Rational drug design approach for overcoming drug resistance: application to pyrimethamine resistance in malaria

Pyrimethamine acts by selectively inhibiting malarial dihydrofolate reductase-thymidylate synthase (DHFR-TS). Resistance in the most important human parasite, Plasmodium falciparum, initially results from an S108N mutation in the DHFR domain, with additional mutation (most commonly C59R or N51I or both) imparting much greater resistance. From a homology model of the 3-D structure of DHFR-TS, rational drug design techniques have been used to design and subsequently synthesize inhibitors able to overcome malarial pyrimethamine resistance. Compared to pyrimethamine (Ki 1.5 nM) with purified recombinant DHFR fromP. falciparum, the Ki value of the m-methoxy analogue of pyrimethamine was 1.07 nM, but against the DHFR bearing the double mutation (C59R + S108N), the Ki values for pyrimethamine and the m-methoxy analogue were 71.7 and 14.0 nM, respectively. The m-chloro analogue of pyrimethamine was a stronger inhibitor of both wild-type DHFR (with Ki 0.30 nM) and the doubly mutant (C59R +S108N) purified enzyme (with Ki 2.40 nM). Growth of parasite cultures of P. falciparum in vitro was also strongly inhibited by these compounds with 50% inhibition of growth occurring at 3.7 microM for the m-methoxy and 0.6 microM for the m-chloro compounds with the K1 parasite line bearing the double mutation (S108N + C59R), compared to 10.2 microM for pyrimethamine. These inhibitors were also found in preliminary studies to retain antimalarial activity in vivo in P. berghei-infected mice.     

Testing candidate genes that may affect susceptibility to leprosy

Due to increased chloroquine resistance, the antifolate/sulpha drug combinations are becoming increasingly important in the chemotherapy of falciparum malaria. However, point mutations in the dihydrofolate reductase gene lead to resistance to the antifolate drugs. We therefore investigated the prevalence of the 6 reported point mutations in this gene among field isolates of Plasmodium falciparum from Kenya, to determine if the mutations correlated with resistance to pyrimethamine and the biguanides cycloguanil and chlorcycloguanil. We used a mutation-specific polymerase chain reaction technique to test for these reported mutations in 21 Kenyan isolates and 4 reference lines. We also amplified and directly sequenced the dihydrofolate reductase coding sequence from these parasites to confirm the results and test for other possible mutations. Of the reported mutations, we found S108N, which is the central mutation of pyrimethamine resistance, and mutations N51I and C59R, which modulate the levels of resistance and may confer decreases in response to cycloguanil that are folate and p-aminobenzoic acid dependent. No isolate possessed the paired point mutations S108T and A16V, or I164L and S108N, which have been associated with cycloguanil resistance in previous studies. These results provided supportive evidence for the combined use of a cycloguanil-class drug (e.g., chlorproguanil) and a sulpha drug (e.g., dapsone) against P.falciparum malaria in Kenya.     

Production and systemic absorption of toxic byproducts of tissue combustion during laparoscopic surgery

Recently the efficacy of sulfadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Tanzania has been seriously compromised by the development of resistance. The occurrence of active site mutations in the Plasmodium falciparum gene sequence coding for dihydrofolate reductase (DHFR) is known to confer resistance to pyrimethamine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results confirm the occurrence of one or more DHFR mutations in all the samples, but no relationship was found with the presence of parasites in the blood at day 7. The results suggest that alterations in the coding region for dihydropteroate synthetase (DHPS), the enzyme target for sulfadoxine, should be studied in order to predict resistance to the S/P combination. It has been proposed earlier that sulfadoxine could itself act on DHFR, because of a false dihydrofolate produced by drug metabolism through DHPS and dihydrofolate synthase. The results of this treatment study suggest that such a possibility is unlikely.     

Polymorphisms in the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) genes of Plasmodium falciparum and in vivo resistance to sulphadoxine/pyrimethamine in isolates from Tanzania

The efficacy of sulphadoxine/pyrimethamine (S/P) in treatment of uncomplicated falciparum malaria in Africa is increasingly compromised by development of resistance. The occurrence of mutations associated with the active site sequence in the Plasmodium falciparum genes coding for dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) is associated with in vitro resistance to pyrimethamine and sulphadoxine. This study investigates the occurrence of these mutations in infected blood samples taken from Tanzanian children before treatment with S/P and their relationship to parasite breakthrough by day 7. The results show that alleles of DHPS (436-alanine, 437-alanine and 540-lysine) were significantly reduced in prevalence on day 7 after S/P treatment. In this area, a DHPS with 436-serine, 437-glycine and 540-glutamate appears to play a major role in resistance to S/P in vivo. Evidence for the influence of mutations in the DHFR gene in this investigation is not clear, probably because of the high prevalence of 'resistance-related' mutations at day 0 in the local parasite population. For apparently the same reason, it was not possible to show a statistical association between S/P resistance and the presence of particular polymorphisms in the DHFR and DHPS genes before treatment.     

Cycloguanil and its parent compound proguanil demonstrate distinct activities against Plasmodium falciparum malaria parasites transformed with human dihydrofolate reductase

The lack of suitable antimalarial agents to replace chloroquine and pyrimethamine/sulfadoxine threatens efforts to control the spread of drug-resistant strains of the malaria parasite Plasmodium falciparum. Here we describe a transformation system, involving WR99210 selection of parasites transformed with either wild-type or methotrexate-resistant human dihydrofolate reductase (DHFR), that has application for the screening of P. falciparum-specific DHFR inhibitors that are active against drug-resistant parasites. Using this system, we have found that the prophylactic drug cycloguanil has a mode of pharmacological action distinct from the activity of its parent compound proguanil. Complementation assays demonstrate that cycloguanil acts specifically on P. falciparum DHFR and has no other significant target. The target of proguanil itself is separate from DHFR. We propose a strategy of combination chemotherapy incorporating the use of multiple parasite-specific inhibitors that act at the same molecular target and thereby maintain, in combination, their effectiveness against alternative forms of resistance that arise from different sets of point mutations in the target. This approach could be combined with traditional forms of combination chemotherapy in which two or more compounds are used against separate targets.     

Transformation with human dihydrofolate reductase renders malaria parasites insensitive to WR99210 but does not affect the intrinsic activity of proguanil

Increasing resistance of Plasmodium falciparum malaria parasites to chloroquine and the dihydrofolate reductase (DHFR) inhibitors pyrimethamine and cycloguanil have sparked renewed interest in the antimalarial drugs WR99210 and proguanil, the cycloguanil precursor. To investigate suggestions that WR99210 and proguanil act against a target other than the reductase moiety of the P. falciparum bifunctional DHFR-thymidylate synthase enzyme, we have transformed P. falciparum with a variant form of human DHFR selectable by methotrexate. Human DHFR was found to fully negate the antiparasitic effect of WR99210, thus demonstrating that the only significant action of WR99210 is against parasite DHFR. Although the human enzyme also resulted in greater resistance to cycloguanil, no decrease was found in the level of susceptibility of transformed parasites to proguanil, thus providing evidence of intrinsic activity of this parent compound against a target other than DHFR. The transformation system described here has the advantage that P. falciparum drug-resistant lines are uniformly sensitive to methotrexate and will complement transformation with existing pyrimethamine-resistance markers in functional studies of P. falciparum genes. This system also provides an approach for screening and identifying novel DHFR inhibitors that will be important in combined chemotherapeutic formulations against malaria.     

Molecular epidemiology of malaria in Yaoundé, Cameroon. III. Analysis of chloroquine resistance and point mutations in the multidrug resistance 1 (pfmdr 1) gene of Plasmodium falciparum

It has been postulated that chloroquine resistance may be associated with a single point mutation at codon 86 of the Plasmodium falciparum multidrug resistance 1 (pfmdr 1) gene. Using a simple and rapid molecular technique involving polymerase chain reaction and restriction fragment length polymorphism, the frequency of the Asn-to-Tyr mutation associated with chloroquine resistance was established among 129 clinical isolates obtained from indigenous patients in Yaoundé, Cameroon. The results showed that 110 of 129 isolates display a mutant codon. The other clinical isolates had either a pure wild-type Asn-86 codon (n = 12) or mixed Asn/Tyr alleles (n = 7). In vitro drug assays were performed to compare the genotype and phenotype in 102 clinical isolates. Of these isolates, 86 displayed pure Tyr-86 mutant codon; 48 (56%) mutant isolates were chloroquine-resistant (50% inhibitory concentration [IC50] > 100 nM), as expected, but 38 (44%) mutant isolates were chloroquine-sensitive (IC50 < 100 nM). Three chloroquine-resistant isolates and seven chloroquine-sensitive parasites carried a wild-type Asn-86 codon. Mixed alleles were found in six isolates (four chloroquine-sensitive and two chloroquine-resistant isolates). Our results did not confirm previous observations on the possible association between chloroquine resistance phenotype and genotype based on the pfmdr 1 gene.     

A mutation in the 14 alpha-demethylase gene of Uncinula necator that correlates with resistance to a sterol biosynthesis inhibitor

We investigated the molecular basis of resistance of the obligate biotrophic grape powdery mildew fungus Uncinula necator to sterol demethylation-inhibiting fungicides (DMIs). The sensitivity of 91 single-spore field isolates of U. necator to triadimenol was assessed by using a leaf disc assay. Resistance factors (RF) ranged from 1.8 to 26.0. The gene encoding the target of DMIs (eburicol 14 alpha-demethylase) from five sensitive and seven resistant isolates was cloned and sequenced. A single mutation, leading to the substitution of a phenylalanine residue for a tyrosine residue at position 136, was found in all isolates exhibiting an RF higher than 5. No mutation was found in sensitive or weakly resistant (RF, < 5) isolates. An allele-specific PCR assay was developed to detect the mutation. Among the 91 isolates tested, only isolates with RF higher than 5 carried the mutation. Three of the 19 resistant isolates and all sensitive and weakly resistant isolates did not possess the mutation. The mutation at codon 136 is thus clearly associated with high levels of resistance to triadimenol.     

Characterization of the rpoB gene mutations in clinical isolates of rifampicin-resistant Mycobacterium tuberculosis

OBJECTIVES: Characterization and frequency of the rpoB gene mutations associated with rifampin resistance in Mycobacterium tuberculosis clinical isolates in Sevilla. METHODS: Characterization of rpoB mutations in 21 rifampicin-resistant strains of M. tuberculosis isolated during a three-year period (1994-1996) by three different molecular methods: a nonradioactive Single-strand conformation polymorphism (SSCP) analysis, DNA sequence analysis and a commercial method the line probe assay InnoLiPA. RESULTS: Five distinct rpoB mutations were identified. Ser531-->Leu mutation was detected in 14 strains (66.7%), H526-->Asp in 3 strains (14.3%), Ans512-->Ser in 1 strain (4.8%), Glu513-->Leu in 1 strain (4.8%). A nine nucleotide deletion (codon 510-513) was found in one strain (4.8%) while in the remaining resistant strain (4.8%) no mutation was detected. CONCLUSIONS: The frequency of the different mutations found in the rpoB gene, associated with rifampicin resistance in Mycobacterium tuberculosis clinical isolates in Seville, are similar to those previously reported. However, two new mutations has been detected: a nine nucleotide deletion (codon 510-513), and the Asn512-->Ser point mutation. The characterization of the mutations in the rpoB gene could serve as epidemiological marker for the rifampicin resistant clinical isolates of M. tuberculosis.     

Cerebrovascular effects of analgosedation

The in vitro drug sensitivity of three dihydrofolate reductase inhibitors (pyrimethamine, cycloguanil, trimethoprim) was determined against 29 strains and isolates of Plasmodium falciparum by an isotopic semi-microtest. Trimethoprim is less active than pyrimethamine or cycloguanil and its activity is correlated with that of two other inhibitors, suggesting cross-resistance in vitro among the dihydrofolate reductase inhibitors.     

Allelic exchange at the endogenous genomic locus in Plasmodium falciparum proves the role of dihydropteroate synthase in sulfadoxine-resistant malaria

We have exploited the recently developed ability to trans- fect the malaria parasite Plasmodium falciparum to investigate the role of polymorphisms in the enzyme dihydropteroate synthase (DHPS), identified in sulfadoxine-resistant field isolates. By using a truncated form of the dhps gene, specific mutations were introduced into the endogenous gene by allelic replacement such that they were under the control of the endogenous promoter. Using this approach a series of mutant dhps alleles that mirror P.falciparum variants found in field isolates were found to confer different levels of sulfadoxine resistance. This analysis shows that alteration of Ala437 to Gly (A437G) confers on the parasite a 5-fold increase in sulfadoxine resistance and addition of further mutations increases the level of resistance to 24-fold above that seen for the transfectant expressing the wild-type dhps allele. This indicates that resistance to high levels of sulfadoxine in P.falciparum has arisen by an accumulation of mutations and that Gly437 is a key residue, consistent with its occurrence in most dhps alleles from resistant isolates. These studies provide proof that the mechanism of resistance to sulfadoxine in P.falciparum involves mutations in the dhps gene and determines the relative contribution of these mutations to this phenotype.     

Response of Plasmodium falciparum to chloroquine and Fansidar in vivo and chloroquine and amodiaquine in vitro in Uganda

The response of P. falciparum to chloroquine and pyrimethamine-sulfadoxine in vivo and chloroquine and amodiaquine in vitro was investigated in parasitaemic school children from six locations. Mean parasite sensitivity to chloroquine at day 7 was 74% (range 61-97) with parasite clearance rates between 2-3 days and complete defervescence in 85% of febrile children. Sensitivity declined in the four sites followed up to day 14 to 45% (range 37-53). Parasites were significantly more sensitive to pyrimethamine/sulfadoxine at 5/6 sites (100% day 7) but 5% of subjects became parasitaemic by day 14. In vitro isolates were significantly less sensitive to chloroquine than to amodiaquine with a mean 99% effective concentration of 348 mumol/L compared to 6.44 mumol/L. Clearly the role of chloroquine as the primary therapy for uncomplicated P. falciparum malaria should be reconsidered especially in the light of increasing disease severity and resurgence. Amodiaquine may be suitable alternative with pyrimethamine/sulfadoxine as second line and for more severe malaria prior to referral. The cost of alternative antimalarials and the dynamic and deteriorating pattern of resistance are powerful arguments for more objective slide diagnosis to minimise drug pressure and a regular drug sensitivity surveillance system. We believe that the latter should concentrate on measuring clinical drug efficacy in symptomatic outpatients rather than in asymptomatic children while the former needs more pragmatic and economical strategies possibly centred on seasonality and risk.     

A PCR-based method to characterise and identify benzimidazole resistance in Helminthosporium solani

Control of Helminthosporium solani, the cause of silver scurf in potato tubers, has been impaired by selection of benzimidazole-resistant strains as a result of repeated use of the fungicide thiabendazole. Identification of thiabendazole-resistant strains of H. solani by conventional techniques takes several weeks. Primers designed from conserved regions of the fungal beta-tubulin gene were used to PCR amplify and sequence a portion of the gene. A point mutation was detected at codon 198 in thiabendazole-resistant isolates causing a change in the amino acid sequence from glutamic acid to alanine or glutamine. Species-specific PCR primers designed to amplify this region were used in conjunction with a restriction endonuclease to cause cleavage in sensitive isolates only and thus provide a rapid diagnostic test to differentiate field isolates.     

Sequence variation of the hydroxymethyldihydropterin pyrophosphokinase: dihydropteroate synthase gene in lines of the human malaria parasite, Plasmodium falciparum, with differing resistance to sulfadoxine

Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur-based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine-resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (CH2OH-H2pterinPP kinase) at its 5' proximal end. This domain is unusually large, with two long insertions relative to other CH2OH-H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high-level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations at positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs that are conserved across all other known H2Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide-resistant pathogenic bacteria. Copy-number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines.     

Mutations of K-ras oncogene and absence of H-ras mutations in squamous cell carcinomas of the lung

Mutations of the K-ras gene have been implicated in the pathogenesis of human lung adenocarcinomas. In most studies published so far, squamous cell lung cancers harbored ras mutations only exceptionally or no mutations were detected at all. We have examined 141 lung tumor DNA samples for mutations in codons 12, 13, and 61 of K-ras and H-ras oncogenes. A large panel of 118 squamous cell carcinomas was included in the study. For K-ras codon 12, we used a sensitive two-step PCR-restriction fragment length polymorphism method which detects <1% of mutated DNA in the sample. K-ras mutations were found in 17 tumors (12%; 14 in codon 12 and 3 in codon 13). Among 19 adenocarcinomas, mutation was revealed in 7 samples (37%). Of these, one sample harbored two point mutations in codon 12. Nine mutational events were found in squamous cell carcinomas (8%, one adenosquamous carcinoma included, all in codon 12). Of four large cell carcinomas, one contained a mutation. Mutant-enriched PCR products harboring mutations were directly sequenced. Fifteen mutational events were G-->T transversions or G-->A transitions, one was a G-->C transition, and one sample revealed a frameshift deletion of one G from codon 12. Similar mutational spectrum was found in both squamous cell carcinomas and adenocarcinomas, suggesting similar carcinogenic pathways in both histological types of the tumor. The presence of mutations did not correlate with the stage of the disease. Moreover, we analyzed all samples for mutations in codons 12, 13, and 61 of the H-ras gene. We found only one mutation in codon 12. Thus, H-ras mutations apparently play an inferior role in lung carcinogenesis. We conclude that mutations of the K-ras oncogene can play a role in the development of not only lung adenocarcinomas but also of a subset (about 8%) of squamous cell carcinomas.     

The regulatory mechanism of phosphatidylinositol 3-kinase by insulin in 3T3 L1 fibroblasts: phosphorylation-independent activation of phosphatidylinositol 3-kinase

Progress in understanding the basis of resistance to rifampicin (RifR) has allowed molecular tests for the detection of drug-resistant tuberculosis to be developed. One hundred thirteen strains of Mycobacterium tuberculosis isolated from patients with multidrug resistant tuberculosis (MDR-TB) were investigated for genotypic analysis of RifR by polymerase chain reaction-heteroduplex formation (PCR-HDF) and characterization of mutations by automated DNA sequencing of the rpoB gene. A subset of isolates (22) representative of different mutations as confirmed by sequence analysis were also evaluated by the Line Probe Assay (LiPA). In 106 of the RifR strains, 24 mutations within an 81-bp region of the rpoB gene affecting 13 amino acids were observed. Most isolates (7/8) harboring Leu533 --> Pro codon mutation required minimum inhibitory concentrations (MICs) of < or = 8 microg/ml. There was geographic variation in the frequency of occurrence of particular rpoB mutations, with the Ser531 --> Leu/Trp codon mutation found in 59/113 of isolates. Although there are certain limitations in the use of both the rapid PCR-HDF diagnostic assay and the LiPA for the detection of rifampicin susceptibility of M. tuberculosis, these provide important and convenient tools for identifying and managing patients with MDR-TB.     

Hepatitis B virus genomic sequence in the circulation of hepatocellular carcinoma patients: comparative analysis of 40 full-length isolates

We determined full-length nucleotide sequence of hepatitis B virus (HBV) genome in sera from 40 Japanese patients with HBsAg-positive hepatocellular carcinoma (HCC), in order to obtain information on HCC-specific characteristics, if any, of the HBV genome. Direct sequencing of the long distance PCR products starting from 50 microliters of serum samples revealed that 95% of our isolates were of genotype C, and that mutations and deletions/insertions were very common. With respect to envelope protein genes, deletions and missense mutations were frequent in preS2, and the determinant a domain of HBsAg was rich in "antibody-escape" mutations. Within the precore/core region, the most remarkable mutation was the replacement of proline of wild type by other amino acids at codon 130 of the core gene, which was found in 58% of our isolates, while precore-stop mutation was found in 45%. Most interestingly, however, about 90% of our isolates had mutations at nt positions 1762 (A-to-T) and 1764 (G-to-A) within the core promoter, which had been implicated in "e-suppressive" phenotype of HBV genome. G-to-A at nt 1613 and C-to-T at nt 1653 within enhancer II and T-to-C/A at nt 1753 within core promoter were also evident: 38%, 53%, and 40%, respectively. It was interesting that some of the characteristics observed in our isolates form HCC patients had been previously implicated in fulminant hepatitis and/or acute exacerbation of chronic hepatitis.     

NMR spectroscopy of human post mortem cerebrospinal fluid: distinction of Alzheimer''s disease from control using pattern recognition and statistics

Mutations of ras oncogenes in 37 human stomach cancers and 13 adenomas were investigated with regard to the histological phenotypes using polymerase chain reaction (PCR), allele-specific oligonucleotide hybridization and/or direct sequencing of the PCR products. The ras mutation was found only in one case (2.7%), the histology of which was poorly differentiated adenocarcinoma. We found no mutation in stomach adenomas. The mutation consisted of a guanine-to-adenine transition in the first base of codon 13 of c-Ki-ras which replaced wild-type glycine with serine, indicating that a putative glycine-to-aspartic acid change is not necessarily the critical event for c-Ki-ras gene activation in codon 13. These results further confirm the infrequency of ras mutation in stomach tumors and also suggest that ras mutations are not specific to the differentiated type of stomach cancer.     

Transformation of Plasmodium falciparum malaria parasites by homologous integration of plasmids that confer resistance to pyrimethamine

Plasmodium falciparum malaria parasites were transformed with plasmids containing P. falciparum or Toxoplasma gondii dihydrofolate reductase-thymidylate synthase (dhfr-ts) coding sequences that confer resistance to pyrimethamine. Under pyrimethamine pressure, transformed parasites were obtained that maintained the transfected plasmids as unrearranged episomes for several weeks. These parasite populations were replaced after 2 to 3 months by parasites that had incorporated the transfected DNA into nuclear chromosomes. Depending upon the particular construct used for transformation, homologous integration was detected in the P. falciparum dhfr-ts locus (chromosome 4) or in hrp3 and hrp2 sequences that were used in the plasmid constructs as gene control regions (chromosomes 13 and 8, respectively). Transformation by homologous integration sets the stage for targeted gene alterations and knock-outs that will advance understanding of P. falciparum.