The goal of this research is to evaluate inosine monophosphate dehydrogenase (IMPDH, EC 1.1.1.205) as a target for chemotherapy in Pneumocystis carinii. This enzyme is the rate-limiting step in purine biosynthesis, and its inhibition limits cells for guanine nucleotides, unless active purine salvage can overcome the metabolic blockade. Two known inhibitors of IMPDH (mycophenolic acid and tiazofurin) are strong inhibitors of P. carinii growth in short-term culture; this and other evidence suggests purine salvage in P. carinii may be insufficient to overcome IMPDH inhibition. The hypothesis to be tested is: Mycophenolic acid and tiazofurin are potent inhibitors of P. carinii growth in culture because they and/or their metabolites specifically inhibit P. carinii IMPDH, creating an insurmountable metabolic blockade because salvage pathways cannot supply adequate XMP or GMP to support proliferation of the organism.
Specific aims are: 1) Purify recombinant P. carinii and human IMPDH; 2) Characterize recombinant P. carinii IMPDH, with human IMPDH as a reference; 3) Assess effects of inhibitors of recombinant P. carinii IMPDH on proliferation of P. carinii in short term culture; 4) Assess uptake and metabolic effects, on P. carinii and on a mammalian cell line, of IMPDH inhibitors 5) Evaluate potential selectivity by comparing activity of inhibitors against recombinant E. coli Sphi1101, which lacks native E. coli IMPDH, reconstructed to contain P. carinii IMPDH or human IMPDH. To accomplish these goals, recombinant P. carinii IMPDH will be purified by affinity chromatography and characterized, emphasizing kinetics of inhibition with mycophenolic acid, tiazofurin and 12 of their analogs. The goal of these first 2 aims is to compare the catalytic properties of P. carinii IMPDH to human type I enzyme in order to evaluate the potential for developing selective inhibitors of the enzyme. The goal of the last 3 aims is to evaluate effects of IMPDH inhibitors on the whole organism, which will reveal the activity of inhibitors requiring metabolic activation. In addition, purine salvage will be measured directly; various purines will also be tested for the ability to reverse growth inhibition caused by IMPDH inhibitors. Establishing IMPDH as a target for chemotherapy in P. carinii opens the way for developing new agents effective against this important pathogen affecting immunosuppressed patients.
| Sullivan Jr, William J; Dixon, Stacy E; Li, Catherine et al. (2005) IMP dehydrogenase from the protozoan parasite Toxoplasma gondii. Antimicrob Agents Chemother 49:2172-9 |
