Pneumocystis carinii (pc) is a major cause of opportunistic infection and mortality in immuno-suppressed patients, particularly those with AIDS. Antifolate drugs, usually consisting of a sulfonamide in combination with an inhibitor of the enzyme dihydrofolate reductase (DHFR), have been the most effective anti-Pneumocystis carinii agents in clinical use to date. However, their use has been limited by problems of toxicity and resistance. The enzyme dihydrofolate reductase has recently been isolated, cloned and sequenced from Pneumocystis carinii. Antifolate binding affinity data measured against pcDHFR show that antibacterials such as trimethoprim and pyrimethamine are effective inhibitors, as are lipophilic antifolates such as trimetrexate and piritrexim which are second generation anticancer agents. It is proposed that binding selectivity of pc responsive antifolates is a result of specific enzyme interactions with bound inhibitors. We will test this hypothesis by comparison of the three dimensional crystal structures of both human and pcDHFR complexed with the cofactor NADPH and pc selective antifolates being synthesized in the Gangjee and Rosowsky laboratories, and, to examine whether observed structural changes are correlated to inhibitor selectivity. Analysis of these data will provide molecular level details of inhibitor-enzyme geometry, hydrogen bonding, conformation and the role of specific active site residues, especially the contribution by the substitution of F31I and N64F between human and pcDHFR, in modulating pc selectivity. X-ray crystallographic techniques using molecular replacement methods will be used to solve these crystal structures. The rationale for this proposed research is that differences in the structural requirements for antifolate binding to human and Pneumocystis carinii DHFR can be exploited for structure-based drug design of new antifolates with potential as AIDS adjuvants. Since selectivity apparently requires only small changes in enzyme-inhibitor geometry, we propose to look for subtle differences in a series of carefully determine crystal structures of DHFR complexes with pc selective antifolates. Thus knowledge of the three dimensional structure of enzyme-inhibitor complexes are required to define the mechanism of pc selectivity and action. Dr. Sherry Queener, Indiana University, will supply samples of pcDHFR and measure the pc activity of new antifolates.
Showing the most recent 10 out of 14 publications
