Phosphorylation-dependent BRD4 dimerization and implications for therapeutic inhibition of BET family proteins

Bromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains. Phosphorylation by casein kinase II (CK2) regulates BRD4 function, is necessary for active transcription and is involved in resistance to BRD4 drug inhibition in triple-negative breast cancer. Here, we provide the first biophysical analysis of BRD4 phospho-regulation. Using integrative structural biology, we show that phosphorylation by CK2 modulates the dimerization of human BRD4. We identify two conserved regions, a coiled-coil motif and the Basic-residue enriched Interaction Domain (BID), essential for the BRD4 structural rearrangement, which we term the phosphorylation-dependent dimerization domain (PDD). Finally, we demonstrate that bivalent inhibitors induce a conformational change within BRD4 dimers in vitro and in cancer cells. Our results enable the proposal of a model for BRD4 activation critical for the characterization of its protein-protein interaction network and for the development of more specific therapeutics.

Comprehensive Investigation of Stereoselective Food Drug Interaction Potential of Resveratrol on Nine P450 and Six UGT Isoforms in Human Liver Microsomes

The stereoselectivity of the food drug inhibition potential of resveratrol on cytochrome P450s and uridine 5′-diphosphoglucuronosyl transferases was investigated in human liver microsomes. Resveratrol enantiomers showed stereoselective inhibition of CYP2C9, CYP3A, and UGT1A1. The inhibitions of CYP1A2, CYP2B6, and CYP2C19 by resveratrol were stereo-nonselective. The estimated Ki values determined for CYP1A2 were 13.8 and 9.2 μM for trans- and cis-resveratrol, respectively. Trans-resveratrol noncompetitively inhibited CYP3A and UGT1A1 activities with Ki values of 23.8 and 27.4 μM, respectively. Trans-resveratrol inhibited CYP1A2, CYP2C19, CYP2E1, and CYP3A in a time-dependent manner with Ki shift values >2.0, while cis-resveratrol time-dependently inhibited CYP2C19 and CYP2E1. The time-dependent inhibition of trans-resveratrol against CYP3A4, CYP2E1, CYP2C19, and CYP1A2 was elucidated using glutathione as a trapping reagent. This information helped the prediction of food drug interaction potentials between resveratrol and co-administered drugs which are mainly metabolized by UGT1A1, CYP1A2, CYP2C19, CYP2E1, and CYP3A.

Synergistic block of SARS-CoV-2 infection by combined drug inhibition of the host entry factors PIKfyve kinase and TMPRSS2 protease

Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike S protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic, and through an ill-defined mechanism prevents in vitro infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ∼5-10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both, a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2 virus. The substantial ∼5-fold or more decrease of half maximal effective concentrations (EC 50 values) suggests a plausible treatment strategy based on the combined use of these inhibitors. IMPORTANCE Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the coronavirus disease 2019 (COVID-2019) global pandemic. There are ongoing efforts to uncover effective antiviral agents that could mitigate the severity of the disease by controlling the ensuing viral replication. Promising candidates include small molecules that inhibit the enzymatic activities of host proteins, thus preventing SARS-CoV-2 entry and infection. They include Apilimod, an inhibitor of PIKfyve kinase and camostat mesylate and nafamostat mesylate, inhibitors of TMPRSS2 protease. Our research is significant for having uncovered an unexpected synergism in the effective inhibitory activity of apilimod used together with camostat mesylate or with nafamostat mesylate.

Gain-of-Function Mutant TP53 R248Q Overexpressed in Epithelial Ovarian Carcinoma Alters AKT-Dependent Regulation of Intercellular Trafficking in Responses to EGFR/MDM2 Inhibitor

As the most common gene mutation found in cancers, p53 mutations are detected in up to 96% of high-grade serous ovarian carcinoma (HGSOC). Meanwhile, mutant p53 overexpression is known to drive oncogenic phenotypes in cancer patients and to sustain the activation of EGFR signaling. Previously, we have demonstrated that the combined inhibition of EGFR and MDM2-p53 pathways, by gefitinib and JNJ-26854165, exerts a strong synergistic lethal effect on HGSOC cells. In this study, we investigated whether the gain-of-function p53 mutation (p53R248Q) overexpression could affect EGFR-related signaling and the corresponding drug inhibition outcome in HGSOC. The targeted inhibition responses of gefitinib and JNJ-26854165, in p53R248Q-overexpressing cells, were extensively evaluated. We found that the phosphorylation of AKT increased when p53R248Q was transiently overexpressed. Immunocytochemistry analysis further showed that upon p53R248Q overexpression, several AKT-related regulatory proteins translocated in unique intracellular patterns. Subsequent analysis revealed that, under the combined inhibition of gefitinib and JNJ-26854165, the cytonuclear trafficking of EGFR and MDM2 was disrupted. Next, we analyzed the gefitinib and JNJ-26854165 responses and found differential sensitivity to the single- or combined-drug inhibitions in p53R248Q-overexpressing cells. Our findings suggested that the R248Q mutation of p53 in HGSOC caused significant changes in signaling protein function and trafficking, under EGFR/MDM2-targeted inhibition. Such knowledge could help to advance our understanding of the role of mutant p53 in ovarian carcinoma and to improve the prognosis of patients receiving EGFR/MDM2-targeted therapies.

Phosphorylation-dependent BRD4 dimerization and implications for therapeutic inhibition of BET family proteins

Bromodomain-containing protein 4 (BRD4) is an epigenetic reader and oncology drug target that regulates gene transcription through binding to acetylated chromatin via bromodomains (BD). Phosphorylation by casein kinase II (CK2) regulates BRD4 function, is necessary for active transcription and is involved in resistance to BRD4 drug inhibition in triple-negative breast cancer. Here, we provide the first biophysical analysis of BRD4 phospho-regulation. Using integrative structural biology, we show that phosphorylation by CK2 modulates the dimerization of human BRD4. We identify two conserved regions, a coiled-coil motif and the Basic-residue enriched Interaction Domain (BID), essential for the BRD4 structural rearrangement, which we term the phosphorylation-dependent dimerization domain (PDD). Finally, we demonstrate that bivalent inhibitors induce a conformational change within BRD4 dimers in vitro and in cancer cells. Our results enable the proposal of a new model for BRD4 activation critical for the characterization of its protein-protein interaction network and for the development of new specific therapeutics.

Antibody Therapy Targeting Cancer-Specific Cell Surface Antigen AGR2

For anterior gradient 2 (AGR2), normal cells express the intracellular form iAGR2 localized to the endoplasmic reticulum while cancer cells express the extracellular form eAGR2 localized on the cell surface and secreted. Antibodies targeting eAGR2+ cancer cells for eradication will spare normal cells. Two AGR2 monoclonal antibodies, P1G4 and P3A5, were shown to recognize specifically eAGR2+ pancreatic tumors implanted in mice. In addition, P1G4 showed enhancement in drug inhibition of tumor growth. Human:mouse chimeric antibodies of IgG1, IgG2, IgG4 were generated for both antibodies. These human IgG were shown to lyse eAGR2+ prostate cancer cells in vitro with human serum. AGR2 has an important function in distal spread of cancer cells, and is highly expressed in prostate, pancreatic, bladder metastases. Therefore, immunotherapy based on AGR2 antibody-mediated ADCC and CDC is highly promising. Cancer specificity of eAGR2 predicts possibly minimal collateral damage to healthy tissues and organs. Moreover, AGR2 therapy, once fully developed and approved, can be used to treat other solid tumors since AGR2 is an adenocarcinoma antigen found in many common malignancies.