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Mutations in Plasmodium falciparum dihydropteroate synthase have been linked to resistance to the antimalarial drug, sulfadoxine, which competes with the dihydropteroate synthase substrate, p-aminobenzoate. In an effort to evaluate the role of these mutations in a simple model system, we have expressed six relevant alleles of the P. falciparum dihydropteroate synthase gene in Escherichia coli. When each construct was produced in a dihydropteroate synthase disrupted E. coli strain that required thymidine, the thymidine requirement was lost, indicating heterologous complementation had occurred. In the presence of sulfadoxine, the growth of the strain with the wild-type dihydropteroate synthase allele was inhibited while those containing each of the five mutant alleles grew, indicating that these mutations can confer sulfadoxine resistance in E. coli. When tested against twelve additional 'sulfa' drugs a variety of responses were obtained. All strains were resistant to sulfadiazine, but the wild-type allele conferred sensitivity to all other sulfa drugs. Three alleles conferred resistance to dapsone, a drug that is to be targetted for a new regime of malaria treatment in Africa. All mutant alleles remained sensitive to sulfachloropyridazine and sulfacetamide. These results suggest new drugs that could be tried for effective malaria treatment.

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.

The nature of the interactions between Plasmodium falciparum dihydrofolate reductase (pfDHFR) and antimalarial antifolates, i.e., pyrimethamine (Pyr), cycloguanil (Cyc) and WR99210 including some of their analogues, was investigated by molecular modeling in conjunction with the determination of the inhibition constants (Ki). A three-dimensional structural model of pfDHFR was constructed using multiple sequence alignment and homology modeling procedures, followed by extensive molecular dynamics calculations. Mutations at amino acid residues 16 and 108 known to be associated with antifolate resistance were introduced into the structure, and the interactions of the inhibitors with the enzymes were assessed by docking and molecular dynamics for both wild-type and mutant DHFRs. The Ki values of a number of analogues tested support the validity of the model. A 'steric constraint' hypothesis is proposed to explain the structural basis of the antifolate resistance.

The safety and immunogenicity of REMUNE, an HIV-specific immune based therapy for HIV infection, was evaluated in a cohort of 30 HIV infected subjects in Thailand. This therapy utilizes a gp120 depleted inactivated virus (HZ321), which exhibits a high degree of conservation with the core antigens of both type B' and E strains of HIV, the predominant Thailand isolates. The treatment was well tolerated, with no serious adverse events reported over the course of the 4-month trial. Treatment in which four doses were administered with REMUNE appeared to boost HIV-specific immune responses, with approximately 75% of the treated subjects demonstrating an increase in either the repertoire or the intensity of the serological response to HIV as measured by Western blot. CD4%, viral load, and weight remained stable over the course of the 4-month study relative to baseline values. Viral subtyping of this cohort revealed a predominance of type 'E'. These data suggest that REMUNE is safe and immunogenic in seropositive Thai subjects and supports further study of the therapeutic potential of REMUNE to treat HIV-1 infection.