinone's selective recognition by BrD1 over BrD2. Molecular dynamics simulations, free energy calculations and conformational analyses of the apo-BRD4-BrD1|2 and BRD4-BrD1|2/Olinone complexes showed that Olinone's selectivity is facilitated by five key residues: Leu92 in BrD1|385 in BrD2 of ZA loop, Asn140|433, Asp144|His437 and Asp145|Glu438 of BC loop, and Ile146|Va149 of helix C. Furthermore, the difference in hydrogen bonds number and in mobility of the ZA and BC loops of the acetyl-lysine binding site between BRD4 BrD1/Olinone and BrD2/Olinone complexes also contribute to the difference in Olinone's binding affinity and selectivity towards BrD1 over BrD2. Altogether, our computer-aided molecular design techniques can effectively guide the development of smallmolecule BRD4 BrD1 inhibitors, explain their selectivity origin, and further open doors to the design of new therapeutically-improved derivatives.
Bromodomain-containing proteins are readers of acetylated lysine and play important roles in cancer. Bromo-domain-containing protein 7 (BRD7) has been implicated in multiple malignancies; however, there are no selective chemical probes to study its function in disease. Using crystal structures of BRD7 and BRD9 bromodomains (BDs) bound to BRD9-selective ligands, we identified a binding pocket exclusive to BRD7. We synthesized a se-ries of ligands designed to occupy this binding region and identified two BRD7-selective inhibitors, 1-78 and 2-77, that bind with nanomolar affinity to the BRD7 BD. Our binding mode analyses indicate that these ligands oc-cupy a uniquely accessible binding cleft in BRD7 and maintain key interactions with the asparagine and tyrosine residues critical for acetylated lysine binding. Finally, we validated the utility and selectivity of the compounds in cell-based models of prostate cancer.
This study aims to predict structural stability changes and to identify the molecular determinants of ligand-bromodomain complex interactions that undergo mutations in the ligand binding site (LBS) using computational chemistry methods. The stability changes of the complexes were investigated using Molecular Dynamics (MD) simulations during a 25 ns production run. The identification of molecular interaction determinants was performed by decoding the interaction fingerprint, utilizing the output of trajectory data of the MD simulations, which were converted into a series of pdb files along the time step. The system preparations were done using CHARMM-GUI. The MD simulations were carried out using the GROMACS program. Protein-ligand interaction fingerprints (PLIF) were identified using the PyPLIF-HIPPOS program. This study successfully predicted the stability of both wild-type and mutated ligand-bromodomain complex structures, where the W81A mutation led to a decrease in complex stability. The key residues and non-hydrophobic interaction types responsible for the stabilities were identified as TRP81 aromatic edge-to-face, TYR139 aromatic edge-to-face, and TYR139 aromatic face-to-face.