Devika S. Kumar

Histone deacetylases (HDACs) are pivotal epigenetic regulators that modulate diverse cellular pathways by removing acetyl groups from lysine residues on both histone and non-histone proteins. Histone deacetylase 11 (HDAC11), the sole member of class IV HDACs, exhibits both deacetylation and fatty acid deacylation activities. Accumulating evidence implicates HDAC11 as a key epigenetic regulator of fundamental cellular processes, including metabolism, immune responses, and tissue development. Dysregulation of HDAC11 activity has been associated with inflammatory diseases, metabolic disorders, neurodegenerative conditions, and cancer, highlighting its potential as a therapeutic target. Although several HDAC11-specific inhibitors have been identified, none have progressed to clinical development. In this study, we aimed to discover HDAC11-selective inhibitors by integrating in silico and in vitro validation approaches. Homology modelling of the HDAC11 structure was conducted, followed by model validation, structure-based virtual screening, molecular dynamics (MD) simulations, and binding free energy calculations. We identified and validated three lead compounds and their intermediates using biochemical and cell-based assays. Fluorescence-based and HPLC-based enzymatic assays demonstrated potent inhibition of both the deacetylase and deacylase activities of HDAC11, with Inhibitor 6 and Inhibitor 3 exhibiting the strongest effects among the six compounds tested. Further, a decrease in lipid accumulation, reduced stability of the HDAC11 substrate SHMT2, as determined by immunoblot analysis and decreased cell viability, as assessed by MTT assay, confirmed HDAC11 inhibition in cellular models. The study shows that new HDAC11 inhibitors significantly reduce the viability of breast cancer cells and induce apoptosis; inhibitor 6, in particular, showed high potency, similar to the reference compound SIS-17. Flow cytometry showed that treated MDA-MB-231 cells exhibited cell-cycle arrest and increased apoptosis, a finding further confirmed by Annexin V/PI staining. Molecular analysis showed that BAX increased while BCL2 decreased, indicating that apoptotic pathways were activated in novel compound-treated MDA-MB-231 cells. The results suggest that inhibiting HDAC11 is an effective way to induce cancer cell death and provide a basis for further assessment of these compounds as potential treatments for breast cancer. Collectively, this study identifies novel zinc-chelating HDAC11 inhibitors containing a nitro-sp² group, providing promising candidates for further therapeutic development.

Introduction: Protein Kinase C alpha (PKCα), a key regulator of cellular signaling, is frequently dysregulated in breast cancer. Despite its clinical relevance, there is a dearth of selective and safe PKCα inhibitors. This study aims to identify and characterize novel PKCα inhibitors using computational and experimental methodologies. Methods: A structure-based virtual screening of the Enamine Hinge Binders Library against the PKCα receptor (PDB ID: 3IW4) was performed using the Glide module of Schrödinger Suite. Analysis of docking scores, protein-ligand interactions, binding free energies, and ADMET profiles helped identify lead candidates. The stability of protein-ligand complexes was confirmed using MD simulations. The functional activity of identified candidates was validated using a kinase assay, and cell death was analyzed using an MTT assay on breast cancer cell lines. Results: Compounds Z3077775938 (P1) and Z22177390 (P2) exhibited strong binding and stability within the hinge-binding region of PKCα and showed favorable drug-likeness profiles. Structural analysis revealed P1 and P2 as novel scaffolds. The kinase assay revealed dosedependent inhibition of PKCα’s ATP-binding activity with IC50 values of P1 and P2 as 0.92 nM and 15.27 nM, respectively. Both compounds induced dose-dependent cytotoxicity in breast cancer cells. Discussion: This study identified two novel, structurally diverse PKCα inhibitors. Their low IC50 values and dose-dependent cytotoxicity, as observed in both ER+ and triple-negative breast cancer cell lines, validate their functional efficacy and highlight therapeutic potential for breast cancer treatment Conclusion: Overall, this study identified two PKCα inhibitors that provide a foundation for further optimization as selective therapeutic agents in PKCα-driven malignancies