Elucidating the Role of Protein–Protein Interactions in Modulating Inhibitor Affinity and Release Mechanisms in Serine Arginine Protein Kinase

Protein-protein (pp) interactions make up a varied class of potential therapeutic targets for malignancy, which cause disturbances in the alternating splicing of SR proteins by SRPK due to nuclear accumulation. SRPK1 phosphorylates several serine residues in the RS domain of ASF/SF2, a classic SR protein. Substrate selectivity is dependent on protein interactions beyond the kinase active site. The RS domain's phosphorylation cycle needs strong, sustained SRPK-SR binding. In light of this evidence, the present research investigates the dynamics of protein-protein complexes bound to nucleotide triphosphates and inhibitor molecules. The influence of protein-protein interactions and the role of small molecules at the binding site, exerting competitive inhibition, were examined using classical molecular dynamics simulations complemented by statistical analysis. Trajectory visualization and analysis revealed differential ASF-SRPK binding in the presence of ATP and MSC1186 (the most recent SRPK inhibitor). In fact, conformational changes and binding orientations of small molecules, together with ASF-SRPK interactions, are interdependent in sustaining biological functions. Free energy of binding of the small molecules at the active pocket was empirically obtained from the Generalized Born Implicit Solvation Model with details of the residue-wise contribution toward binding. A precise absolute binding free energy approach, based on streamlined alchemical free energy perturbation, was applied to evaluate the binding affinity at the competitive pocket and yielded results consistent with experimental data. Enhanced sampling method─"random accelerated molecular dynamics (RAMD)", including PMF evaluation and path analysis─was employed to explore the egression route and investigate the exit dynamics of small molecules from the binding pocket. This study lays the groundwork for novel therapeutic design methodologies while improving our molecular-level understanding of protein-protein-small-molecule interactions linked to carcinogenesis.