Worachart Sirawaraporn

Molecular characterization of dihydrofolate reductase in relation to antifolate resistance in Plasmodium vivax

The genes encoding the wild-type and six (five single and one double) mutant dihydrofolate reductase (DHFR) domains of the human malaria parasite, Plasmodium vivax (Pv), were cloned and expressed in Escherichia coli. The catalytic activities and the kinetic parameters of the purified recombinant wild-type and the mutant PvDHFRs were determined. Generally, all the PvDHFR mutants yielded enzymes with poorer catalytic activities when compared to the wild type enzyme. The widely used antifolates, pyrimethamine and cycloguanil, were effective inhibitors of the wild-type PvDHFR, but were approximately 60 to >4000 times less active against the mutant enzymes. In contrast to the analogous S108N mutation of Plasmodium falciparum DHFR (PfDHFR), the single S117N mutation in PvDHFR conferred approximately 4000- and approximately 1600-fold increased resistance to pyrimethamine and cycloguanil, respectively, compared to the wild-type PvDHFR. The S58R+S117N double mutant PvDHFR was 10- to 25-fold less resistant than the S117N mutant to the inhibitors, but also exhibited higher kcat/Km value than the single mutant. The antifolate WR99210 was equally effective against both the wild-type and SP21 (S58R+S117N) mutant DHFRs, but was much less effective against some of the single mutants. Data on kinetic parameters and inhibitory constant suggest that the wild-type P. vivax is susceptible to antimalarial antifolates and that point mutations in the DHFR domain of P. vivax are responsible for antifolate resistance.

Antimalarial Drug Development and New Targets

The Molecular Approaches to Malaria (MAM2000) conference, Lorne, Australia, 2-5 February 2000, brought together world-class malaria research scientists. The development of new tools and technologies - transfection, DNA microarrays and proteomic analysis - and the availability of DNA sequences generated by the Malaria Genome Project, along with more classic approaches, have facilitated the identification of novel drug targets, the development of new antimalarials and the generation of a deeper understanding of the molecular mechanism(s) of drug resistance in malaria. It is hoped that combinations of these technologies could lead to strategies that enable the development of effective, efficient and affordable new drugs to overcome drug-resistant malaria, as discussed at MAM2000 and outlined here by Ian Macreadie and colleagues.

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). Mutants conferring resistance to antimalarial antifolates were selected under the condition that inhibited the growth of the wild-type pfDHFR. All obtained Pyr resistant mutants possessed S108 mutation, in combination with common mutations of N51I, C59R and I164L previously found in the field. New Pyr resistant mutants with novel mutations (K27T, N121D, N144K and V213E) not found in the field were also identified. Exposure of the randomly mutated pfDHFR libraries to WR99210 or SO3 resulted in selection of novel single and multiple mutants including D54N, F58L and a combination of C50R, K181R, T219P and K227E, which exhibited 2- to over 2000-fold increase in resistance against antifolates. Kinetic analysis of these mutants suggested that apart from the active site residues that are crucial for DHFR activity, residues remote from the binding pocket also play essential roles in substrate and inhibitor binding.

Specificity in structure-based drug design: Identification of a novel, selective inhibitor of Pneumocystis carinii dihydrofolate reductase†

Specificity is an important aspect of structure‐based drug design. Distinguishing between related targets in different organisms is often the key to therapeutic success. Pneumocystis carinii is a fungal opportunist which causes a crippling pneumonia in immunocompromised individuals. We report the identification of novel inhibitors of P. cariniidihydrofolate reductase (DHFR) that are selective versus inhibition of human DHFR using computational molecular docking techniques. The Fine Chemicals Directory, a database of commercially available compounds, was screened with the DOCK program suite to produce a list of potential P. carinii DHFR inhibitors. We then used a postdocking refinement directed at discerning subtle structural and chemical features that might reflect species specificity. Of 40 compounds predicted to exhibit anti‐PneumocystisDHFR activity, each of novel chemical framework, 13 (33%) show IC50 values better than 150 μM in an enzyme assay. These inhibitors were further assayed against human DHFR: 10 of the 13 (77%) bind preferentially to the fungal enzyme. The most potent compound identified is a 7 μM inhibitor of P. carinii DHFR with 25‐fold selectivity. The ability of molecular docking methods to locate selective inhibitors reinforces our view of structure‐based drug discovery as a valuable strategy, not only for identifying lead compounds, but also for addressing receptor specificity. Proteins 29:59–67, 1997.

Kinetic and molecular properties of dihydrofolate reductase from pyrimethamine-sensitive and pyrimethamine-resistant Plasmodium chabaudi

Dihydrofolate reductase (5,6,7,8-tetrahydrofolate: NADP+ oxidoreductase, EC 1.5.1.3) was partially purified from a cloned strain of pyrimethamine-sensitive Plasmodium chabaudi and a drug-resistant clone derived from it. A molecular weight of approximately 120000 was estimated by gel filtration for enzyme from both pyrimethamine-sensitive and resistant parasites. The specific activities of the crude enzyme at pH 7.4 were 2.7 +/- 0.8 and 1.4 +/- 0.6 nmol min-1 mg-1 protein for sensitive and resistant strains, respectively. Methotrexate titration (pH 7.4, 37 degrees C) indicated that the apparent turnover number of the enzyme from the sensitive parasites was 1229 +/- 322 mol min-1 mol-1 compared with 1238 +/- 179 mol min-1 mol-1 for the enzyme from the resistant parasites. There was therefore no significant difference in the amounts of the enzyme from both sources. The Km value for dihydrofolate (9.3 microM) of the enzyme from the drug-sensitive parasites at pH 7.4 was lower than that from the resistant parasites by a factor of approximately 4. The Km values for NADPH of the enzyme from both sources were similar. Inhibition by pyrimethamine of the enzyme from the sensitive parasites was competitive with dihydrofolate, with Ki of 0.26 nM. By contrast, noncompetitive inhibition was observed for the enzyme from the resistant parasites, with Kis of 50 nM and Kii of 33 nM. The enzyme from drug-sensitive and drug-resistant parasites had different activity profiles with respect to pH and temperature. Moreover, the former was more sensitive to heat denaturation than the latter. From these results, it was concluded that the major basis for drug resistance is not an increase in enzyme content, but a large decrease in drug binding with the structurally different enzyme.

Synthesis of Solution-Phase Combinatorial Library of 4,6-Diamino-1,2-dihydro-1,3,5-triazine and Identification of New Leads Against A16V+S108T Mutant Dihydrofolate Reductase of Plasmodium falciparum

An efficient method to synthesize solution-phase combinatorial library of 1-aryl-4,6-diamino-1,2-dihydro-1,3,5-triazine was developed. The strategy involved an acid-catalyzed cyclocondensation between arylbiguanide hydrochlorides and carbonyl compounds in the presence of triethyl orthoacetate as water scavenger. A 96-membered combinatorial library was constructed from 6 aryl biguanides and 16 carbonyl compounds. Screening of the library by iterative deconvolution method revealed two candidate leads which are equally active against wild-type Plasmodium falciparum dihydrofolate reductase, but are about 100-fold more effective against the A16V+S108T mutant enzyme as compared to cycloguanil.

Chemical synthesis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene

Plasmodium falciparum dihydrofolate reductase-thymidylate synthase (DHFR-TS) is a well-known target for pyrimethamine and cycloguanil. The low amounts of enzyme obtainable from parasites or the currently available heterologous expression systems have thus far hindered studies of this enzyme. The 1912-base pair P. falciparum DHFR-TS gene was designed based on E. coli codon preference with unique restriction sites evenly placed throughout the coding sequence. The gene was designed and synthesized as three separated domains: the DHFR domain, the junctional sequence, and the TS domain. Each of these domains contained numerous unique restriction sites to facilitate mutagenesis. The three domains were assembled into a complete DHFR-TS gene which contained 30 unique restriction sites in the coding sequence. The bifunctional DHFR-TS was expressed from the synthetic gene as soluble enzyme in E. coli about 10-fold more efficiently than from the wild-type sequence. The DHFR-TS from the synthetic gene had kinetic properties similar to those of the wild-type enzyme and represents a convenient source of protein for further study. The unique restriction sites in the coding sequence permits easy mutagenesis of the gene which should facilitate further understanding of the molecular basis of antifolate resistance in malaria.

Dihydrofolate reductase and antifolate resistance in malaria

The dihydrofolate reductase (DHFR, EC 1.5.1.3) domain of Plasmodium falciparum bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is an attractive target of two important antifolate antimalarials: pyrimethamine (Pyr) and cycloguanil (Cyc). Over recent years, knowledge of malarial DHFR and mechanism(s) of antifolate resistance have increased substantially. These observations have provided an important framework for better understanding the molecular basis of antifolate resistance in malaria. This article provides a brief review and update on molecular aspects relevant to antifolate resistance in malaria.

Eosin B as a Novel Antimalarial Agent for Drug-Resistant Plasmodium falciparum

ABSTRACT 4′,5′-Dibromo-2′,7′-dinitrofluorescein, a red dye commonly referred to as eosin B, inhibits Toxoplasma gondii in both enzymatic and cell culture studies with a 50% inhibitory concentration (IC50) of 180 μM. As a non-active-site inhibitor of the bifunctional T. gondii dihydrofolate reductase-thymidylate synthase (DHFR-TS), eosin B offers a novel mechanism for inhibition of the parasitic folate biosynthesis pathway. In the present study, eosin B was further evaluated as a potential antiparasitic compound through in vitro and cell culture testing of its effects on Plasmodium falciparum. Our data revealed that eosin B is a highly selective, potent inhibitor of a variety of drug-resistant malarial strains, with an average IC50 of 124 nM. Furthermore, there is no indication of cross-resistance with other clinically utilized compounds, suggesting that eosin B is acting via a novel mechanism. The antimalarial mode of action appears to be multifaceted and includes extensive damage to membranes, the alteration of intracellular organelles, and enzymatic inhibition not only of DHFR-TS but also of glutathione reductase and thioredoxin reductase. In addition, preliminary studies suggest that eosin B is also acting as a redox cycling compound. Overall, our data suggest that eosin B is an effective lead compound for the development of new, more effective antimalarial drugs.

Cheaper faster drug development validated by the repositioning of drugs against neglected tropical diseases

There is an urgent need to make drug discovery cheaper and faster. This will enable the development of treatments for diseases currently neglected for economic reasons, such as tropical and orphan diseases, and generally increase the supply of new drugs. Here, we report the Robot Scientist ‘Eve’ designed to make drug discovery more economical. A Robot Scientist is a laboratory automation system that uses artificial intelligence (AI) techniques to discover scientific knowledge through cycles of experimentation. Eve integrates and automates library-screening, hit-confirmation, and lead generation through cycles of quantitative structure activity relationship learning and testing. Using econometric modelling we demonstrate that the use of AI to select compounds economically outperforms standard drug screening. For further efficiency Eve uses a standardized form of assay to compute Boolean functions of compound properties. These assays can be quickly and cheaply engineered using synthetic biology, enabling more targets to be assayed for a given budget. Eve has repositioned several drugs against specific targets in parasites that cause tropical diseases. One validated discovery is that the anti-cancer compound TNP-470 is a potent inhibitor of dihydrofolate reductase from the malaria-causing parasite Plasmodium vivax.