Structural Insight into Effective Inhibitors' Binding to Dihydrofolate Reductase Thymidylate Synthase.

Pyrimethamine (Pyr), a known dihydrofolate reductase (DHFR) inhibitor, has long been used to treat toxoplasmosis caused by (Tg) infection. However, Pyr is effective only at high doses with associated toxicity to patients, calling for safer alternative treatments. In this study, we investigated a series of Pyr analogues, previously developed as DHFR inhibitors of bifunctional DHFR-thymidylate synthase (PfDHFR-TS), for their activity against DHFR-TS (TgDHFR-TS).

Flexible diaminodihydrotriazine inhibitors of Plasmodium falciparum dihydrofolate reductase: Binding strengths, modes of binding and their antimalarial activities.

A series of flexible diaminodihydrotriazines or cycloguanil (Cyc) analogues are developed and shown to inhibit P. falciparum dihydrofolate reductase (PfDHFR) of the wild type or those carrying either single (S108N), double (C59R + S108N and A16V + S108T), triple (N51I + C59R + S108N and C59R + S108N + I164L) or quadruple (N51I + C59R + S108N + I164L) mutations, responsible for antifolate resistance. The flexibility of the side chain at position N has been included in the design so as to avoid unfavourable steric interaction with the side chain of residue 108 of the resistant mutants.

Hybrid Inhibitors of Malarial Dihydrofolate Reductase with Dual Binding Modes That Can Forestall Resistance.

The S108N mutation of dihydrofolate reductase (DHFR) renders malaria parasites resistant to pyrimethamine through steric clash with the rigid side chain of the inhibitor. Inhibitors with flexible side chains can avoid this clash and retain effectiveness against the mutant. However, other mutations such as N108S reversion confer resistance to flexible inhibitors.

Characterization of Plasmodium knowlesi dihydrofolate reductase-thymidylate synthase and sensitivity to antifolates.

Malaria caused by an infection of Plasmodium knowlesi can result in high parasitemia and deaths. Therefore, effective and prompt treatment is necessary to reduce morbidity and mortality. The study aims to characterize P.

Malarial dihydrofolate reductase as a paradigm for drug development against a resistance-compromised target.

Malarial dihydrofolate reductase (DHFR) is the target of antifolate antimalarial drugs such as pyrimethamine and cycloguanil, the clinical efficacy of which have been compromised by resistance arising through mutations at various sites on the enzyme. Here, we describe the use of cocrystal structures with inhibitors and substrates, along with efficacy and pharmacokinetic profiling for the design, characterization, and preclinical development of a selective, highly efficacious, and orally available antimalarial drug candidate that potently inhibits both wild-type and clinically relevant mutated forms of Plasmodium falciparum (Pf) DHFR. Important structural characteristics of P218 include pyrimidine side-chain flexibility and a carboxylate group that makes charge-mediated hydrogen bonds with conserved Arg122 (PfDHFR-TS amino acid numbering).

Particular interaction between pyrimethamine derivatives and quadruple mutant type dihydrofolate reductase of Plasmodium falciparum: CoMFA and quantum chemical calculations studies.

Comparative molecular field analysis (CoMFA) was performed on twenty-three pyrimethamine (pyr) derivatives active against quadruple mutant type (Asn51Ile, Cys59Arg, Ser108Asn, Ile164Leu) dihydrofolate reductase of Plasmodium falcipaarum (PfDHFR). The represented CoMFA models were evaluated based on the various three different probe atoms, C(sp3) (+1), O(sp3) (-1) and H (+1), resulting in the best model with combined three types of probe atoms. The statistical results were r(2)(cv) = 0.

Evaluation of the activities of pyrimethamine analogs against Plasmodium vivax and Plasmodium falciparum dihydrofolate reductase-thymidylate synthase using in vitro enzyme inhibition and bacterial complementation assays.

Pyrimethamine analogs were examined as potential agents against vivax malaria using a bacterial surrogate system carrying Plasmodium vivax dihydrofolate reductase-thymidylate synthase (PvDHFR-TS), in which the PvDHFR complemented chemically knocked out host dihydrofolate reductase. The system was initially tested with P. falciparum dihydrofolate reductase-thymidylate synthase and was found to have good correlation with the parasite-based system.

Crystal structure of dihydrofolate reductase from Plasmodium vivax: pyrimethamine displacement linked with mutation-induced resistance.

Pyrimethamine (Pyr) targets dihydrofolate reductase of Plasmodium vivax (PvDHFR) as well as other malarial parasites, but its use as antimalarial is hampered by the widespread high resistance. Comparison of the crystal structures of PvDHFR from wild-type and the Pyr-resistant (SP21, Ser-58 --> Arg + Ser-117 --> Asn) strain as complexes with NADPH and Pyr or its analog lacking p-Cl (Pyr20) clearly shows that the steric conflict arising from the side chain of Asn-117 in the mutant enzyme, accompanied by the loss of binding to Ser-120, is mainly responsible for the reduction in binding of Pyr. Pyr20 still effectively inhibits both the wild-type and SP21 proteins, and the x-ray structures of these complexes show how Pyr20 fits into both active sites without steric strain.

Stoichiometric selection of tight-binding inhibitors by wild-type and mutant forms of malarial (Plasmodium falciparum) dihydrofolate reductase.

A simple method for screening combinatorial and other libraries of inhibitors of malarial (Plasmodium falciparum) dihydrofolate reductase (PfDHFR) has been developed, based on the affinities of the inhibitors with the enzyme. In the presence of limiting amounts of the enzyme, a number of inhibitors in the library were bound to extents reflecting the relative binding affinities. Following ultrafiltration and guanidine hydrochloride treatment to release bound inhibitors, the amounts of free and bound inhibitors could be determined by high-performance liquid chromatography and liquid chromatography-mass spectrometry.

Inhibitors of multiple mutants of Plasmodium falciparum dihydrofolate reductase and their antimalarial activities.

Novel analogues of pyrimethamine (Pyr) and cycloguanil (Cyc) have been synthesized and tested as inhibitors of Plasmodium falciparum dihydrofolate reductase carrying triple (N51I+C59R+S108N, C59R+S108N+I164L) and quadruple (N51I+C59R+S108N+I164L) mutations responsible for antifolate resistance. The inhibitors were designed to avoid steric clash of the p-Cl group of the inhibitors with the side chain of Asn108, augmented by additional mutations of the resistant mutants. Cycloguanil derivatives were also designed to avoid steric clash with the side chain of Val16 in the A16V+S108T mutant.

Ecdysteroids from a Zoanthus sp.

A new ecdysteroid, zoanthusterone, has been isolated from a marine zoanthid, Zoanthus sp. Ten known ecdysteroids, ponasterone A, 20-hydroxyecdysone 2-acetate, viticosterone E, integristerone A 25-acetate, 2-deoxy-20-hydroxyecdysone, ecdysone, ajugasterone C, dacryhainansterone, inokosterone, and 20-hydroxyecdysone, have also been isolated. This is the first report of ecdysteroids in a Zoanthus species.

Development of 2,4-diaminopyrimidines as antimalarials based on inhibition of the S108N and C59R+S108N mutants of dihydrofolate reductase from pyrimethamine-resistant Plasmodium falciparum.

The reduced binding of pyrimethamine to Ser108Asn (S108N) mutants of parasite dihydrofolate reductase (DHFR), which forms the basis of resistance of Plasmodium falciparum to pyrimethamine, is largely due to steric constraint imposed by the bulky side chain of N108 on Cl of the 5-p-Cl-phenyl group. This and other S108 mutants with bulky side chains all showed reduced binding to pyrimethamine and cycloguanil. Less effect on binding to some bulky mutants was observed for trimethoprim, with greater flexibility for the 5-substituent.

Novel antifolate resistant mutations of Plasmodium falciparum dihydrofolate reductase selected in Escherichia coli.

A simple and effective system has been developed from which a number of Plasmodium falciparum dihydrofolate reductase (pfDHFR) mutants conferring resistance to antifolates were randomly generated and characterized. The system exploited error-prone PCR to generate random mutations in the pfDHFR. Using the synthetic gene encoding for wild-type and quadruple mutant (N51I+C59R+S108N+I164L) pfDHFRs as templates, mutants resistant to pyrimethamine (Pyr), m-Cl analogue of Pyr (SO3) and WR99210 were selected by bacterial complementation system in which the endogenous DHFR activity of bacterial host cells, but not of Plasmodium, is selectively inhibited by trimethoprim (Tmp).