Lectina quimérica biespecífica: estratégia terapêutica para o câncer de pâncreas

Abstract

Tumor cells present modified glycans on their surface, resulting from mutations that affect cellular metabolism and that are not present in healthy cells. Lectins, proteins that interact specifically and selectively with carbohydrates, recognize modified glycans and induce cell death in several types of cancer. Some frequently modified glycans include N-glycans (such as high mannose) and O-glycans (such as T and Tn antigens). In this study, bioinformatics analyses and recombinant DNA technology were used to construct, characterize and test a chimeric lectin with antitumor activity. This chimera consists of a mutant of the banana lectin (Musa acuminata) and Xerocomus chrysenteron mushroom lectin (XCL), presenting affinity for both high mannose and T and Tn antigens. Docking studies contributed to the interaction affinity analysis with the described ligands, and the chimera presented greater binding affinity towards T antigen and high mannose with five branches. Molecular dynamics allowed the evaluation of the stability of the macromolecule for 100 ns, which demonstrated little variation in RMSD, RMSF and radius of gyration at the tested temperature, also maintaining the expected conformation. The best protocol for recombinant production included testing in different strains, temperatures and concentrations of the inducing agent. The 32 kDa chimeric protein was produced in the E. coli Origami and RIL strains and its isolated portions were produced in the RIL strain. The expression induction protocol at 16 °C overnight produced the highest amount of the chimera, which was soluble in D-PBS after dialysis. Its antitumor activity was tested in 2D and 3D cell cultures using the CFPAC-1 lineage of pancreatic cancer, demonstrating a marked antiproliferative effect in the 2D culture and reduced action in the 3D culture. Likely pathways of action of the chimeric lectin were evaluated using known markers of apoptosis and autophagy, which revealed that the chimera acts on cell death by a distinct mechanism from that presented by its isolated portions. This is a pioneer study in the field of lectinology and presents a potential broad-spectrum antitumor therapy derived from natural sources and produced in large quantities through recombinant expression.