Characterization of furoxan derivative compounds as efflux pump inhibitors in Mycobacterium tuberculosis and study of the efflux and halotolerance influence in the resistance profiles of the bacillus

Author(s)

Leonardo Biancolino Marino

• Document type: Doctoral Thesis
• Press: Araraquara. 2016-03-24.
• Institution: Universidade Estadual Paulista (Unesp). Faculdade de Ciências Farmacêuticas. Araraquara
• Defense date: 2016-02-29
• Advisor: Clarice Queico Fujimura Leite; Luiz Pedro Sorio de Carvalho; Cleslei Fernando Zanelli

Abstract

Tuberculosis (TB) is an infectious disease caused mainly by Mycobacterium tuberculosis bacillus, presenting 9.5 million of new cases and 1.5 million of deaths in 2014. Two aggravating factors are associated with these high statistical indexes: a co-infection with HIV ("Human Immunodeficiency Virus") and the increasing emergence of resistant strains represented by MDR ("multi-drug resistant") and XDR ("extreme drug resistant") strains. The bacillus resistance manifests from acquired events (such as acquisition of chromosomal mutations in specific genes related to drug metabolism) or intrinsic, as a highly hydrophobic cell wall (due to the large amount of mycolic acids) and the action of efflux pumps (responsible for drug extrusion). Thus, the thesis project evaluated the expression of five genes encoding efflux pumps by RT-qPCR technique in 12 clinical isolates against rifampicin, the main anti-TB drug used in the standard treatment. Results indicated that these proteins contribute to the emergence of resistance, indicated by overexpression of these genes after treatment. The second part of the study evaluated the activity of 15 furoxan and benzofuroxan derivative compounds for inhibitory capacity against the M. tuberculosis, cytotoxicity in VERO cells, as well as ethidium bromide accumulation and efflux assays. Aiming evaluate their mechanism of action, microarray and assays of nitric oxide release were also done, as they are supposed nitric oxide donors and may present an action by oxidative stress and/or the activity efflux pumps inhibition. Results indicated that the class of compounds is promising for the treatment, with good inhibitory activity against bacillus and substantial degree of selectivity. Mechanism of action assays indicated activity independent of efflux inhibition, of isoniazid subunit’ or nitric oxide release. Microarray pointed to changes in expression of ribosomal proteins, similar to what occurs with aminoglycosides. Finally, the third part of the study demonstrated that the halotolerance developed by M. tuberculosis might cause the emergence of drug resistant bacilli, based on the different concentrations of sodium chloride that the bacillus faces during its cycle. The results indicate a need for reflection on the new guidelines for testing new TB drugs. The results pointed to an increase in the minimal inhibitory concentrations of various anti-TB drugs, when the bacillus was subjected to the sodium chloride concentration found in macrophages, indicating a need for reflection on the guidelines for discovery of new anti-TB drugs. (AU)

• FAPESP's process: 11/21232-1 - Evaluation of the involvement of eIF5A in the secretory pathway in Saccharomyces cerevisiae
• Grantee: Leonardo Biancolino Marino
• Support Opportunities: Scholarships in Brazil - Doctorate (Direct)