Kinetic and structural analysis for potent antifolate inhibition of Pneumocystis jirovecii, Pneumocystis carinii, and human dihydrofolate reductases and their active-site variants.

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 (Ki, 2.7 nM) compared to its selectivity for hDHFR (Ki, 24.4 nM), whereas there is only a 2.3-fold selectivity for pcDHFR (Ki, 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 Ki 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 Ki 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.

Clinical validation of cutoff target ranges in newborn screening of metabolic disorders by tandem mass spectrometry: a worldwide collaborative project.

PURPOSE: To achieve clinical validation of cutoff values for newborn screening by tandem mass spectrometry through a worldwide collaborative effort. METHODS: Cumulative percentiles of amino acids and acylcarnitines in dried blood spots of approximately 25­30 million normal newborns and 10,742 deidentified true positive cases are compared to assign clinical significance, which is achieved when the median of a disorder range is, and usually markedly outside, either the 99th or the 1st percentile of the normal population. The cutoff target ranges of analytes and ratios are then defined as the interval between selected percentiles of the two populations. When overlaps occur, adjustments are made to maximize sensitivity and specificity taking all available factors into consideration. RESULTS: As of December 1, 2010, 130 sites in 45 countries have uploaded a total of 25,114 percentile data points, 565,232 analyte results of true positive cases with 64 conditions, and 5,341 cutoff values. The average rate of submission of true positive cases between December 1, 2008, and December 1, 2010, was 5.1 cases/day. This cumulative evidence generated 91 high and 23 low cutoff target ranges. The overall proportion of cutoff values within the respective target range was 42% (2,269/5,341). CONCLUSION: An unprecedented level of cooperation and collaboration has allowed the objective definition of cutoff target ranges for 114 markers to be applied to newborn screening of rare metabolic disorders.

N9-substituted 2,4-diaminoquinazolines: synthesis and biological evaluation of lipophilic inhibitors of pneumocystis carinii and toxoplasma gondii dihydrofolate reductase.

N9-substituted 2,4-diaminoquinazolines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). Reduction of commercially available 2,4-diamino-6-nitroquinazoline 14 with Raney nickel afforded 2,4,6-triaminoquinazoline 15. Reductive amination of 15 with the appropriate benzaldehydes or naphthaldehydes, followed by N9-alkylation, afforded the target compounds 5- 13. In the 2,5-dimethoxybenzylamino substituted quinazoline analogues, replacement of the N9-CH 3 group of 4 with the N9-C2H5 group of 8 resulted in a 9- and 8-fold increase in potency against pcDHFR and tgDHFR, respectively. The N9-C2H5 substituted compound 8 was highly potent, with IC50 values of 9.9 and 3.7 nM against pcDHFR and tgDHFR, respectively. N9-propyl and N9-cyclopropyl methyl substitutions did not afford further increases in potency. This study indicates that the N9-ethyl substitution is optimum for inhibitory activity against pcDHFR and tgDHFR for the 2,4-diaminoquinazolines. Selectivity was unaffected by N9 substitution.

Synthesis and biological evaluation of 2,4-diamino-6-(arylaminomethyl)pyrido[2,3-d]pyrimidines as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase and as antiopportunistic infection and antitumor agents.

A series of 2,4-diamino-6-(arylaminomethyl)pyrido[2,3-d]pyrimidines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc), Toxoplasma gondii (tg), and rat liver (rl) dihydrofolate reductase (DHFR) and as inhibitors of the growth of tumor cell lines in culture. Compounds 4-15 were designed as part of a continuing effort to examine the effects of substitutions at the 5-position, in the two-atom bridge, and in the side chain phenyl ring on structure-activity/selectivity relationships of 2,4-diaminopyrido[2,3-d]pyrimidines against a variety of DHFRs. Reductive amination of the common intermediate 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile 16 with the appropriate anilines afforded the target compounds 4-12. Nucleophilic substitution or reductive methylation afforded the N10-methyl target compounds 13-15. As predicted, compounds 4-15 were, in general, less potent against all three DHFRs compared to the corresponding 2,4-diamino-5-methyl analogues previously reported; however, the greater decrease in potency against rlDHFR compared to pcDHFR and tgDHFR resulted in appreciable selectivity toward pathogenic DHFRs from different pathogens. The 2',5'-dichloro analogue 8 showed selectivity ratios (IC(50) against rlDHFR/IC(50) against pcDHFR or tgDHFR) of 15.7 and 23 for pcDHFR and tgDHFR, respectively. Thus, the selectivity of 8 for pcDHFR is higher than the first line clinical agent trimethoprim (TMP). In a P. carinii cell culture study, analogue 8 exhibited 88% cell growth inhibition at a concentration of 10 muM and afforded marginal effects in an in vivo study in the T. gondii mouse model. Selected compounds were evaluated in the National Cancer Institute (NCI) in vitro preclinical antitumor screening program and inhibited the growth of tumor cells in culture at micromolar to submicromolar concentrations and were selected for evaluation in a NCI in vivo hollow fiber assay.