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Studies of the mechanism and inhibition of dihydroorotate dehydrogenase from Leishmania major

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Author(s):
Renata Almeida Garcia Reis
Total Authors: 1
Document type: Doctoral Thesis
Press: Ribeirão Preto.
Institution: Universidade de São Paulo (USP). Faculdade de Ciências Farmacêuticas de Ribeirão Preto (PCARP/BC)
Defense date:
Examining board members:
Maria Cristina Nonato Costa; Hamilton Cabral; Artur Torres Cordeiro; Marcos Vicente de Albuquerque Salles Navarro
Advisor: Maria Cristina Nonato Costa
Abstract

Dihydroorotate dehydrogenases (DHODHs) catalyze the fourth and only redox reaction in the de novo pyrimidine biosynthetic pathway, the oxidation of dihydroorotate (DHO) to orotate (ORO) through a mechanism dependent on the FMN cofactor and electron acceptors. The significance of pyrimidine for cell proliferation and maintenance determine DHODH as potential therapeutic target. DHODHs have been distinguished in two major classes based on sequence and structural comparisons: class 1, which is further divided into subclasses 1A and 1B, and class 2. The model protein in our studies on trypanosomatid class 1A DHODHs is the enzyme from Leishmania major (LmDHODH). The essential question in this study was to contribute for the understanding about the catalytic and inhibition mechanisms of class 1A DHODHs . First, steady-state and pre-steady state kinetic approaches have been used to characterize the catalytic cycle of LmDHODH and have allowed the comparison between the global cycle and each independent half-reaction. Also, the interaction of LmDHODH with the products of the reaction was analyzed by kinetic, spectral titration and isothermal titration calorimetry assays. After topographical analysis based on the LmDHODH crystallographic structure, different mutants were constructed and structurally and kinetically characterized. For inhibitor search, chemical libraries of compounds were screened by using biolayer interferometry binding and enzymatic assays. Based on our results, for the first time, steady-state kinetics analysis of class 1A allowed the observation of a positive cooperativity for DHO binding with a Hill coefficient (h) value close to 2. Moreover, our results show that the degree of cooperativity in DHO binding is affected by ORO. Comparison between steady and pre-steady-state parameters together with studies of interaction for LmDHODH with both products, suggests that ORO release is the rate-limiting step in overall catalysis with is not true to the human enzyme (class 2 DHODH). The hybrid approach used to characterize LmDHODH mutants allowed us to propose that the cooperative behavior is related with the cross-talk between the two active sites mediated by the dimer interface. Also, the conformational dynamics of the catalytic loop is very relevant in the global mechanism of the reaction and there is a correlation between catalytic loop rearrangement and dimer interface. Our results bring an important contribution for a better understanding of the detailed catalytic mechanism adopted by class 1A DHODHs. They also reveal key differences in the enzymatic mechanism adopted by class 1A and class 2 DHODHs, to be exploited in the development of potent and selective LmDHODH inhibitors. Regarding to inhibitors search, few hits were obtained by using screening of Drug Discovery Unit (DDU) fragment library with biolayer interferometry binding assay. Also, an end-point enzymatic assay was developed for LmDHODH and the optimisation carried out at the DDU provided a reliable HTP assay using both DDU\'s fragment library and small diversity set. Hits were validated using a potency assay with good inhibitors found using both libraries. The screens identified a number of series as starting points for medicinal chemistry and structural investigations. Two hits identified from small diversity set exhibit great IC50 values, 260 nM and 1.07 ?M. (AU)