Identification of Novel PKC Alpha Inhibitors: Structural and Functional Characterization Using In Silico and In Vitro Studies.

Abstract

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.