Parasitic protozoa rely on purine salvage pathways for which the enzyme hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT; hereafter, purine phosphoribosyltransferase (PPRT)) is essential. The genus Trypanosoma causes debilitating human diseases of high morbidity. Neither vaccines nor useful therapies exist. Trypanosoma cruzi causes Chagas?s Disease in Central and South America, with over 40 cases recently reported in Texas. Trypanosoma brucei rhodosiense and Trypanosoma brucei gambiense cause African sleeping sickness. Parasitic protozoa including T. cruzi and T. brucei are incapable of purine biosynthesis de novo and make nucleotides and nucleosides by purine salvage pathways. PPRT catalyzes the formation of GMP, IMP, and XMP from 5-phospho-ribose 1-pyrophosphate (PRPP) and the respective bases, guanine, hypoxanthine, and xanthine. Plasmodium falciparum, causes the most virulent form of malaria, and is also a purine auxotroph for which the action of PPRT is the only physiological path for hypoxanthine incorporation into the nucleotide pool. Transition-state analogue inhibitors (TSAIs) based on transition states for related phosphoribosyltransferases are inhibitors of P. falciparum PPRT. Cell-permeable prodrugs blocked the proliferation of P. falciparum in culture and showed selectivity vs. human HGPRT, despite the high structural similarity and active-site conservation of the enzymes. This research will design, synthesize, and characterize both in vitro and in vivo novel inhibitors of the PPRTs from P. falciparum, T. brucei ssp. and T. cruzi. Transition-state methodology, quantum computational chemistry, X-ray structure-based inhibitor design and expert chemical synthesis will be used for inhibitor development. Existing lead compounds of sub-nanomolar potency for PPRT from P. falciparum will initiate and inform our inhibitor program. Crystal structures have been reported for T. cruzi PPRT, but no potent inhibitors have been defined. No inhibitor development for T. brucei has been reported, and PPRTs have not been kinetically characterized from T. brucei. The generation of new lead compounds is anticipated to proceed rapidly for P. falciparum PPRT and emerge for Trypanosoma PPRTs once the transition-state structures of these enzymes have been solved. Optimized inhibitors which bind each of the target PPRTs will be evaluated by X-ray crystallography, to provide a refinement guide for chemistry. Potent and selective (vs. human HGPRT) inhibitors of the PPRTs will be evaluated in cell cultures of P. falciparum, T. cruzi, and T. brucei ssp for parasiticidal activity. The most effective of these will be evaluated in murine models of Malaria, Chagas?s disease and African sleeping sickness. Importantly, a successful outcome from this proposal could lead to new therapeutic agents to treat three diseases which comprise unmet or under-met medical needs. This development plan uses transition state theory to meet the NIH goals of reducing the lead time for drug development.
We will use kinetic isotope effects and computational chemistry to characterize the transition-state structure of the recombinant hypoxanthine-guanine phosphoribosyltransferases (purine phosphoribosyltransferase or PPRT) of Plasmodium falciparum, Trypanosoma cruzi and Trypanosoma brucei to guide the design, synthesis, and in vitro and cellular characterization of transition-state analogue inhibitors of PPRTs. Their anti-protozoal activity will be determined in cellular cultures and animal models of malaria, Chagas?s disease, and African sleeping sickness. This frontier technology of drug design has the promise to shorten the time to effective therapeutic agents for neglected tropical diseases.
| Evans, Gary B; Tyler, Peter C; Schramm, Vern L (2018) Immucillins in Infectious Diseases. ACS Infect Dis 4:107-117 |
| Ducati, Rodrigo G; Firestone, Ross S; Schramm, Vern L (2017) Kinetic Isotope Effects and Transition State Structure for Hypoxanthine-Guanine-Xanthine Phosphoribosyltransferase from Plasmodium falciparum. Biochemistry 56:6368-6376 |
