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

Abstract

Dihydrofolate reductase (DHFR) is the target of trimethoprim (TMP), which has been widely used in combination with sulfa drugs for treatment and prophylaxis of Pneumocystis carinii pneumonia. While the ratderived P. carinii DHFR has been well characterized, kinetic studies of human-derived P. carinii DHFR, which differs from rat-derived P. carinii DHFR by 38% in amino acid sequence, have not been reported to date. Here we report on the expression and kinetic characterization of the recombinant human-derived P. carinii DHFR. The 618-bp coding sequence of the human-derived P. carinii DHFR gene was expressed in Escherichia coli. As determined by sodium dodecyl sulfate-polyacrylamide gel eletrophoresis, the purified enzyme had a molecular mass of 25 kDa, consistent with that predicted from the DNA sequence. Kinetic analysis showed that the K m values for dihydrofolate and NADPH were 2.7 ؎ 0.3 and 14.0 ؎ 4.3 M, respectively, which are similar to those reported for rat-derived P. carinii DHFR. Inhibition studies revealed that both TMP and pyrimethamine were poor inhibitors of human-derived P. carinii DHFR, with K i values of 0.28 ؎ 0.08 and 0.065 ؎ 0.005 M, respectively, while trimetrexate and methotrexate were potent inhibitors, with K i values of 0.23 ؎ 0.03 and 0.016 ؎ 0.004 nM, respectively. The availability of purified recombinant enzyme in large quantities should facilitate the identification of antifolate inhibitors with greater potency and higher selectivity for humanderived P. carinii DHFR.Pneumocystis carinii pneumonia (PCP) remains a leading cause of morbidity and mortality in AIDS. Currently, one of the most widely used agents for treatment and prophylaxis of this infection is the combination of trimethoprim (TMP) and sulfamethoxazole (SMX). TMP inhibits dihydrofolate reductase (DHFR) (EC 1.5.1.3), which catalyzes the reduction of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate in the presence of NADPH and is essential for biosynthesis of thymidylate, purine nucleotides, and several amino acids. 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. A. Kovacs, Letter, Lancet 351:1631, 1998) and Europe (11) and have been associated with prophylaxis and/or treatment failures of TMP-SMX, suggesting that P. carinii is developing resistance to sulfa drugs. In contrast, the DHFR gene did not show any mutations suggestive of drug resistance (21). This may reflect an absence of drug pressure on DHFR and supports the concept that TMP contributes little to the efficacy of the TMP-S...

Dihydrofolate reductase gene polymorphisms in Pneumocystis carinii f. sp. hominis in Japan

“…The DHFR nucleotide sequence was identical in 37 isolates from 35 patients except one with a single synonymous change, despite prior exposure to TMP in 15 patients [11]. Consistently, TMP was reported to be a poor inhibitor of P. carinii DHFR compared with the inhibition by other agents [14][15][16]. The present study found four substitution sites with two non-synonymous and two synonymous changes in the coding region of DHFR.…”

Kinetic and Structural Analysis for Potent Antifolate Inhibition of Pneumocystis jirovecii, Pneumocystis carinii, and Human Dihydrofolate Reductases and Their Active-Site Variants

“…Although TMP-SMX is still the most effective first-line therapy, more than one-third of patients experience dose-limiting toxicity (1)(2)(3)(4)(5)(6). Failure of therapy or prophylaxis with TMP is common, but attempts to link these failures to variants of P. jirovecii dihydrofolate reductase (pjDHFR) that lead to alternative forms of the enzyme have been inconclusive (7,8). To date, most structural and drug design efforts have focused on the DHFR from Pneumocystis carinii (pcDHFR), the organism originally isolated from rat lungs (9)(10)(11)(12).…”

Towards New Antifolates Targeting Eukaryotic Opportunistic Infections

“…As discussed in the introduction, TMP-SMZ is used prophylactically for Pneumocystis pneumonia, despite the weak affinity of TMP for Pneumocystis DHFR (IC 50 ϭ 4.8 M for P. jirovecii DHFR) (18). Since the propargyl-based antifolates are significantly more potent than TMP against the fungal C. glabrata DHFR, they may also be effective against the fungal Pneumocystis DHFR.…”

“…In fact, studies have reported that mutations conferring resistance to TMP-SMZ arise in DHPS, not in DHFR (17,28). In contrast, when the DHFR inhibitor pyrimethamine, which is four times more potent than TMP (18), is used in combination with sulfadiazine against pneumocystis, mutations arise in DHFR as well as DHPS (20). A logical conclusion of these studies is that the low potency of TMP against P. jirovecii DHFR is preventing it from reaching its full potential as an effective therapy for this eukaryotic opportunistic pathogen.…”

“…Even the use of TMP-SMZ as a prophylactic agent against pneumocystis relies heavily on the sulfa component (31) and only on relatively weak binding between TMP and P. jirovecii DHFR (6,18). In fact, studies have reported that mutations conferring resistance to TMP-SMZ arise in DHPS, not in DHFR (17, 28).…”