BROMODOMAINS

Bromodomains are chromatin interaction domains that can control gene expression by directing assembly of transcriptional complexes on chromatin, doing so by binding acyl-lysine residues on histones and transcription factors. The importance of the normal function of these proteins is highlighted by their implication in several diseases, including cancer and type II diabetes.

We seek to characterize the key aspects of bromodomain protein structure, and to develop chemical tools to study their function. To do this, we use a wide variety of techniques such as protein-detected Nuclear Magnetic Resonance (NMR) spectroscopy, structure-activity relationship (SAR), organic synthesis, Isothermal Titration Calorimetry (ITC), and x-ray crystallography, among other biophysical assays to explore the unique structural aspects of these interactions as well the affinity and selectivity.

STRUCTURE-INFORMED BROMODOMAIN STUDIES

Despite clear implications in numerous inflammation-related diseases, such as cancer, diabetes, and heart disease, much remains unknown regarding bromodomain structure, function, and potential drug targets. We approach these unknowns using methodology developed in the MCW Program in Chemical Biology and Mellowes Center Structural Genomics Unit. We develop novel inhibitors and chemical probes, and investigate disease-associated mutations.

SELECTIVE TARGETING of PBRM1

Focusing on the Polybromo-1 (PBRM1) protein, which is implicated in cancer, we noted that no selective inhibitors yet existed to target individual bromodomains. Of the six connected bromodomains, we developed a selective inhibitor for the second (BD2) (Shishodia et al, 2022). This was done using our innovative NMR fragment screening pipeline (Egner, 2017) coupled with functional studies. This represents a major advance in studying this protein implicated in cancer, as it is the first selective inhibitor for any of the PBRM1 bromodomains.

TARGETING BRD4 by COVALENT MODIFICATION

BRD4, a BET family bromodomain protein containing two bromodomains, is a promising target for cancer and other inflammation-related diseases. However, selective inhibitors and probes remain elusive. We screened a cysteine-reactive drug fragment library against BRD4 and found binding to a unique cysteine on the second bromomdomain of BRD4. Coupling this fragment to the pan-BET inhibitor JQ1 allowed for covalent and selective modification of BRD4 (Olp et al, 2020).

BROMODOMAIN "READERS" of DISTINCT ACYLATIONS

Bromodomain proteins are known as epigenetic "readers," and their role in binding to acetylated lysine residues on histone proteins is well-described. However, since 1963, when lysine acetylation was first described, numerous distinct acylation marks on lysines have been recently discovered. Levels of different acylated-CoA species can be linked to the metabolome, thus posing a potential link between metabolism and regulation of transcription by bromodomain binding. We seek to understand the significance of bromodomain protein interactions with the broad range of acylations and the downstream effects on transcriptional regulation.

MULTIPLE ACYLATIONS TUNE HISTONE H4 BINDING TO BRD4

We investigated whether BET family bromodomain proteins (BRD2, BRD3, BRD4, and BRDT) could interact with metabolically-derived lysine acylations (Olp et al, 2017). In N-terminal BET bromodomains (BD1) bound acetylated and propionylated peptides; all other acylations inhibited binding. This suggests the levels of acylated-CoA tune or block recruitment of BET bromodomains to histones, linking metabolism to transcription.

SELECTED REFERENCES

Shishodia S, Nunez R, Stormier BP, Bursch KL, Goetz CJ, Olp MD, Jensen DR, Fenske TG, Ordonez-Rubiano SC, Blau ME, Roach MK, Peterson FC, Volkman BF, Dykhuizen EC, Smith BC. Selective and Cell-Active PBRM1 Bromodomain Inhibitors Discovered through NMR Fragment Screening. Journal of Medicinal Chemistry. 2022 October. doi: 10.1021/acs.jmedchem.2c00864

Olp MD, Cui H, Pomerantz WC, Smith BC. "Pharmacological Regulation and Functional Significance of Chromatin Binding by BET Tandem Bromodomains." In: Protein-Protein Interaction Regulators. Roy S, Fu H, editors. Cambridge, UK: Royal Society of Chemistry; 2021. Chapter 9; p.209-248.

Olp MD, Sprague DJ, Goetz CJ, Kathman SG, Wynia-Smith SL, Shishodia S, Summers SB, Xu Z, Statsyuk AV, Smith BC. “Covalent-Fragment Screening of BRD4 Identifies a Ligandable Site Orthogonal to the Acetyl-Lysine Binding Sites.” ACS Chemical Biology. 2020 March 23. PMID 32149490

Fahey JM, Stancill JS, Smith BC, Girotti AW. “Antagonistic effects of nitric oxide in a glioblastoma photodynamic therapy model: mitigation by BET bromodomain inhibitor JQ1”. Journal of Biological Chemistry. 2018, 293, 5345-59.

Egner JM, Jensen DR, Olp MD, Kennedy NW, Volkman BF, Peterson FC, Smith BC, Hill RB. “Development and Validation of 2D Difference Intensity Analysis for Chemical Library Screening by Protein-Detected NMR Spectroscopy”. ChemBioChem. 2017, 19, 448-58. Appeared as the Cover Article

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