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The aim of this study is to address the challenge of the expression of recombinant atypical protein kinase C (aPKCs) in a bacterial system and understand the binding interactions of novel anti-cancer inhibitors of aPKCs. The Protein kinase Cs (PKCs) expression at a high rate is a key indicator of cancer cell proliferation and survival. In particular, the over-expressions of aPKCs were found to be directly associated with various cancer cells in benign and malignant stages. Several cancer growth blockers (inhibitors) are available for targeted therapy. However, these inhibitors had to pass through rigorous in silico screening, in vitro, and in vivo assays before FDA approval. During the characterization studies of the protein, insights into binding interactions between aPKCs and drug molecules are crucial. For successive studies such as for biophysical characterization, the recombinant bioactive full-length PKC- ι (an aPKC) expression in the bacterial system is still a challenge. In addition, the binding interaction studies of aPKCs with inhibitors in physiological conditions providing structural details are limited to some X-ray crystallography structures and computational studies. Here we addressed these two challenges and report a protocol detailing the bioactive full-length and catalytic domain of PKC- ι expression and purification from the bacterial system. The further characterization of the recombinant protein by the determination of melting temperature, functional activity, and circular dichroism reported here provides important information on the characteristics of the protein in its native state. Moreover, we show the chemical interactions of five novel anti-cancer inhibitors ICA-1S (Nucleosidic homolog) 5-amino-1-((1R,2S,3S,4R)-2,3-dihydroxy-4-methylcyclopentyl)-1H-imidazole-4-carboxamide), ICA-1T (Phosphorylated nucleosidic homolog of ICA-1S, [4-(5-amino-4-carbamoylimidazol-1-yl)-2,3-dihydroxycyclopentyl] methyl dihydrogen phosphate), ACPD (2-acetyl-1,3-cyclopentanedione), DNDA (3,4-diaminonaphthalene-2,7-disulfonic acid) and ζ(ZETA)-Stat (8-hydroxy-1,3,6- naphthalenetrisulfonic acid) to locate their regions of binding on the catalytic domain of the aPKCs (PKC- ι and PKC- ζ). We have utilized the chemical shift changes obtained from various multi-dimensional Nuclear Magnetic Resonance (NMR) experiments. Moreover, we have applied the isothermal calorimetry method to understand the biophysical properties of the aPKCs in presence of these inhibitors. The observed biophysical properties of the protein and chemical shift perturbations in the amide backbone of the catalytic domain upon binding shed light on the molecular recognition process, binding characteristics, and atomic-level structural details of the aPKCs. Citation Format: Radwan Ebna Noor, Tracess B. Smalley, Mildred Acevedo-Duncan. A biophysical investigation into the binding interactions of novel anti-cancer inhibitors of atypical protein kinase C (aPKCs). [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3844. |
