Estudo estrutural da enzima purina nucleosídeo fosforilase (E.C. 2.4.2.1) de Mycobacterium tuberculosis

Abstract

Tuberculosis re-emerged in the mid 80s and currently about two million people die each year due to this disease. The resurgence of tuberculosis has become a threat to public health. The high susceptibility of HIV-infected patients and the proliferation of multi-drug resistant strains have created the need to develop new therapies. However, no new class of drugs against tuberculosis was developed in the last 45 years. Thus, it becomes imminent need to develop new antituberculosis agents. As purine metabolism could be implicated in mycobacterial latency the purine nucleoside phosphorylase (EC 2.4.2.1) has been proposed as target for development of antibacterial drugs. PNP catalyzes the reversible cleavage in the presence of inorganic phosphate of N-ribosidic bonds of the purine nucleosides and deoxynucleosides, except adenosine. This reaction generates the purine base and ribose (deoxyribose)-1-phosphate. PNP is specific for purine nucleosides in the β-configuration and cleaves the glycosidic bond with inversion of configuration to produce α-ribose-1-phosphate. In this work PNP was crystallized in association with inosine, hypoxanthine and acyclovir, substrate, product and inhibitor, respectively, and data were collected using synchrotron radiation. In addition, molecular dynamics simulations and isothermal titration calorimetry experiments were used to provide detailed information on the dynamic properties and to investigate the profile and thermodynamic affinities of MtPNP associated with these molecules. With obtained results we hope to contribute to the search of new selective inhibitors for MtPNP, since differences between the mycobacterial and human enzyme binding sites have been also identified, making structure-based drug design feasible.