Phosphopantothenoylcysteine synthetase (PPCS) is the second enzyme in the universal Coenzyme A (CoA) biosynthetic pathway. PPCS is responsible for catalyzing the condensation of 4’-phosphopantothenate (PPA) and L-cysteine via nucleotide triphosphate activation of PPA. PPCSs have been broadly classified into three types (Type I-III) based upon expression profile and nucleotide triphosphate specificity. Type I PPCSs are found in a majority of bacteria and archaea, utilize CTP for activation of PPA in the first half reaction, and are expressed as the C-terminal domain of a fusion protein with phosphopantothenoylcysteine decarboxylase (PPCDC). Type II PPCSs are in eukaryotes, utilize both CTP and ATP, and expressed separately from PPCDC as a monofunctional enzyme. Type III PPCSs are found in certain bacteria, utilize CTP, and are expressed as a monofunctional enzyme. Based upon the difference in nucleotide triphosphate specificity and PPCS type between human and bacteria, PPCS was chosen for exploration as a possible novel antibacterial target.
Four mimics of the activated intermediate produced from the first half reaction catalyzed by PPCS were synthesized in twelve steps in average of 18% overall yield. These four intermediate mimics were tested in vitro for PPCS inhibition against PPCS from E. coli, E. faecalis, S. pneumoniae, and human. IC50s were obtained for all four intermediate mimics and the best mimic had a Ki of 24 nM against efPPCS. The best intermediate mimic displayed low nanomolar potency versus the bacterial forms of PPCS while displaying 100-1000 fold selectivity for the bacterial PPCS over human PPCS. Further, three of the intermediate mimics were used in a structural study to elucidate how they bind within the PPCS active site. The co-crystal structures of PPCS and the three intermediate mimics were solved to 2.11-2.37 Å. Analogs of PPA where the carboxylate was replaced with either an amine or thiol. The phosphorylated thiol PPA mimic was found to act as a competitive inhibitor of PPCS with respect to PPA with a Ki of 12 M. This study shows that it is possible to selectively inhibit bacterial PPCS over human PPCS and thus PPCS represents an antibacterial target worthy of further investigation.
Ph.D.
Medicinal Chemistry
University of Michigan, Horace H. Rackham School of Graduate Studies
http://deepblue.lib.umich.edu/bitstream/2027.42/78970/1/jamesdo_1.pdf