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.     

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.     

Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase

We have overexpressed the gene for dihydrofolate reductase (DHFR) from Thermotoga maritima in Escherichia coli and characterized the biochemical properties of the recombinant protein. This enzyme is involved in the de novo synthesis of deoxythymidine 5'-phosphate and is critical for cell growth. High levels of T. maritima DHFR in the new expression system conferred resistance to high levels of DHFR inhibitors which inhibit the growth of non-recombinant cells. The enzyme was purified to homogeneity in the following two steps: heat treatment followed by affinity chromatography or cation-exchange chromatography. Most of the biochemical properties of T. maritima DHFR resemble those of other bacterial or eukaryotic DHFRs, however, some are unique to T. maritima DHFR. The pH optima for activity, Km for substrates, and polypeptide chain length of T. maritima DHFR are similar to those of other DHFRs. In addition, the secondary structure of T. maritima DHFR, as measured by circular dichroism, is similar to that of other DHFRs. Interestingly, T. maritima DHFR exhibits some characteristics of eukaryotic DHFRs, such as a basic pI, an excess of positively charged residues in the polypeptide chain and activation of the enzyme by inorganic salts and urea. Unlike most other DHFRs which are monomeric or part of a bifunctional DHFR-thymidylate synthase (TS) enzyme, T. maritima DHFR seems to generally form a dimer in solution and is also much more thermostable than other DHFRs. It may be that dimer formation is a key factor in determining the stability of T. maritima DHFR.     

6-substituted 2,4-diamino-5-methylpyrido[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases from Pneumocystis carinii and Toxoplasma gondii and as antitumor agents

The synthesis and biological activity of 15 6-substituted 2,4-diamino-5-methylpyrido[2,3-d]-pyrimidines are reported. These compounds were synthesized in improved yields by modifications of procedures previously reported by us. Specifically, dimethoxyphenyl-substituted compounds with H and CH3 at the N-10 position and trimethoxyphenyl-substituted compounds with N-10 ethyl, isopropyl, and propargyl moieties were synthesized. These compounds were evaluated as inhibitors of dihydrofolate reductases (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and rat liver, and selected analogues were evaluated as inhibitors of the growth of T. gondii and tumor cells in culture. All the compounds showed increased selectivity (vs rat liver DHFR) for T. gondii DHFR compared to trimetrexate. In general, for the trimethoxy-substituted analogues, increasing the size of the N-10 substituent from a methyl group to larger groups resulted in a decrease in selectivity and potency for both P. carinii and T. gondii DHFR. For the dimethoxy-substituted analogues, N-10 methylation in general decreased potency but increased selectivity for T. gondii DHFR. In an attempt to improve the cell penetration of these analogues, the N-10 naphthyl-substituted analogues were also synthesized. These analogues displayed excellent cell penetration and inhibition of T. gondii cells in culture. Further, these analogues were potent inhibitors of the growth of tumor cells in the preclinical in-vitro screening program of the National Cancer Institute with IC50s in the nanomolar range.     

How do mutations at phenylalanine-153 and isoleucine-155 partially suppress the effects of the aspartate-27-->serine mutation in Escherichia coli dihydrofolate reductase?

Several second-site suppressors of the D27S lesion in Escherichia coli dihydrofolate reductase (DHFR) have been identified. The activity of the primary mutant, D27S DHRF, was found to be greatly decreased at pH 7.0, consistent with aspartic acid-27 being critically involved in proton donation during catalysis. Partial suppressors of the D27S mutation have been selected by their ability to confer an increased resistance to trimethoprim upon host E. coli; the suppressors have been identified as F153S or I155N substitutions. D27S+F153S and D27S+I155N DHFRs display 2-3-fold increases in kcat over D27S DHFR values, but only the F153S mutation decreases the Km for dihydrofolate by a factor of 2. Neither double mutant approaches wild-type DHFR activity. Unexpectedly, Phe153 and Ile155 occur on the surface of the protein and are approximately 8 and 14 A distant from the active site. Ile155 is a member of a beta-bulge. A previously identified suppressing mutation, F137S, occurs nearby and is also a member of the same beta-bulge [Howell et al. (1990) Biochemistry 29, 8561-8569]. Clustering of these three second-site mutations indicates this area of the structure may be important in protein function. Conformational changes due to the presence of these suppressing mutations are likely as the F153S and I155N mutations do not affect hydride-transfer rates upon introduction in wild-type DHFR and alterations in circular dichroism spectra are associated with the double-mutant DHFRs.     

In vitro activities of several diaminomethylpyridopyrimidines against Mycobacterium avium complex

Three recently synthesized dihydrofolate reductase (DHFR) inhibitors designated SoRI 8890, 8895, and 8897 were evaluated for their in vitro activities against 25 isolates of Mycobacterium avium complex. The MICs at which 50 and 90% of isolates were inhibited were 1 and 2, 4 and 8, and 4 and 8 microgram/ml for SoRI 8890, 8895, and 8897, respectively. Although the addition of dapsone at 0.5 microgram/ml did not significantly enhance the in vitro activities of these compounds, their activities alone were comparable to, if not better than, results seen with other DHFR inhibitors, such as pyrimethamine or WR99210.     

2,4-Diaminothieno[2,3-d]pyrimidine lipophilic antifolates as inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase

Ten previously unreported 2,4-diaminothieno[2,3-d]pyrimidine lipophilic dihydrofolate reductase inhibitors were synthesized as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. Pivaloylation of 2,4-diamino-5-methylthieno[2,3-d]pyrimidine followed by dibromination with N-bromosuccinimide in the presence of benzoyl peroxide gave 2,4-bis(pivaloylamino)-6-bromo-5-(bromomethyl)thieno[2,3-d]pyrimid ine, which after condensation with substituted anilines or N-methylanilines and deprotection with base yielded 2,4-diamino-6-bromo-5-[(substituted anilino)methyl]thieno[2,3-d]pyrimidines. Removal of the 6-bromo substituent was accomplished with sodium borohydride and palladium chloride. The reaction yields were generally good to excellent. The products were tested as inhibitors of dihydrofolate reductase (DHFR) from P. carinii, T. gondii, and rat liver. Although the IC50 could not be reached for the 6-unsubstituted compounds because of their extremely poor solubility, three of the five 6-bromo derivatives were soluble enough to allow the IC50 to be determined against all three enzymes. 2,4-Diamino-5-[3,5-dichloro-4-(1-pyrrolo)anilino]methyl]- 6-bromothieno[2,3-d]pyrimidine was the most active of the 6-bromo derivatives, with an IC50 of 7.5 microM against P. carinii DHFR, but showed no selectivity for either P. carinii or T. gondii DHFR relative to the enzyme from rat liver.     

In search of circular permuted variants of Escherichia coli dihydrofolate reductase

A circularized form of a Cys-free mutant of Escherichia coli dihydrofolate reductase (DHFR) was used to search for a proteolytic site that gave new N- and C-termini on circularized DHFR with enzyme activity. Of the six site-specific proteolytic enzymes tested, three proteases, Achromobacter protease I (lysine-specific endopeptidase), asparaginylendopeptidase, and Staphylococcus aureus V8 protease, cleaved a single site of the circularized DHFR to form circular permuted variants. Twenty-four possible sites for cleavage were found formation of eight circular permuted variants was suggested by results of N-terminal sequence analysis of the linearized proteins isolated by gel filtration in the presence of 5 M guanidine hydrochloride. Mapping of the predicted cleavage sites on the DHFR molecule suggested that they were not all at a specific loop and, therefore, there are many possible circular permuted variants.     

Synthesis and biological activities of tricyclic conformationally restricted tetrahydropyrido annulated furo[2,3-d]pyrimidines as inhibitors of dihydrofolate reductases

The synthesis of seven 2,4-diamino-5,6,7,8-tetrahydro-7-substituted pyrido[4',3':4,5]furo[2,3-d]pyrimidines 1-6 are reported as nonclassical antifolate inhibitors of dihydrofolate reductase (DHFR) and compound 7 as a classical antifolate inhibitor of tumor cells in culture. The compounds were designed as conformationally restricted analogues of trimetrexate. The synthesis was accomplished from the cyclocondensation of 3-bromo-4-piperidone with 2, 4-diamino-6-hydroxypyrimidine to afford regiospecifically 2, 4-diamino-5,6,7,8-tetrahydropyrido[4',3':4,5]furo[2, 3-d]pyrimidine-7-hydrobromide (16). This in turn was alkylated with the appropriate benzyl halide to afford the target compounds 1-6. The classical antifolate 7 utilized 4-(chloromethyl)benzoyl-l-glutamic acid diethyl ester (17) instead of the benzyl halide for alkylation, followed by saponification to afford 7. Compounds 1-6 showed moderate inhibitory potency against DHFR from Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and rat liver. The classical analogue 7 was 88-fold more potent against M. avium DHFR than against rat liver DHFR. The classical analogue was also inhibitory against the growth of tumor cells, CCRF-CEM, and FaDu, in culture.     

Selective Pneumocystis carinii dihydrofolate reductase inhibitors: design, synthesis, and biological evaluation of new 2,4-diamino-5-substituted-furo[2,3-d]pyrimidines

Nonclassical antifolates, 2,4-diamino-5-substituted-furo[2, 3-d]pyrimidines 3-12 with bridge region variations of C8-S9, C8-N9, and C8-O9 and 1-naphthyl, 2-naphthyl, 2-phenoxyphenyl, 4-phenoxyphenyl, and 2-biphenyl side chains were synthesized as phenyl ring appended analogues of previously reported 2, 4-diamino-5-(anilinomethyl)furo[2,3-d]pyrimidines. The phenyl ring appended analogues were designed to specifically interact with Phe69 of dihydrofolate reductase (DHFR) from Pneumocystis carinii (pc) to afford selective inhibitors of pcDHFR. Additional substituted phenyl side chains which include 2,5-dichloro, 3,4-dichloro, 3,4,5-trichloro, 3-methoxy, and 2,5-dimethoxy analogues 13-17 were also synthesized. The compounds were prepared by nucleophilic displacement of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine(2) with the appropriate thiol, amine, or naphthol. Compound 2 was obtained from 2,4-diamino-6-hydroxypyrimidine and 1, 3-dichloroacetone. The compounds were evaluated as inhibitors against DHFR from P. carinii, Toxoplasma gondii, and rat liver. Two analogues, 2,4-diamino-5-[(2'-naphthylthio)methyl]furo[2, 3-d]pyrimidine (5) and 2,4-diamino-5-[(2'-phenylanilino)methyl]furo[2,3-d]pyrimidine (11) showed significant selectivity and potency for pcDHFR compared to trimethoprim. The X-ray crystal structure of 5 with pcDHFR was also carried out, which corroborated the design rationale and indicated a hydrophobic interaction of the naphthalene ring of 5 and Phe69 of pcDHFR which is responsible, in part, for the more than 18-fold selectivity of 5 for pcDHFR as compared with rat liver DHFR.     

A new procedure for improving the predictiveness of CoMFA models and its application to a set of dihydrofolate reductase inhibitors

A new automated procedure to improve the predictive quality of CoMFA models for both training and test sets is described. A model of greater consistency is generated by performing small reorientations of the underlying molecules for which too low activities are calculated. In order to predict activities of test compounds, the most similar molecules in the previously optimized model are identified and used as a basis for the prediction. This method has been applied to two independent sets of dihydrofolate reductase inhibitors (80 compounds each, serving as training sets), resulting in a significant increase of the cross-validated r2 value. For both models, the predictive r2 value for a test set consisting of 70 compounds was improved substantially.     

Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system

In an attempt to introduce a large peptide that is not normally translocated across membranes into the cytosol of eukaryotic cells, we created a new chimeric protein termed CEDH between Pseudomonas aeruginosa exotoxin A (ETA) and a variant enzyme of Mus musculus dihydrofolate reductase (DHFR) with reduced affinity for antifolates, ETA(1-413).DHFR(1-187).ETA(609-613). We have defined, genetically constructed and expressed the chimeric protein in Escherichia coli. We showed that the CEDH chimeric protein, purified to homogeneity on an immunoaffinity resin, confers a methotrexate-resistant phenotype to Chinese hamster ovary cells. Furthermore, the chimeric protein allowed the growth of dihydrofolate reductase-deficient Chinese hamster ovary cells in the absence of hypoxanthine and thymidine. These results demonstrated that the chimeric protein exhibited enzyme activity and possessed the tightly folded native structure, and that the DHFR protein can be selectively internalized and translocated via domains of exotoxin A. These data show that the ETA system is an efficient system for the delivery of a variety of large polypeptides into the cytosol without stress to the target cells, and extends the use of this delivery system to proteins that are not normally translocated across membranes.     

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.     

Overexpression and large-scale purification of recombinant hamster polymorphic arylamine N-acetyltransferase as a dihydrofolate reductase fusion protein

N-Acetyltransferases (NATs) are enzymes that catalyze the detoxification and/or bioactivation of a variety of xenobiotics. Rapid kinetic, biophysical, structural, and bioactivation studies on NATs require quantities of purified enzyme capable of being obtained only through recombinant DNA technology. This laboratory has previously developed a protein expression and purification system in which NATs are expressed as proteins fused to a FLAG octapeptide followed by a thrombin-cleavage site to allow liberation of the rNAT. Typically, however, only 0.5-1.5 mg of the recombinant NAT's could be readily purified in a single isolation sequence by immunoaffinity chromatography. Therefore, the expression system was modified by inserting the L54F dihydrofolate reductase (DHFR) mutant gene sequence between the FLAG octapeptide and the thrombin-cleavage site. Expression was carried out with TOPP3 Escherichia coli cells. The new purification methodology utilizes the unique pH dependence of binding to a methotrexate (MTX)-affinity column by the L54F DHFR mutant. Unfortunately, the affinity chromatography strategy did not work satisfactorily. Although the specific activity of the purified rNAT2 was comparable to that of NAT2 obtained from hamster tissue, only 3% of the activity was recovered. The apparent cause of the low recovery is the unanticipated irreversible binding of rNAT2 to MTX. Ion-exchange chromatography was investigated as an alternative purification method. An initial DEAE anion-exchange column resulted in partial purification of the fusion protein. The fusion protein was cleaved with thrombin and reapplied to a DEAE anion-exchange column. The second DEAE column resulted in not only the separation of rNAT2-70D from FLAG-L54F DHFR, but also the purification of rNAT2-70D to near homogeneity. Application of the nearly homogeneous rNAT2-70D to a gel-filtration column resulted in recovery of homogeneous protein. The ion-exchange method of purifying rNAT2-70D is inexpensive and simple and yields more than 8 mg of pure enzyme from 1 liter of cell culture.     

GroEL-mediated folding of structurally homologous dihydrofolate reductases

Using stopped-flow fluorescence techniques, we have examined both the refolding and unfolding reactions of four structurally homologous dihydrofolate reductases (murine DHFR, wild-type E. coli DHFR, and two E. coli DHFR mutants) in the presence and absence of the molecular chaperonin GroEL. We show that GroEL binds the unfolded conformation of each DHFR with second order rate constants greater than 3 x 10(7) M(-1)s(-1) at 22 degrees C. Once bound to GroEL, the proteins refold with rate constants similar to those for folding in the absence of GroEL. The overall rate of formation of native enzyme is decreased by the stability of the complex between GroEL and the last folding intermediate. For wild-type E. coli DHFR, complex formation is transient while for the others, a stable complex is formed. The stable complexes are the same regardless of whether they are formed from the unfolded or folded DHFR. When complex formation is initiated from the native conformation, GroEL binds to a pre-existing non-native conformation, presumably a late folding intermediate, rather than to the native state, thus shifting the conformational equilibrium toward the non-native species by mass action. The model presented here for the interaction of these four proteins with GroEL quantitatively describes the difference between the formation of a transient complex and a stable complex as defined by the rate constants for release and rebinding to GroEL relative to the rate constant for the last folding step. Due to this kinetic partitioning, three different mechanisms can be proposed for the formation of stable complexes between GroEL and either murine DHFR or the two E. coli DHFR mutants. These data show that productive folding of GroEL-bound proteins can occur in the absence of nucleotides or the co-chaperonin GroES and suggest that transient complex formation may be the functional role of GroEL under normal conditions.     

Characterizing replication intermediates in the amplified CHO dihydrofolate reductase domain by two novel gel electrophoretic techniques

Using neutral/neutral and neutral/alkaline two-dimensional (2-D) gel techniques, we previously obtained evidence that initiation can occur at any of a large number of sites distributed throughout a broad initiation zone in the dihydrofolate reductase (DHFR) domain of Chinese hamster ovary (CHO) cells. However, other techniques have suggested a much more circumscribed mode of initiation in this locus. This dichotomy has raised the issue whether the patterns of replicating DNA on 2-D gels have been misinterpreted and, in some cases, may represent such noncanonical replication intermediates as broken bubbles or microbubbles. In an accompanying study (R. F. Kalejta and J. L. Hamlin, Mol. Cell. Biol. 16:4915-4922, 1996), we have shown that broken bubbles migrate to unique positions in three different gel systems and therefore are not likely to be confused with classic replication intermediates. Here, we have applied a broken bubble assay developed from that study to an analysis of the amplified DHFR locus in CHO cells. This assay gives information about the number and positions of initiation sites within a fragment. In addition, we have analyzed the DHFR locus by a novel stop-and-go-alkaline gel technique that measures the size of nascent strands at all positions along each arc in a neutral/neutral 2-D gel. Results of these analyses support the view that the 2-D gel patterns previously assigned to classic, intact replication bubbles and single-forked structures indeed correspond to these entities. Furthermore, potential nascent-strand start sites appear to be distributed at very frequent intervals along the template in the intergenic region in the DHFR domain.     

History and future of antimicrobial diaminopyrimidines

Numerous pyrimidine and purine analogs were synthesized in the late forties in G.H. Hitchings' group as potential nucleic acid antagonists. Several key observations finally led to the selection of pyrimethamine as an antimalarial and trimethoprim (TMP) as an antibacterial agent: i) 2,4-diamino-5-substituted pyrimidines interfered with folic acid utilization rather than being thymine antagonists as expected; ii) a large degree of selectivity could be obtained by suitable substitution and non-toxic diaminopyrimidines with preferential antimicrobial activity were found; iii) the identification of dihydrofolate reductase (DHFR) as the specific target for aminopterin and methotrexate in 1958 and for TMP in 1965, and the diversity of this enzyme in different species. Although several diaminopyrimidines were initially tested as monotherapies in clinical trials, the pronounced synergism between some of these new compounds and sulfonamides seen against Plasmodium was finally also applied in the development of TMP. Its combination with sulfamethoxazole later proved one of the most successful agents ever developed. Further milestones in the application of antimicrobial DHFR inhibitors were the introduction of TMP alone in 1972, the launch of a new combination of tetroxoprim, a close TMP-analog, with sulfadiazine, and the successful clinical trials with brodimoprim, which proved clinically efficacious and safe with once-daily low dose monotherapy. Efforts to discover new antimicrobial DHFR inhibitors have recently intensified. DHFRs from important gram-positive problem organisms such as S. aureus, S. epidermidis have been cloned and sequenced, as well as DHFRs from opportunistic pathogens such as P. carinii, T. gondii, and of mycobacteria. DHFR crystal structures from several of these organisms are available to aid rational inhibitor design.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

6-Substituted 2,4-diaminopyrido[3,2-d]pyrimidine analogues of piritrexim as inhibitors of dihydrofolate reductase from rat liver, Pneumocystis carinii, and Toxoplasma gondii and as antitumor agents

The synthesis and biological activity are reported for 21 6-substituted 2,4-diaminopyrido[3,2-d]pyrimidine analogues (4-24) of piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) and as antitumor agents. Recombinant DHFR from Pneumocystis carinii (pc) and native DHFR from Toxoplasma gondii (tg) were the target enzymes tested; these organisms are responsible for fatal opportunistic infections in AIDS patients. Rat liver (rl) DHFR served as the mammalian reference enzyme to determine selectivity for the pathogenic DHFR. The synthesis of S9-bridged compounds 4-6 was achieved by aryl displacement of 2,4-diamino-6-chloropyrido[3, 2-d]pyrimidine (27) with thiol nucleophiles. Oxidation of 4-6 with hydrogen peroxide in glacial acetic acid afforded the corresponding sulfone analogues 7-9. The N9-bridged compounds 10-24 were synthesized from their precursor 3-amino-6-(arylamino)-2-pyridinecarbonitriles via a thermal cyclization with chloroformamidine hydrochloride. Unlike the S9-bridged compounds, the arylamino side chains of the N9-bridged analogues were introduced prior to the formation of the 2, 4-diaminopyrido[3,2-d]pyrimidine nucleus. A reversed two-atom-bridged analogue (25) was also synthesized using a synthetic strategy similar to that utilized for compounds 10-24. The IC50 values of these compounds against pcDHFR ranged from 0.0023 x 10(-6) M for 2,4-diamino-6-(N-methyl-3',4'-dimethoxyanilino)pyrido[3, 2-d]pyrimidine (21), which was the most potent, to 90.4 x 10(-6) M for 2,4-diamino-6-(4'-methoxyanilino)pyrido[3,2-d]pyrimidine (12), which was the least potent. The three S9-bridged compounds tested were more potent than the corresponding sulfone-bridged compounds for all three DHFRs. N9-Methylation increased the potency by as much as 17 000-fold (compounds 15 and 21). None of the analogues were selective for pcDHFR. Against tgDHFR the most potent analogue was again 21 with an IC50 value of 0.00088 x 10(-6) M and the least potent was 12 with an IC50 of 2.8 x 10(-6) M. N9-Methylation afforded an increase in potency of up to 770-fold (compound 15 NH vs 21 N-CH3) compared to the corresponding N9-H analogue. In contrast to pcDHFR, several analogues had a greater selectivity ratio for tgDHFR compared to trimetrexate (TMQ) or PTX, most notably 2, 4-diamino-6-[(3',4'- dimethoxyphenyl)thio]pyrido[3,2-d]pyrimidine (4), 2,4-diamino-6-[(2'-methoxyphenyl)sulfonyl]pyrido[3, 2-d]pyrimidine (7), and 2,4-diamino-6-(2', 5'-dimethoxyanilino)pyrido[3,2-d]pyrimidine (14) which combined relatively high potency at 10(-7)-10(-8) M along with selectivity ratios of 3.97, 6.67, and 4.93, respectively. Several analogues synthesized had better selectivity ratios than TMQ or PTX for both pcDHFR and tgDHFR, and the potencies of the N9-methylated compounds were comparable to or greater than that of TMQ or PTX. Selected compounds were evaluated as inhibitors of the growth of a variety of tumor cells in culture. The N9-CH3 analogues were, in general, highly potent with GI50 values in the nanomolar range. The N9-H and S9 analogues were less potent with GI50 values in the millimolar to micromolar range.