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

Abstract

A major concern of immunocompromised patients, in particular those with AIDS, is susceptibility to infection caused by opportunistic pathogens such as Pneumocystis jirovecii, which is a leading cause of pneumonia in immunocompromised patients. We report the first kinetic and structural data for 2,4-diamino-6-[(2=,5=-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine (OAAG324), a potent inhibitor of dihydrofolate reductase (DHFR) from P. jirovecii (pjDHFR), and also for trimethoprim (TMP) and methotrexate (MTX) with pjDHFR, Pneumocystis carinii DHFR (pcDHFR), and human DHFR (hDHFR). OAAG324 shows a 9.0-fold selectivity for pjDHFR (K i , 2.7 nM) compared to its selectivity for hDHFR (K i , 24.4 nM), whereas there is only a 2.3-fold selectivity for pcDHFR (K i , 6.3 nM). In order to understand the determinants of inhibitory potency, active-site mutations of pj-, pc-, and hDHFR were explored to make these enzymes more like each other. The most unexpected observations were that the variant pcDHFR forms with K37Q and K37Q/F69N mutations, which made the enzyme more like the human form, also made these enzymes more sensitive to the inhibitory activity of OAAG324, with K i values of 0.26 and 0.71 nM, respectively. A similar gain in sensitivity was also observed for the hDHFR N64F variant, which showed a lower K i value (0.58 nM) than native hDHFR, pcDHFR, or pjDHFR. Structural data are reported for complexes of OAAG324 with hDHFR and its Q35K and Q35S/N64F variants and for the complex of the K37S/F69N variant of pcDHFR with TMP. These results provide useful insight into the role of these residues in the optimization of highly selective inhibitors of DHFR against the opportunistic pathogen P. jirovecii.

Trimethoprim Resistance of Dihydrofolate Reductase Variants from Clinical Isolates of Pneumocystis jirovecii

“…The homology data for the pjDHFR complex with OAAG324 ( Fig. 1) and NADPH reveals that this compound is displaced above the plane of the pyrido [2,3-d]pyrimidine ring compared to the crystal structure data (15). Similar modeling of pjDHFR with TMP and NADPH is consistent with the crystal structure of pcDHFR with TMP and NADPH (18).…”

“…1) to assess whether resistance to one antifolate was likely to confer resistance to the class. OAAG324 was chosen as the experimental antifolate because it differed significantly in structure from trimethoprim (14) and the compound was already known to be longer and more flexible than trimethoprim, such that the dichlorophenyl ring can adopt alternate conformations within the active site (15).…”

“…The deduced structure of P. jirovecii DHFR was calculated by homology modeling to the crystal structure of the P. carinii DHFR ternary complex with methotrexate and NADPH (15)(16)(17). The homology data for the pjDHFR complex with OAAG324 ( Fig.…”

Pneumocystis jirovecii Rtt109, a Novel Drug Target for Pneumocystis Pneumonia in Immunosuppressed Humans

“…1, top). The moderate divergence between these two closely related species is not unexpected, as several features in the mitochondrial DNA of P. murina and P. carinii diverge from those of P. jirovecii (56), and this pattern of sequence similarity has also been observed between P. carinii and P. jirovecii dihydrofolate reductases (47,57). In addition, there appears to be significant genetic diversity at several regions among P. jirovecii isolates, based on genetic analyses (58)(59)(60)(61).…”

“…Pneumocystis is challenging to treat with standard antifun-gals, and the use of the available agents can be limited by drugrelated toxicities. Accordingly, efficacious anti-Pneumocystis agents with minimal human toxicities still represent an unmet clinical need, despite significant efforts over the course of several decades (44)(45)(46)(47)(48)(49).…”