Biophysical Insights into the Binding Interactions of Inhibitors (ICA-1S/1T) Targeting Protein Kinase C-ι

The overexpression of atypical protein kinase C-iota (PKC-ι) is a biomarker for carcinogenesis in various cells, such as glioma, ovarian, renal, etc. manifesting as a potential drug target. In previous in vitro studies, ICA-1S and ICA-1T, an experimental candidate for inhibiting PKC-ι, has demonstrated its specificity and promising efficacy against various cancer cell types. Moreover, the in vivo studies have demonstrated low toxicity levels in acute and chronic murine models. Despite these prior developments, the binding affinities of the inhibitors were never thoroughly explored from a biophysical perspective. Here, we present the biophysical characterizations of PKC-ι in combination with ICA-1S/1T. Various methods based on light scattering, intrinsic fluorescence, thermal denaturation, and heat exchange techniques were applied. The biophysical characteristics, including particle sizing, thermal unfolding, aggregation profiles, enthalpy, entropy, free energy changes, and binding affinity (Kd) of the PKC-ι in the presence of ICA-1S were observed. The studies indicate the presence of domain-specific stabilities in the protein-ligand complex. Moreover, the results indicate a spontaneous reaction with an entropic gain, resulting in a possible entropy-driven hydrophobic interaction in the binding pocket. Altogether, the biophysical approaches employed in this research reveal important insights into the binding interactions of PKC-ι and its inhibitors ICA-1S/1T.