Yongyuth Yuthavong

The antimalarial action on Plasmodium falciparum of qinghaosu and artesunate in combination with agents which modulate oxidant stress

The antimalarial activity of qinghaosu (artemisinine) against Plasmodium falciparum in culture was enhanced by increased oxygen tension. Artesunate, a qinghaosu derivative, showed synergistic effects with miconazole, and with doxorubicin, both of which have been suggested to exert their chemotherapeutic effect through increasing the oxidant stress. In contrast, catalase, dithiothreitol and alpha-tocopherol reduced the effectiveness of qinghaosu in vitro. These results suggest that the action of qinghaosu and artesunate might involve increase in oxidant stress on the infected red cells.

Antimalarial sesquiterpenes from tubers of Cyperus rotundus: structure of 10,12-peroxycalamenene, a sesquiterpene endoperoxide.

Activity-guided investigation of Cyperus rotundus tubers led to the isolation of patchoulenone, caryophyllene alpha-oxide, 10,12-peroxycalamenene and 4,7-dimethyl-1-tetralone. The antimalarial activities of these compounds are in the range of EC50 10(-4)-10(-6) M, with the novel endoperoxide sesquiterpene, 10,12-peroxycalamenene, exhibiting the strongest effect at EC50 2.33 x 10(-6) M.

AN OVERVIEW OF CHEMOTHERAPEUTIC TARGETS FOR ANTIMALARIAL DRUG DISCOVERY

The need for new antimalarials comes from the widespread resistance to those in current use. New antimalarial targets are required to allow the discovery of chemically diverse, effective drugs. The search for such new targets and new drug chemotypes will likely be helped by the advent of functional genomics and structure-based drug design. After validation of the putative targets as those capable of providing effective and safe drugs, targets can be used as the basis for screening compounds in order to identify new leads, which, in turn, will qualify for lead optimization work. The combined use of combinatorial chemistry--to generate large numbers of structurally diverse compounds--and of high throughput screening systems--to speed up the testing of compounds--hopefully will help to optimize the process. Potential chemotherapeutic targets in the malaria parasite can be broadly classified into three categories: those involved in processes occurring in the digestive vacuole, enzymes involved in macromolecular and metabolite synthesis, and those responsible for membrane processes and signalling. The processes occurring in the digestive vacuole include haemoglobin digestion, redox processes and free radical formation, and reactions accompanying haem release followed by its polymerization into haemozoin. Many enzymes in macromolecular and metabolite synthesis are promising potential targets, some of which have been established in other microorganisms, although not yet validated for Plasmodium, with very few exceptions (such as dihydrofolate reductase). Proteins responsible for membrane processes, including trafficking and drug transport and signalling, are potentially important also to identify compounds to be used in combination with antimalarial drugs to combat resistance.

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). An analogous interaction of P218 with human DHFR is disfavored because of three species-dependent amino acid substitutions in the vicinity of the conserved Arg. Thus, P218 binds to the active site of PfDHFR in a substantially different fashion from the human enzyme, which is the basis for its high selectivity. Unlike pyrimethamine, P218 binds both wild-type and mutant PfDHFR in a slow-on/slow-off tight-binding mode, which prolongs the target residence time. P218, when bound to PfDHFR-TS, resides almost entirely within the envelope mapped out by the dihydrofolate substrate, which may make it less susceptible to resistance mutations. The high in vivo efficacy in a SCID mouse model of P. falciparum malaria, good oral bioavailability, favorable enzyme selectivity, and good safety characteristics of P218 make it a potential candidate for further development.

A Genetically Hard-Wired Metabolic Transcriptome in Plasmodium falciparum Fails to Mount Protective Responses to Lethal Antifolates

Genome sequences of Plasmodium falciparum allow for global analysis of drug responses to antimalarial agents. It was of interest to learn how DNA microarrays may be used to study drug action in malaria parasites. In one large, tightly controlled study involving 123 microarray hybridizations between cDNA from isogenic drug-sensitive and drug-resistant parasites, a lethal antifolate (WR99210) failed to over-produce RNA for the genetically proven principal target, dihydrofolate reductase-thymidylate synthase (DHFR-TS). This transcriptional rigidity carried over to metabolically related RNA encoding folate and pyrimidine biosynthesis, as well as to the rest of the parasite genome. No genes were reproducibly up-regulated by more than 2-fold until 24 h after initial drug exposure, even though clonal viability decreased by 50% within 6 h. We predicted and showed that while the parasites do not mount protective transcriptional responses to antifolates in real time, P. falciparum cells transfected with human DHFR gene, and adapted to long-term WR99210 exposure, adjusted the hard-wired transcriptome itself to thrive in the presence of the drug. A system-wide incapacity for changing RNA levels in response to specific metabolic perturbations may contribute to selective vulnerabilities of Plasmodium falciparum to lethal antimetabolites. In addition, such regulation affects how DNA microarrays are used to understand the mode of action of antimetabolites.

Basis for antifolate action and resistance in malaria

Resistance to antifolates of the malaria parasite Plasmodium falciparum stems from stepwise mutations of the target enzyme dihydrofolate reductase (DHFR). New drugs can be developed against resistant parasites, which are assumed to have limited possibilities in mutations. Mechanisms of resistance other than reduced binding of inhibitors to mutant enzymes may be possible and need to be further explored. New synergistic combinations of drugs targeting DHFR and dihydropteroate synthase may be employed, with new provisions against development of resistance.

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.

Mitochondria as the site of action of tetracycline on Plasmodium falciparum

Rhodamine 123 (Rh 123) was used as a fluorescent probe for the mitochondria of the malarial parasite Plasmodium falciparum. On treatment with tetracycline in vitro, a marked decrease in the percentage of parasites with Rh123 fluorescence in the mitochondria was observed in parallel with an increase in the percentage of parasites with abnormal morphology during onset of decrease in parasitemia. Similar results were obtained, over a shorter time period, with 2,4-dinitrophenol. However, the percentage of parasites with fluorescence did not decrease with increase in parasite abnormal morphology or decrease in parasitemia on treatment with pyrimethamine or cycloheximide. Isoelectric focusing-SDS gel electrophoresis of radiolabelled parasite proteins showed two components of 95 and 85 kDa, the synthesis of which was sensitive to tetracycline, but not cycloheximide. It is concluded that tetracycline exerts its action through the effect on parasite mitochondria and mitochondrial protein synthesis.

Malarial (Plasmodium falciparum) dihydrofolate reductase-thymidylate synthase: structural basis for antifolate resistance and development of effective inhibitors.

Dihydrofolate reductase-thymidylate synthase (DHFR-TS) from Plasmodium falciparum, a validated target for antifolate antimalarials, is a dimeric enzyme with interdomain interactions significantly mediated by the junction region as well as the Plasmodium-specific additional sequences (inserts) in the DHFR domain. The X-ray structures of both the wild-type and mutant enzymes associated with drug resistance, in complex with either a drug which lost, or which still retains, effectiveness for the mutants, reveal features which explain the basis of drug resistance resulting from mutations around the active site. Binding of rigid inhibitors like pyrimethamine and cycloguanil to the enzyme active site is affected by steric conflict with the side-chains of mutated residues 108 and 16, as well as by changes in the main chain configuration. The role of important residues on binding of inhibitors and substrates was further elucidated by site-directed and random mutagenesis studies. Guided by the active site structure and modes of inhibitor binding, new inhibitors with high affinity against both wild-type and mutant enzymes have been designed and synthesized, some of which have very potent anti-malarial activities against drug-resistant P. falciparum bearing the mutant enzymes.

Depression of Plasmodium falciparum dihydroorotate dehydrogenase activity in in vitro culture by tetracycline

The activity of Plasmodium falciparum dihydroorotate dehydrogenase, a particulate, electron transport-linked enzyme involved in de novo pyrimidine synthesis, was depressed when the parasite was cultured in the presence of a therapeutic concentration of tetracycline over a 96 h period. There was no direct inhibitory effect of the antibiotic on the enzyme activity. The activity of glutamate dehydrogenase, which is cytoplasmic in the parasite, was unaffected by tetracycline over the same period. Dihydroorotate dehydrogenase activity was substantially recovered when electron acceptors were added. It is suggested that the effect of tetracycline is manifested at the level of the dehydrogenase and/or the electron transport chain linked to this enzyme.