Activity of antifolates against Pneumocystis carinii dihydrofolate reductase and identification of a potent new agent.

Abstract

The therapy of Pneumocystis carinii (PC) pneumonia is often unsuccessful, particularly in patients with acquired immune deficiency syndrome (AIDS). Because of difficulties in growing the organism in vitro or obtaining purified organisms, current treatment choices have been made with little information on the metabolic effects of therapeutic agents on PC. This report quantitates the effects of the commonly used antifolates as well as the classic antineoplastic antifolate methotrexate and a lipid-soluble analogue, trimetrexate, on the target enzyme, dihydrofolate reductase (DHFR), in the PC organisms. Trimethoprim and pyrimethamine were found to be weak inhibitors (ID50 = 39,600 and 2,800 nM, respectively), while methotrexate and trimetrexate were potent reductase inhibitors (ID50 = 1.4 and 26.1 nM, respectively). transport studies with radiolabeled compounds showed that compounds with the classic folate structure (methotrexate and leucovorin) were not taken up by the intact PC organisms. In contrast, trimetrexate exhibited rapid uptake. These results suggest a major therapeutic advantage may be gained by combining a potent, readily transported PC DHFR inhibitor such as trimetrexate with the reduced folate leucovorin to achieve a highly potent antiprotozoan effect while preventing toxicity to mammalian cells.

Expression and Characterization of Recombinant Human-Derived Pneumocystis carinii Dihydrofolate Reductase

“…Compared to the reported K i values of these inhibitors for human DHFR (4), only TMP showed a relatively favorable selectivity for human-derived P. carinii DHFR (the K i ratio for human versus human-derived P. carinii DHFR is 3.0), while the other three inhibitors appeared to be selective for human DHFR (the K i ratios for human versus human-derived P. carinii DHFR varied from 0.008 for trimetrexate to 0.5 for methotrexate). This is consistent with the inhibition profiles of rat-derived P. carinii DHFR described previously (2,6,7,9,22,25).…”

“…Despite its obvious efficacy, this combination is complicated by frequent toxic and allergic side effects (19); moreover, there are increasing concerns about whether TMP truly contributes to the activity of this combination against P. carinii. It has been shown in vitro that TMP is a poor inhibitor of rat-derived P. carinii DHFR (2,6,7,9,22,25) and that TMP alone is ineffective in the treatment of rat PCP (16,26). Recently, mutations in the P. carinii dihydropteroate synthase gene, the target of sulfamides, have been reported in the United States (15,21; Q. Mei, S. Gurunathan, H. Masur, and J.…”

“…While the rat-derived P. carinii DHFR has been well characterized in terms of its molecular and kinetic properties (2,6,7,9,17,18,22,25), little is known about the human-derived P. carinii DHFR, which we have recently cloned and which differs from the rat-derived P. carinii DHFR by 38% in amino acid sequence (21). For designing potential antifolates for treatment of humans, the ideal target should be the DHFR of human P. carinii, since that is the pathogen for humans.…”

Trimethoprim resistance

“…Bacterial pathogens known to be intrinsically resistant to TMP are fewer than susceptible ones. TMP is also active against certain types of malaria (18,54) and, in combination with sulfonamides, against Pneumocystis carinii (67), although TMP alone has only very weak activity against the DHFR of P. carinii (2).…”

Pneumocystis carinii, an opportunist in immunocompromised patients

“…Since the adoption of pentamidine for prophylaxis, many reports of disseminated Pneumocystis infection in patients receiving aerosolized pentamidine prophylaxis suggest that it only suppresses development of pulmonary infection (142,151,170 (3), and the drugs were tested in animal models. Two potent dihydrofolate reductase inhibitors, trimetrexate and piritrexim, showed anti-Pneumocystis activity in animal models (100,144) and clinical trials (2,163).…”