Refolding of Recombinant Pneumocystis carinii Dihydrofolate Reductase and Characterization of the Enzyme

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).…”

“…2) and NADPH were determined to be 2.7 Ϯ 0.3 and 14.0 Ϯ 4.3 M, respectively. These values are similar (within severalfold) to those of rat-derived P. carinii, for which the K m values were reported to be 1.77 to 17.7 and 1.38 to 40.2 M for dihydrofolate and NADPH, respectively (7,18,22,25).…”

“…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.…”

On the use of PIPSA to Guide Target‐Selective Drug Design

“…For example, DHFRs from species that form the conserved salt bridge to the nitrogens of DHF by glutamate (human Glu 30) are separated from those using aspartate (E. coli Asp 27) ( Figure 2 and Figure 4). Furthermore, an interesting clustering can be found [28] Mus musculus Chordata W1 D3 N1 0.30 1.36 [29] Homo sapiens Chordata W1 D3 N1 0.11 2.5 [30] Bos taurus Chordata W1 D3 N1 2.3 33 [31] Sus scrofa Chordata W1 D3 N1 0.74 3.22 [29] Gallus gallus Chordata W1 D3 N1 0.15 1.8 [32] Thermotoga maritima Thermotogae W1 D1 N1 0.3 4.0 [33] Salmonella typhimurium Proteobacteria W1 D1 N1 0.4 n. d. [34] Escherichia coli Proteobacteria W1 D2 N4 0.27 1.05 [35] Saccharomyces cerevisiae Ascomycota W2 D4 N2 13 45 [36] Candida albicans Ascomycota W2 D4 N2 2.7 3.6 [5] Pneumocystis carinii Ascomycota W2 D4 N1 2.3 3.0 [37] Glycine max Magnoliophyta W2 D4 N1 35 415 [36] Daucus carota Magnoliophyta W2 D4 N1 3.7 2.2 [38] Neisseria gonorrhoeae Proteobacteria W2 D1 N1 2.6 10 [39] Mycobacterium tuberculosis Actinobacteria W2 D3 N1 4.5 4.2 [40] Heliothis virescens Arthropoda W3 D3 N1 7.5 6.7 [41] Lactobacillus casei Firmicutes W3 D1 N3 0.36 0.78 [42] Crithidia fasciculata Euglenozoa W4 D1 N1 1.1 2.7 [43] Leishmania major Euglenozoa W4 D1 N3 1.3 0.9 [44] [a] The clustering gives four clusters based on the PIPSA analysis with the human template structure (see Figures 2 and 4). Table 1 and middle panel of Figure 2; D1: triangles, D2: squares, D3: circles, D4: diamonds).…”