Targeting pyrimidine synthesis in Plasmodium falciparum with transition state analogues of orotate phosphoribosyltransferase
MetadataShow full item record
Orotate phosphoribosyltransferase (OPRT) catalyzes the reversible formation of orotidine 5'-monophosphate (OMP) from alpha-D-phosphoribosylpyrophosphate (PRPP) and orotate, the 5th step in de novo pyrimidine synthesis. Plasmodium falciparum, the most virulent protozoan parasite responsible for malaria, requires de novo pyrimidine synthesis as it does not encode enzymes for pyrimidine salvage. The essential function of OPRT in parasites ( PfOPRT) makes it a target for antimalarial drugs. All rapidly proliferating cells, including human cancer cells, require robust de novo pyrimidine synthesis. Human OPRT (HsOPRT) is thus a target for anticancer drugs. This work characterizes PfOPRT and HsOPRT as potential drug targets. Transition state analogues provide a validated approach to drug design as agents with high potency and selectivity against targets. Using kinetic isotope effects (KIEs) and quantum chemical calculations, we have solved PfOPRT and HsOPRT transition states structures with different substrate analogues. PfOPRT and HsOPRT display similar late transition states with full dissociation of orotates, well-developed ribocations and partial participation of nucleophiles. Isotope-edited Fourier transform infrared (FTIR) difference spectra combined with homology modeling and computational analysis establishes a reaction coordinate with leaving group activation by multiple hydrogen-bond interactions and migration of the ribocation toward enzyme-bound trianionic PPi. Using isotopically labeled OMP and orotidine, binding isotope effects (BIEs) of PfOPRT and HsOPRT were measured with and without sulfate, a PPi analogue. Large normal [1'-3H] BIEs reveal ground state destabilization upon formation of the Michaelis complexes of PfOPRT and HsOPRT. Transition state analogues were designed and synthesized to mimic the geometrical and electrostatic features of the PfOPRT and HsOPRT transition states. In vitro assays with recombinant OPRTs show that these transition state analogues display Ki values as low as 40 nM against PfOPRT and HsOPRT. Transition state analysis of PfOPRT and HsOPRT provide new insight into OPRT catalytic mechanism and lead us to the path for design of powerful and specific OPRT inhibitors as antimalarials and anticancer agents.
- Theses and Dissertations