Biophysical studies of glucose-6-phosphate dehydrogenase inhibitors complexes

Abstract

According to the World Health Organization (WHO) it is estimated that 7-8 million people are infected with Trypanosoma cruzi worldwide. Despite the socioeconomic and public health impacts of Chagas disease, the available drugs lack desired safety and efficacy profiles, creating an urgent need for new therapeutics. The enzyme glucose-6-phosphate dehydrogenase (G6PDH) catalyzes the first limiting step of the pentose phosphate pathway in the parasite and is an attractive target for the development of trypanocidal drugs.Our group has established production systems for the T. cruzi and human G6PDHs, and using the parasite enzyme a high throughput screening (HTS) was performed allowing the identification of low micromolar quinazolinone and thieno-pyrimidine inhibitors. These compounds showed anti-trypanosomal activity in-vitro, and for quinazolinone derivatives were observed EC50´s of the same order of magnitude as benznidazole. Like classical G6PDH steroidal inhibitors, quinazolinones and thieno-pyrimidines act through a reversible uncompetitive mechanism, yet showed an unprecedented selectivity towards the parasite G6PDH. Our proposal aims to determine the condition where stable G6PDH-inhibitor complexes are formed, to evaluate ligand-binding thermodynamics, to map out the uncompetitive binding site of G6PDHs, and to identify the determinants of specificity. For this purpose fluorescence-based thermal shift, isothermal titration calorimetry, and protein X-ray crystallography experiments will be carried out. For both parasite and human G6PDH, X-ray structures were determined but no protein-inhibitor complexes have been obtained so far. These studies will be executed under the supervision of Prof. Dr. Bill Hunter at the University of Dundee, Scotland. (AU)