Investigação do potencial impacto de substituições em subtipos de cruzipaínas nas interações com inibidores

Abstract

Despite being discovered in 1909, Chagas disease remains a major public health and social problem, particularly in Latin America. The available treatments have limited efficacy in the chronic phase of the disease and are associated with significant adverse effects. In this context, cruzain (cruzipain 1), a cysteine protease essential for the survival of Trypanosoma cruzi, has been explored as a therapeutic target for the development of new inhibitors. However, despite its potential, the different subtypes of cruzipain are still poorly studied, especially regarding the structural variations that may influence the interaction with ligands. In this study, we employed computational approaches to investigate the impact of substitutions in cruzipain subtypes on the interaction with inhibitors described for cruzain. To achieve this, we established a molecular docking protocol, validated through enrichment and redocking analyses. The enrichment analysis revealed that the program was able to discriminate between active compounds and decoys (AUC = 0.83), while redocking showed a success rate of 52%, demonstrating the protocol’s viability for studying cruzipains. Structural models of the different subtypes were generated by comparative modeling and used in docking simulations. The results indicated that substitutions in the S2 and S3 subsites had a greater impact on the obtained poses, with bulky residues such as Phe61 partially obstructing these regions and influencing ligand positioning. Additionally, rescoring analyses showed that substitutions in the active site also modified the interaction profile with the inhibitors, resulting in variations in the estimated energy values between cruzipain subtypes. Molecular dynamics simulations of ligand 16 with cruzipains revealed variations in complex stability. In cruzipain 2, the inhibitor remained in the active site throughout the entire simulation. In contrast, the presence of Phe61 and Trp67 in cruzipain 3.7 led to reduced stability, reflected by the loss of inhibitor interaction with the active site. In cruzipain 3.9, in which only Trp67 is present, the impact on stability was less pronounced, with the inhibitor remaining in the active site for a longer simulation period. In the complex with cruzipain 4, the inhibitor exhibited high instability in the active site across all replicas. Notably, the substitution of Glu208 by Gly resulted in the loss of a hydrogen bond with the phenolic ring of the inhibitor, which may have contributed to the lower stability observed. These findings contribute to the understanding of the structural differences between cruzipain subtypes and their potential impact on interactions with inhibitors, aiding in the development of therapeutic strategies against Chagas disease.