Computational modeling of novel inhibitory peptides targeting proteoglycan like region of carbonic anhydrase IX and in vitro validation in HeLa cells

Abstract Carbonic anhydrase IX (CAIX) is a tumour-associated, hypoxia-induced, membrane-bound metallo-enzyme which catalyzes the reversible hydration of carbon dioxide (CO2) to bicarbonate (HCO3−) and proton (H+) ions. Over expression of CAIX is observed in cancers of colon, lung, kidney, breast, etc. CAIX plays a vital role in maintaining favourable intracellular pH for tumour cell growth and extracellular acidification which in-turn leads to drug resistance and spread of factors influencing tumour invasion. The N-terminal proteoglycan (PG) – like fragment of CAIX is unique to this isoform and is considered as potential druggable hotspot. Recently, M75 monoclonal antibody targeting the LPGEEDLPG epitope of PG like region has been proposed to reduce cellular adhesion in cancer cells. LPGEEDLPG fragment in complex with M75 has been crystallized and it serves as a strong base for development of peptide inhibitors based on interacting interfaces. Thus, in this study, an in-depth analysis of intermolecular interactions in LPGEEDLPG-M75 complex was carried out by implementing extensive molecular dynamics simulations, binding free energy calculations so as to infer the major determinant fragments of M75 that can be used as peptide inhibitors targeting PG region. Based on these analyses, 3 peptides (Pep1, Pep2 and Pep3) were synthesized and validated by in vitro assays involving cytotoxicity assessment, CAIX inhibition analysis through Direct and Indirect functional assays, and inhibition of Cell adhesion in HeLa cells. The results reveal Pep1 to be a promising inhibitor as it could efficiently modulate CAIX mediated pH homeostasis and cell adhesion in cancer cells. Communicated by Ramaswamy H. Sarma