Combined Approaches for Drug Design Points the Way to Novel Proline Racemase Inhibitor Candidates to Fight Chagas’ Disease

Abstract

Chagas’ disease is caused by Trypanosoma cruzi, a protozoan transmitted to humans by blood-feeding insects, blood transfusion or congenitally. Previous research led us to discover a parasite proline racemase (TcPRAC) and to establish its validity as a target for the design of new chemotherapies against the disease, including its chronic form. A known inhibitor of proline racemases, 2-pyrrolecarboxylic acid (PYC), is water-insoluble. We synthesized soluble pyrazole derivatives, but they proved weak or inactive TcPRAC inhibitors. TcPRAC catalytic site is too small and constrained when bound to PYC to allow efficient search for new inhibitors by virtual screening. Forty-nine intermediate conformations between the opened enzyme structure and the closed liganded one were built by calculating a transition path with a method we developed. A wider range of chemical compounds could dock in the partially opened intermediate active site models in silico. Four models were selected for known substrates and weak inhibitors could dock in them and were used to screen chemical libraries. Two identified soluble compounds, (E)-4-oxopent-2-enoic acid (OxoPA) and its derivative (E)-5-bromo-4-oxopent-2-enoic acid (Br-OxoPA), are irreversible competitive inhibitors that presented stronger activity than PYC on TcPRAC. We show here that increasing doses of OxoPA and Br-OxoPA hamper T. cruzi intracellular differentiation and fate in mammalian host cells. Our data confirm that through to their binding mode, these molecules are interesting and promising as lead compounds for the development of chemotherapies against diseases where active proline racemases play essential roles.

Phylogenetic and syntenic data support a single horizontal transference to a Trypanosoma ancestor of a prokaryotic proline racemase implicated in parasite evasion from host defences

“…Taking into account that Tc PRAC-A and TcPRAC-B participate in T. cruzi development in vertebrates and vectors and have been incriminated as host defence factors [ 9 - 12 , 19 , 22 , 55 ], it is tempting to speculate whether DTU-specific Tc PRAC enzymes can contribute to differential degrees of metacyclogenesis, parasitemias and virulence. T. cruzi of different DTUs interacts differently with the host, induces distinct immune responses and infections ranging from highly lethal to virtually asymptomatic, contributing to variable clinical forms of Chagas disease.…”

Designed mono- and di-covalent inhibitors trap modeled functional motions for Trypanosoma cruzi proline racemase in crystallography

“…The structure also revealed that the cyclopentane moiety of the inhibitor could adopt multiple conformations, suggesting that space was available for further chemical modulations. Interestingly, the conformation of the active site in the co-crystal structures with our inhibitors was highly similar to that of the transitional intermediate models built to identify the first inhibitors by virtual screening [ 17 ]. This could be viewed as a demonstration of the relevance of molecular modeling in enlarging chemical space search in drug design.…”

“…Inactivation kinetics were performed at different inhibitor concentrations as described previously [ 17 , 26 , 27 ]. A program developed in-house was used to fit the data [ 17 , 26 , 27 ]. As global fitting appeared difficult and was unsuccessful, single exponential models were simply fit to the individual kinetics.…”

“…This suggested that the enzyme function involved an opening/closing mechanism with intermediate forms that could be used to identify and design novel types of inhibitors. To test this hypothesis, we modeled plausible intermediates of the functional opening/closing motions of Tc PRAC [ 17 ]. This strategy allowed an expansion of the chemical space used in our virtual screening procedure and led us to identify two inhibitors of the enzyme, (E)-4-Oxo-pent-2-enoic acid (OxoPA) [ 18 ] and its derivative (E)-5-Bromo-4-oxo-pent-2-enoic acid (BrOxoPA) which were far more potent than PYC [ 17 ].…”

The Dialogue of the Host-Parasite Relationship:Leishmaniaspp. andTrypanosoma cruziInfection

“…The TcPRAC is a promising target for the development of a new therapy against Chagas disease since parasites are no longer viable when PRAC genes are knocked down or more virulent if PRAC genes are over expressed [ 220 ]. Two compounds, which are irreversible competitive inhibitors of TcPRAC, were able to inhibit the mammalian host cell infection [ 221 ].…”