Structural insights into the molecular basis responsible for the effects of immobilization on the kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi and human

Abstract

O desenvolvimento de métodos rápidos e eficazes para a identificação de novas moléculas bioativas é fundamental para o processo de descoberta e planejamento de fármacos. A integração de um sistema de cromatografia liquida de alta eficiência (CLAE), com biorreatores como fase estacionária (IMER) é uma estratégia atrativa e versátil para a triagem de coleções de compostos visando à identificação de novos agentes terapêuticos. Os parâmetros cinéticos da enzima imobilizada gliceraldeído-3-fosfato desidrogenase (GAPDH) de Trypanosoma cruzi e humana foram determinados (T. cruzi:; humana:) e comparados com aqueles observados em solução (T. cruzi:). Os resultados indicaram uma diminuição na afinidade das enzimas imobilizadas, entretanto, os elementos estruturais necessários para o processo de reconhecimento molecular e atividade biológica permaneceram inalterados, aumentando a estabilidade das enzimas. Além disso, a análise estrutural forneceu dados moleculares importantes envolvidos nos efeitos da imobilização sobre as interações entre ligante e receptor e, consequentemente, sobre a atividade enzimática e parâmetros cinéticos.The development of fast and reliable methods for the identification of new bioactive compounds is of utmost importance to boost the process of drug discovery and development. Immobilized enzyme reactors (IMERs), integrated with high performance liquid chromatography (HPLC), are attractive and versatile tools for screening collections consisting of natural products and synthetic small molecules. Standard kinetic parameters of the immobilized enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from both Trypanosoma cruzi de and human have been determined (T. cruzi:; humana:), and comparisons of these values with those of the parasite and human free enzymes indicate a decrease in the affinity for the immobilized system (T. cruzi:). Interestingly, despite the kinetic differences between the two systems, the immobilized GAPDHs retained the required structural requirements for molecular recognition and biological activity, increasing the stability the enzyme. In the present work, we described an integrated structural analysis which has provided important insights into the molecular basis underlying the effects of immobilization on the ligand-receptor interactions and consequent enzymatic activity and kinetics parameters.

Structure- and ligand-based drug design approaches for neglected tropical diseases

“…Compound 5 was active only against closely related cysteine proteases, however, with significant SI values ranging from 80 (cathepsin-S) to 50.000 (cathepsin-B). The integration of SBDD and LBDD approaches has found its way into drug discovery programs for NTDs [32][33][34][35][36][37][38]. Good examples can be seen in the use of 3D QSAR methods, such as comparative molecular field analysis (CoMFA) [39,40] and comparative molecular similarity index analysis (CoMSIA) [41,42].…”