Efficacies of Lipophilic Inhibitors of Dihydrofolate Reductase against Parasitic Protozoa

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A structurally diverse library of 93 lipophilic di-and tricyclic diaminopyrimidine derivatives was tested for the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of Cryptosporidium parvum (J. R. Vásquez et al., Mol. Biochem. Parasitol. 79:153-165, 1996). In parallel, the library was also tested against human DHFR and, for comparison, the enzyme from Escherichia coli.

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confidence: 99%

Dicyclic and Tricyclic Diaminopyrimidine Derivatives as Potent Inhibitors of Cryptosporidium parvum Dihydrofolate Reductase: Structure-Activity and Structure-Selectivity Correlations

Nelson

Rosowsky

2001

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“…However, drug screening in M. smegmatis has not always been an accurate predictor of activity (28,29) or the mechanism of action (1,22) in M. tuberculosis. In addition, S. cerevisiae has several advantages that we have exploited to develop similar systems for the screening of inhibitors of the DHFR enzymes from P. falciparum, C. parvum, and P. carinii DHFRs (2,19,36,43). First, plasmids that carry a centromere are maintained at one copy per cell in yeasts (37), so the level of expression of an introduced gene can be controlled even on a plasmid.…”

Section: Discussionmentioning

confidence: 99%

“…The first step was to determine the relative potencies of these compounds compared with that of WR99210 by a simple radial assay with the TB-yeast (19). Briefly, TB-yeast was streaked from the center to the edge of a plate to make a template.…”

Section: ϫ10mentioning

confidence: 99%

“…Because screening of drugs with M. tuberculosis cultures is slow, we have developed an alternative strategy: initial screening in an engineered strain of the budding yeast Saccharomyces cerevisiae that is dependent on the M. tuberculosis DHFR for its growth. S. cerevisiae has several advantages that we have exploited to develop similar systems for screening of inhibitors of the DHFR enzymes from P. falciparum, Cryptosporidium parvum, and Pneumocystis carinii (2,19,36,43). We have now constructed a yeast-based system for comparison of compounds that inhibit the DHFR enzyme from M. tuberculosis and used this approach to identify three compounds that are even stronger inhibitors of the M. tuberculosis enzyme than WR99210.…”

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confidence: 99%

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Novel Saccharomyces cerevisiae Screen Identifies WR99210 Analogues That Inhibit Mycobacterium tuberculosis Dihydrofolate Reductase

Gerum

Ulmer

Jacobus

et al. 2002

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The ongoing selection of multidrug-resistant strains of Mycobacterium tuberculosis has markedly reduced the effectiveness of the standard treatment regimens. Thus, there is an urgent need for new drugs that are potent inhibitors of M. tuberculosis, that exhibit favorable resistance profiles, and that are well tolerated by patients. One promising drug target for treatment of mycobacterial infections is dihydrofolate reductase (DHFR; EC 1.5.1.3), a key enzyme in folate utilization. DHFR is an important drug target in many pathogens, but it has not been exploited in the search for drugs effective against M. tuberculosis. The triazine DHFR inhibitor WR99210 has been shown to be effective against other mycobacteria. We show here that WR99210 is also a potent inhibitor of M. tuberculosis and Mycobacterium bovis BCG growth in vitro and that resistance to WR99210 occurred less frequently than resistance to either rifampin or isoniazid. Screening of drugs with M. tuberculosis cultures is slow and requires biosafety level 3 facilities and procedures. We have developed an alternative strategy: initial screening in an engineered strain of the budding yeast Saccharomyces cerevisiae that is dependent on the M. tuberculosis DHFR for its growth. Using this system, we have screened 19 compounds related to WR99210 and found that 7 of these related compounds are also potent inhibitors of the M. tuberculosis DHFR. These studies suggest that compounds of this class are excellent potential leads for further development of drugs effective against M. tuberculosis.The ongoing selection of multidrug-resistant strains of Mycobacterium tuberculosis has markedly reduced the effectiveness of the standard treatment regimens (27). Thus, there is an urgent need for new drugs that are potent inhibitors of M. tuberculosis, that exhibit favorable resistance profiles, and that are well tolerated by patients. One promising drug target for treatment of mycobacterial infections is dihydrofolate reductase (DHFR; EC 1.5.1.3), a key enzyme in folate utilization that catalyzes the reduction of dihydrofolate to tetrahydrofolate. Tetrahydrofolate is necessary for the one-carbon transfer reactions used in the biosynthesis of nucleic and amino acids including thymidylate, adenine, methionine, glycine, and histidine. DHFR inhibitors act by halting the synthesis of these DNA, RNA, and protein subunits, thereby arresting cell growth. Specific competitive inhibitors of DHFR have been used for more than 50 years in combination with sulfa and sulfone compounds as inexpensive effective inhibitors of bacterial and parasitic infections (13, 32). DHFR has distinct advantages as a potential drug target. First, the biochemistry of the folate pathway and the DHFR enzyme are well characterized, and the crystal structures of 10 DHFR enzymes have been solved. Second, this long history means that the safety and selectivity of these inhibitors have been intensively studied (38) and compounds with a higher degree of selectivity for the microbial enzymes than for the human enzym...

“…The yeast was grown in 96-well plates at the indicated concentrations of the drugs dissolved in DMSO. To increase the sensitivity of the yeast to DHFR inhibitors, both the control and the drug-containing wells contained 1 mM sulfanilamide (13). The growth of the yeast in each well was assessed by reading the optical density at 650 nM after approximately 24 h of incubation at 30°C, and the growth in any well was compared with the growth of that strain in solvent alone.…”

Section: Vol 49 2005 De Novo Synthesis Of Novel Antifolate Antimalamentioning

confidence: 99%

2,4-Diaminopteridine-Based Compounds as Precursors for De Novo Synthesis of Antifolates: a Novel Class of Antimalarials

Nduati

Hunt

Kamau

et al. 2005

We have tested the hypothesis that 2,4-diamino-6-hydroxymethyl-pteridine (DAP), 2,4-diaminopteroic acid (DAPA), and 2,4 diamino-N10-methyl-pteroic acid (DAMPA) could be converted into aminopterin (from DAP and DAPA) and methotrexate (from DAMPA), both of which are potent inhibitors of dihydrofolate reductase, a proven drug target for Plasmodium falciparum. DAP, DAPA, and DAMPA inhibited parasite growth in the micromolar range; DAMPA was the most active, with 50% inhibitory concentrations in vitro of 446 ng/ml against the antifolate-sensitive strain and 812 ng/ml against the highly resistant strain under physiological folate conditions. DAMPA potentiates the activity of the sulfone dapsone, an inhibitor of dihydropteroate synthase, but not that of chlorcycloguanil, a known inhibitor of dihydrofolate reductase (DHFR). Experiments with a Saccharomyces cerevisiae strain dependent upon the P. falciparum DHFR enzyme showed that DHFR is a target of DAMPA in that system. We hypothesize that DAMPA is converted to methotrexate by the parasite dihydrofolate synthase, which explains the synergy of DAMPA with dapsone but not with chlorcycloguanil. This de novo synthesis will not occur in the host, since it lacks the complete folate pathway. If this hypothesis holds true, the de novo synthesis of the toxic compounds could be used as a framework for the search for novel potent antimalarial antifolates.Chemotherapy remains one of the most important tools for the management of falciparum malaria. However, malaria control is hampered by the emergence and spread of parasites resistant to almost all available antimalarial drugs. This situation is critical in Africa as a result of the spread of resistance to the combination sulfadoxine-pyrimethamine, an inexpensive treatment widely used in African countries (9, 16-18, 25, 28). As an alternative, a number of combinations with artemisinins are being recommended and implemented, but questions about the cost and the adequacy of the supply of artemisinins and the intrinsic ability of Plasmodium falciparum to select drug-resistant parasite populations underline the need to identify novel agents.