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A polymerase chain reaction (PCR) that amplifies the internal transcribed spacers of rRNA genes of Pneumocystis carinii (Pc-ITS-PCR) was compared with dihydrofolate reductase (DHFR) gene PCR to determine its usefulness in the diagnosis of Pneumocystis carinii pneumonia. All specimens from non-P. carinii pneumonia AIDS patients were both Pc-ITS-PCR- and DHFR PCR-negative. Twenty-six (96%) of 27 bronchoalveolar lavage specimens from P. carinii pneumonia patients were Pc-ITS-PCR-positive, and all 27 of these patients had at least 1 serum sample positive by Pc-ITS-PCR. In contrast, DHFR PCR detected P. carinii in only 4 (17%) of 24 P. carinii-positive lavage specimens and 2 (10%) of the 20 sera from P. carinii pneumonia patients. These results suggest that serum Pc-ITS-PCR may be useful for the diagnosis of P. carinii pneumonia. [ABSTRACT FROM AUTHOR]

To combine the potency of trimetrexate (TMQ) or piritrexim (PTX) with the species selectivity of trimethoprim (TMP), target based design was carried out with the X-ray crystal structure of human dihydrofolate reductase (hDHFR) and the homology model of Pneumocystis jirovecii DHFR (pjDHFR). Using variation of amino acids such as Met33/Phe31 (in pjDHFR/hDHFR) that affect the binding of inhibitors due to their distinct positive or negative steric effect at the active binding site of the inhibitor, we designed a series of substituted-pyrrolo[2,3-d]pyrimidines. The best analogs displayed better potency (IC 50 ) than PTX and high selectivity for pjDHFR versus hDHFR, with 4 exhibiting a selectivity for pjDHFR of 24-fold., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Infection by the parasite Toxoplasma gondii (tg) can lead to toxoplasmosis in immunocompromised patients such as organ transplant, cancer and HIV/AIDS patients. The bifunctional thymidylate synthase-dihydrofolate reductase (TS-DHFR) enzyme is crucial for nucleotide synthesis in T. gondii , and represents a potential target to combat T. gondii infection. While species selectivity with drugs has been attained for DHFR, TS is much more conserved across species and specificity is significantly more challenging. We discovered novel substituted-9 H -pyrimido[4,5- b ]indoles 1 - 3 with single-digit nanomolar K i for tgTS, two of which, 2 and 3 , are 28- and 122-fold selective over human TS (hTS). The synthesis of these compounds, and their structures in complex with tgTS-DHFR are presented along with binding measurements and cell culture data. These results show, for the very first time, that in spite of the high degree of conservation of active site residues between hTS and the parasite TS, specificity has been accomplished via novel structures and provides a new target (TS) for selective drug development against parasitic infections.

Pneumocystis jirovecii is an opportunistic pathogen that causes serious pneumonia in immunosuppressed patients. Standard therapy and prophylaxis include trimethoprim (TMP)-sulfamethoxazole; trimethoprim in this combination targets dihydrofolate reductase (DHFR). Fourteen clinically observed variants of P. jirovecii DHFR were produced recombinantly to allow exploration of the causes of clinically observed failure of therapy and prophylaxis that includes trimethoprim. Six DHFR variants (S31F, F36C, L65P, A67V, V79I, and I158V) showed resistance to inhibition by trimethoprim, with Ki values for trimethoprim 4-fold to 100-fold higher than those for the wild-type P. jirovecii DHFR. An experimental antifolate with more conformational flexibility than trimethoprim showed strong activity against one trimethoprim-resistant variant. The two variants that were most resistant to trimethoprim (F36C and L65P) also had increased Km values for dihydrofolic acid (DHFA). The catalytic rate constant (kcat) was unchanged for most variant forms of P. jirovecii DHFR but was significantly lowered in F36C protein; one naturally occurring variant with two amino acid substitutions (S106P and E127G) showed a doubling of kcat, as well as a Km for NADPH half that of the wild type. The strongest resistance to trimethoprim occurred with amino acid changes in the binding pocket for DHFA or trimethoprim, and the strongest effect on binding of NADPH was linked to a mutation involved in binding the phosphate group of the cofactor. This study marks the first confirmation that naturally occurring mutations in the gene for DHFR from P. jirovecii produce variant forms of DHFR that are resistant to trimethoprim and may contribute to clinically observed failures of standard therapy or prophylaxis.

Opportunistic infections caused by Pneumocystis jirovecii (P. jirovecii, pj), Toxoplasma gondii (T. gondii, tg), and Mycobacterium avium (M. avium, ma) are the principal causes of morbidity and mortality in patients with acquired immunodeficiency syndrome (AIDS). The absence of any animal models for human Pneumocystis jirovecii pneumonia and the lack of crystal structures of pjDHFR and tgDHFR make the design of inhibitors challenging. A novel series of pyrido[2,3-d]pyrimidines as selective and potent DHFR inhibitors against these opportunistic infections are presented. Buchwald-Hartwig coupling reaction of substituted anilines with pivaloyl protected 2,4-diamino-6-bromo-pyrido[2,3-d]pyrimidine was successfully explored to synthesize these analogues. Compound 26 was the most selective inhibitor with excellent potency against pjDHFR. Molecular modeling studies with a pjDHFR homology model explained the potency and selectivity of 26. Structural data are also reported for 26 with pcDHFR and 16 and 22 with variants of pcDHFR.

A major concern of immunocompromised patients, in particular those with AIDS, is susceptibility to infection caused by opportunistic pathogens such as Pneumocystis jirovecii, which is a leading cause of pneumonia in immunocompromised patients. We report the first kinetic and structural data for 2,4-diamino-6-[(2',5'-dichloro anilino)methyl]pyrido[2,3-d]pyrimidine (OAAG324), a potent inhibitor of dihydrofolate reductase (DHFR) from P. jirovecii (pjDHFR), and also for trimethoprim (TMP) and methotrexate (MTX) with pjDHFR, Pneumocystis carinii DHFR (pcDHFR), and human DHFR (hDHFR). OAAG324 shows a 9.0-fold selectivity for pjDHFR (Ki, 2.7 nM) compared to its selectivity for hDHFR (Ki, 24.4 nM), whereas there is only a 2.3-fold selectivity for pcDHFR (Ki, 6.3 nM). In order to understand the determinants of inhibitory potency, active-site mutations of pj-, pc-, and hDHFR were explored to make these enzymes more like each other. The most unexpected observations were that the variant pcDHFR forms with K37Q and K37Q/F69N mutations, which made the enzyme more like the human form, also made these enzymes more sensitive to the inhibitory activity of OAAG324, with Ki values of 0.26 and 0.71 nM, respectively. A similar gain in sensitivity was also observed for the hDHFR N64F variant, which showed a lower Ki value (0.58 nM) than native hDHFR, pcDHFR, or pjDHFR. Structural data are reported for complexes of OAAG324 with hDHFR and its Q35K and Q35S/N64F variants and for the complex of the K37S/F69N variant of pcDHFR with TMP. These results provide useful insight into the role of these residues in the optimization of highly selective inhibitors of DHFR against the opportunistic pathogen P. jirovecii.

Toxoplasma gondii is an obligate intracellular parasite that permanently infects warm-blooded vertebrates through its ability to convert into a latent tissue cyst form. The latent form (bradyzoite) can reinitiate a life-threatening acute infection if host immunity wanes, most commonly in AIDS or organ transplant patients. We have previously shown that bradyzoite development is accompanied by phosphorylation of the parasite eukaryotic initiation factor 2 alpha subunit (eIF2α), which dampens global protein synthesis and reprograms gene expression. In this study, we analyzed the activities of two specific inhibitors of eIF2α dephosphorylation, salubrinal (SAL) and guanabenz (GA). We establish that these drugs are able to inhibit the dephosphorylation of Toxoplasma eIF2α. Our results show that SAL and GA reduce tachyzoite replication in vitro and in vivo. Furthermore, both drugs induce bradyzoite formation and inhibit the reactivation of latent bradyzoites in vitro. To address whether the antiparasitic activities of SAL and GA involve host eIF2α phosphorylation, we infected mutant mouse embryonic fibroblast (MEF) cells incapable of phosphorylating eIF2α, which had no impact on the efficacies of SAL and GA against Toxoplasma infection. Our findings suggest that SAL and GA may serve as potential new drugs for the treatment of acute and chronic toxoplasmosis.

In order to produce a more potent replacement for trimethoprim (TMP) used as a therapy for Pneumocystis pneumonia and targets dihydrofolate reductase from Pneumocystis jirovecii (pjDHFR), it is necessary to understand the determinants of potency and selectivity against DHFR from the mammalian host and fungal pathogen cells. To this end, active site residues in human (h) DHFR were replaced with those from pjDHFR. Structural data are reported for two complexes of TMP with the double mutants Gln35Ser/Asn64Phe (Q35S/N64F) and Gln35Lys/Asn64Phe (Q35K/N64F) of hDHFR that unexpectedly show evidence for the binding of two molecules of TMP: one molecule that binds in the normal folate binding site and the second molecule that binds in a novel subpocket site such that the mutated residue Phe64 is involved in van der Waals contacts to the trimethoxyphenyl ring of the second TMP molecule. Kinetic data for the binding of TMP to hDHFR and pjDHFR reveal an 84-fold selectivity of TMP against pjDHFR (K(i) 49 nM) compared to hDHFR (K(i) 4093 nM). Two mutants that contain one substitution from pj--and one from the closely related Pneumocystis carinii DHFR (pcDHFR) (Q35K/N64F and Q35S/N64F) show K(i) values of 593 and 617 nM, respectively; these K(i) values are well above both the K(i) for pjDHFR and are similar to pcDHFR (Q35K/N64F and Q35S/N64F) (305nM). These results suggest that active site residues 35 and 64 play key roles in determining selectivity for pneumocystis DHFR, but that other residues contribute to the unique binding of inhibitors to these enzymes., (Copyright © 2011 Elsevier Inc. All rights reserved.)

While seeking a new host cell, obligate intracellular parasites, such as the protozoan Toxoplasma gondii, must be able to endure the stress of an extracellular environment. The mechanisms Toxoplasma use to remain viable while deprived of a host cell are not understood. We have previously shown that phosphorylation of Toxoplasma eukaryotic initiation factor-2α (TgIF2α) is a conserved response to stress. Here we report the characterization of Toxoplasma harboring a point mutation (S71A) in TgIF2α that prevents phosphorylation. Results show that TgIF2α phosphorylation is critical for parasite viability because the TgIF2α-S71A mutants are ill-equipped to cope with life outside the host cell. The TgIF2α-S71A mutants also showed a significant delay in producing acute toxoplasmosis in vivo. We conclude that the phosphorylation of TgIF2α plays a crucial role during the lytic cycle by ameliorating the stress of the extracellular environment while the parasite searches for a new host cell.

Twenty-one biguanide and dihydrotriazine derivatives were synthesized and evaluated as inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms: Pneumocystis carinii (pc), Toxoplasma gondii (tg), Mycobacterium avium (ma), and rat liver (rl). The most potent compound in the series was B2-07 with 12 nM activity against tgDHFR. The most striking observation was that B2-07 showed similar potency to trimetrexate, approximately 233-fold improved potency over trimethoprim and approximately 7-fold increased selectivity as compared to trimetrexate against tgDHFR. Molecular docking studies in the developed homology model of tgDHFR rationalized the observed potency of B2-07. This molecule can act as a good lead for further design of molecules with better selectivity and improved potency.

The present work deals with design, synthesis and biological evaluation of novel, diverse compounds as potential inhibitors of dihydrofolate reductase (DHFR) from opportunistic microorganisms; Pneumocystis carinii (pc), Toxoplasma gondii (tg) and Mycobacterium avium (ma). A set of 14 structurally diverse compounds were designed with varying key pharmacophoric features of DHFR inhibitors, bulky distal substitutions and different bridges joining the distal part and 2,4-diaminopyrimidine nucleus. The designed compounds were synthesized and evaluated in enzyme assay against pc, tg and ma DHFR. The rat liver (rl) DHFR was used as mammalian standard. As the next logical step of the project, flexible molecular docking studies were carried out to predict the binding modes of these compounds in pcDHFR active site and the obtained docked poses were post processed using MM-GBSA protocol for prediction of relative binding affinity. The predicted binding modes were able to rationalize the experimental results in most cases. Of particular interest, both the docking scores and MM-GBSA predicted Delta G(bind) were able to distinguish between the active and low active compounds. Furthermore, good correlation coefficient of 0.797 was obtained between the IC(50) values and MM-GBSA predicted Delta G(bind). Taken together, the current work provides not only a novel scaffold for further optimization of DHFR inhibitors but also an understanding of the specific interactions of inhibitors with DHFR and structural modifications that improve selectivity., ((c) 2010 Elsevier Ltd. All rights reserved.)

We report the development of CoMFA analysis models that correlate the 3D chemical structures of 80 compounds with 6-5 fused ring system synthesized in our laboratory and their inhibitory potencies against tgDHFR and rlDHFR. In addition to conventional CoMFA analysis, we used two routines available in the literature aimed at the optimization of CoMFA: all-orientation search (AOS) and cross-validated r(2)-guided region selection (q(2)-GRS) to further optimize the models. During this process, we identified a problem associated with q(2)-GRS routine and modified using two strategies. Thus, for the inhibitory activity against each enzyme (tgDHFR and rlDHFR), five CoMFA models were developed using the conventional CoMFA, AOS optimized CoMFA, the original q(2)-GRS optimized CoMFA and the modified q(2)-GRS optimized CoMFA using the first and the second strategy. In this study, we demonstrate that the modified q(2)-GRS routines are superior to the original routine. On the basis of the steric contour maps of the models, we designed four new compounds in the 2,4-diamino-5-methyl-6-phenylsulfanyl-substituted pyrrolo[2,3-d]pyrimidine series. As predicted, the new compounds were potent and selective inhibitors of tgDHFR. One of them, 2,4-diamino-5-methyl-6-(2',6'-dimethylphenylthio)pyrrolo[2,3-d]pyrimidine, is the first 6-5 fused ring system compound with nanomolar tgDHFR inhibitory activity. The HCl salt of this compound was also prepared to increase solubility. Both forms of the drug were tested in vivo in a Toxoplasma gondii infection mouse model. The results indicate that both forms were active with the HCl salt significantly more potent than the free base., (Copyright 2010 Elsevier Ltd. All rights reserved.)

A novel classical antifolate N-{4-[(2,4-diamino-5-methyl-furo[2,3-d]pyrimidin-6-yl)thio]-benzoyl}-l-glutamic acid 5 and 11 nonclassical antifolates 6-16 were designed, synthesized, and evaluated as inhibitors of dihydrofolate reductase (DHFR) and thymidylate synthase (TS). The nonclassical compounds 6-16 were synthesized from 20 via oxidative addition of substituted thiophenols using iodine. Peptide coupling of the intermediate acid 21 followed by saponification gave the classical analog 5. Compound 5 is the first example, to our knowledge, of a 2,4-diamino furo[2,3-d]pyrimidine classical antifolate that has inhibitory activity against both human DHFR and human TS. The classical analog 5 was a nanomolar inhibitor and remarkably selective inhibitor of Pneumocystis carinii DHFR and Mycobacterium avium DHFR at 263-fold and 2107-fold, respectively, compared to mammalian DHFR. The nonclassical analogs 6-16 were moderately potent against pathogen DHFR or TS. This study shows that the furo[2,3-d]pyrimidine scaffold is conducive to dual human DHFR-TS inhibitory activity and to high potency and selectivity for pathogen DHFR., (Copyright 2009 Elsevier Ltd. All rights reserved.)

To optimize dual receptor tyrosine kinase (RTK) and dihydrofolate reductase (DHFR) inhibition, the E- and Z-isomers of 5-[2-(2-methoxyphenyl)prop-1-en-1-yl]furo[2,3-d]pyrimidine-2,4-diamines (1a and 1b) were separated by HPLC and the X-ray crystal structures (2.0 and 1.4A, respectively) with mouse DHFR and NADPH as well as 1b with human DHFR (1.5A) were determined. The E- and Z-isomers adopt different binding modes when bound to mouse DHFR. A series of 2,4-diaminofuro[2,3-d]pyrimidines 2-13 were designed and synthesized using the X-ray crystal structures of 1a and 1b with DHFR to increase their DHFR inhibitory activity. Wittig reactions of appropriate 2-methoxyphenyl ketones with 2,4-diamino-6-chloromethyl furo[2,3-d]pyrimidine afforded the C8-C9 unsaturated compounds 2-7 and catalytic reduction gave the saturated 8-13. Homologation of the C9-methyl analog maintains DHFR inhibitory activity. In addition, inhibition of EGFR and PDGFR-beta were discovered for saturated C9-homologated analogs 9 and 10 that were absent in the saturated C9-methyl analogs.

To understand the role of specific active site residues in conferring selective dihydrofolate reductase (DHFR) inhibition from pathogenic organisms such as Pneumocystis carinii (pc) or Pneumocystis jirovecii (pj), the causative agent in AIDS pneumonia, it is necessary to evaluate the role of these residues in the human enzyme. We report the first kinetic parameters for DHFR from pjDHFR and pcDHFR with methotrexate (MTX), trimethoprim (TMP), and its potent analogue, PY957. We also report the mutagenesis and kinetic analysis of active site mutant proteins at positions 35 and 64 of human (h) DHFR and the crystal structure determinations of hDHFR ternary complexes of NADPH and PY957 with the wild-type DHFR enzyme, the single mutant protein, Gln35Lys, and two double mutant proteins, Gln35Ser/Asn64Ser and Gln35Ser/Asn64Phe. These substitutions place into human DHFR amino acids found at those sites in the opportunistic pathogens pcDHFR (Q35K/N64F) and pjDHFR (Q35S/N64S). The K(i) inhibition constant for PY957 showed greatest potency of the compound for the N64F single mutant protein (5.2 nM), followed by wild-type pcDHFR (K(i) 22 nM) and then wild-type hDHFR enzyme (K(i) 230 nM). Structural data reveal significant conformational changes in the binding interactions of PY957 in the hDHFR Q35S/N64F mutant protein complex compared to the other hDHFR mutant protein complexes and the pcDHFR ternary complex. The conformation of PY957 in the wild-type DHFR is similar to that observed for the single mutant protein. These data support the hypothesis that the enhanced selectivity of PY957 for pcDHFR is in part due to the contributions at positions 37 and 69 (pcDHFR numbering). This insight will help in the design of more selective inhibitors that target these opportunistic pathogens.

Recent reports of the slow-tight binding inhibition of bovine liver dihydrofolate reductase (bDHFR) in the presence of polyphenols isolated from green tea leaves has spurred renewed interest in the biochemical properties of bDHFR. Earlier studies were done with native bDHFR but in order to validate models of polyphenol binding to bDHFR, larger quantities of bDHFR are necessary to support structural studies. Bovine DHFR differs from its closest sequence homologue, murine DHFR, by 19 amino acids. To obtain the bDHFR cDNA, murineDHFR cDNA was transformed by a series of nested PCRs to reproduce the amino acid coding sequence for bovine DHFR. The bovine liver DHFR cDNA has an open reading frame of 561 base pairs encoding a protein of 187 amino acids that has a high level of conservation at the primary sequence level with other DHFR enzymes, and more so for the amino acid residues in the active site of the mammalian DHFR enzymes. Expression of the bovine DHFR cDNA in bacterial cells produced a stable recombinant protein with high enzymatic activity and kinetic properties similar to those previously reported for the native protein.

N9-substituted 2,4-diaminoquinazolines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). Reduction of commercially available 2,4-diamino-6-nitroquinazoline 14 with Raney nickel afforded 2,4,6-triaminoquinazoline 15. Reductive amination of 15 with the appropriate benzaldehydes or naphthaldehydes, followed by N9-alkylation, afforded the target compounds 5- 13. In the 2,5-dimethoxybenzylamino substituted quinazoline analogues, replacement of the N9-CH 3 group of 4 with the N9-C2H5 group of 8 resulted in a 9- and 8-fold increase in potency against pcDHFR and tgDHFR, respectively. The N9-C2H5 substituted compound 8 was highly potent, with IC50 values of 9.9 and 3.7 nM against pcDHFR and tgDHFR, respectively. N9-propyl and N9-cyclopropyl methyl substitutions did not afford further increases in potency. This study indicates that the N9-ethyl substitution is optimum for inhibitory activity against pcDHFR and tgDHFR for the 2,4-diaminoquinazolines. Selectivity was unaffected by N9 substitution.

Novel classical antifolates (3 and 4) and 17 nonclassical antifolates (11-27) were synthesized as antitumor and/or antiopportunistic infection agents. Intermediates for the synthesis of 3, 4, and 11-27 were 2,4-diamino-5-alkylsubstituted-7H-pyrrolo[2,3-d]pyrimidines, 31 and 38, prepared by a ring transformation/ring annulation sequence of 2-amino-3-cyano-4-alkyl furans to which various aryl thiols were attached at the 6-position via an oxidative addition reaction using I2. The condensation of alpha-hydroxy ketones with malonodinitrile afforded the furans. For the classical analogues 3 and 4, the ester precursors were deprotected, coupled with diethyl-L-glutamate, and saponified. Compounds 3 (IC50 = 60 nM) and 4 (IC50 = 90 nM) were potent inhibitors of human DHFR. Compound 3 inhibited tumor cells in culture with GI50 500-fold selectivity over human DHFR. Analogue 17 was 50-fold more potent than trimethoprim and about twice as selective against T. gondii DHFR.

Background and Methodology: Toxoplasma gondii causes substantial morbidity, mortality, and costs for healthcare in the developed and developing world. Current medicines are not well tolerated and cause hypersensitivity reactions. The dihydrotriazine JPC-2067-B (4, 6-diamino-1, 2-dihydro-2, 2-dimethyl-1-(3'(2-chloro-, 4-trifluoromethoxyphenoxy)propyloxy)-1, 3, 5-triazine), which inhibits dihydrofolate reductase (DHFR), is highly effective against Plasmodium falciparum, Plasmodium vivax, and apicomplexans related to T. gondii. JPC-2067-B is the primary metabolite of the orally active biguanide JPC-2056 1-(3'-(2-chloro-4-trifluoromethoxyphenyloxy)propyl oxy)- 5-isopropylbiguanide, which is being advanced to clinical trials for malaria. Efficacy of the prodrug JPC-2056 and the active metabolite JPC-2067-B against T. gondii and T. gondii DHFR as well as toxicity toward mammalian cells were tested., Principal Findings and Conclusions: Herein, we found that JPC-2067-B is highly effective against T. gondii. We demonstrate that JPC-2067-B inhibits T. gondii growth in culture (IC50 20 nM), inhibits the purified enzyme (IC50 6.5 nM), is more efficacious than pyrimethamine, and is cidal in vitro. JPC-2067-B administered parenterally and the orally administered pro-drug (JPC-2056) are also effective against T. gondii tachyzoites in vivo. A molecular model of T. gondii DHFR-TS complexed with JPC-2067-B was developed. We found that the three main parasite clonal types and isolates from South and Central America, the United States, Canada, China, and Sri Lanka have the same amino acid sequences preserving key binding sites for the triazine., Significance: JPC-2056/JPC-2067-B have potential to be more effective and possibly less toxic treatments for toxoplasmosis than currently available medicines.

Two boron-containing, ortho-icosahedral carborane lipophilic antifolates were synthesized, and the crystal structures of their ternary complexes with human dihydrofolate reductase (DHFR) and dihydronicotinamide adenine dinucleotide phosphate were determined. The compounds were screened for activity against DHFR from six sources (human, rat liver, Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and Lactobacillus casei) and showed good to modest activity against these enzymes. The compounds were also tested for antibacterial activity against L. casei, M. tuberculosis H37Ra, and three M. avium strains and for cytotoxic activity against seven different human tumor cell lines. Antibacterial and cytotoxic activity was modest, with one sample, the closo-carborane 4, showing about 10-fold greater activity. The less toxic nido-carborane 2 was also tested as a candidate for boron neutron capture therapy, but showed poor tumor retention and low selectivity ratios for boron distribution in tumor tissue versus normal tissue.

The classical antifolate N-{4-[(2,4-diamino-5-ethyl-7H-pyrrolo[2,3-d]pyrimidin-6-yl)sulfanyl]benzoyl}-l-glutamic acid (2) and 15 nonclassical analogues (3-17) were synthesized as potential dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. 5-Ethyl-7H-pyrrolo[2,3-d]pyrimidine-2,4-diamine (20) served as the key intermediate to which various aryl thiols and a heteroaryl thiol were appended at the 6-position via an oxidative addition reaction. The classical analogue 2 was synthesized by coupling the benzoic acid derivative 18 with diethyl l-glutamate followed by saponification. The classical compound 2 was an excellent inhibitor of human DHFR (IC50 = 66 nM) as well as a two digit nanomolar (<100 nM) inhibitor of the growth of several tumor cells in culture. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from two pathogens (Toxoplasma gondii and Mycobacterium avium) that cause opportunistic infections in patients with compromised immune systems.

We report that quinoline derivative MC1626, first described as an inhibitor of the histone acetyltransferase (HAT) GCN5, is active against the protozoan parasite Toxoplasma gondii in vitro. However, MC1626 does not inhibit Toxoplasma GCN5 HATs or reduce HAT-mediated activity; rather, this quinoline may target the plastid organelle called the apicoplast.

Six novel C9-methyl-5-substituted-2,4-diaminopyrrolo[2,3-d]pyrimidines 18-23 were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) and as anti-opportunistic agents. These compounds represent the only examples of 9-methyl substitution in the carbon-carbon bridge of 2,4-diaminopyrrolo[2,3-d]pyrimidines. The analogs 18-23 were synthesized in a concise eight-step procedure starting from the appropriate commercially available aromatic methyl ketones. The key step involved a Michael addition reaction of 2,4,6-triaminopyrimidine to the appropriate 1-nitroalkene, followed by ring closure of the nitro adducts via a Nef reaction. The compounds were evaluated as inhibitors of DHFR from Pneumocystis carinii (pc), Toxoplasma gondii (tg), Mycobacterium avium (ma) and rat liver (rl). The biological result indicated that some of these analogs are potent inhibitors of DHFR and some have selectivity for pathogen DHFR. Compound 23 was a two digit nanomolar inhibitor of tgDHFR with 9.6-fold selectivity for tgDHFR.

As part of a search for dihydrofolate reductase (DHFR) inhibitors combining the high potency of piritrexim (PTX) with the high antiparasitic vs mammalian selectivity of trimethoprim (TMP), the heretofore undescribed 2,4-diamino-6-(2',5'-disubstituted benzyl)pyrido[2,3-d]pyrimidines 6-14 with O-(omega-carboxyalkyl) or omega-carboxy-1-alkynyl groups on the benzyl moiety were synthesized and tested against Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium DHFR vs rat DHFR. Three N-(2,4-diaminopteridin-6-yl)methyl)-2'-(omega-carboxy-1-alkynyl)dibenz[b,f]azepines (19-21) were also synthesized and tested. The pyridopyrimidine with the best combination of potency and selectivity was 2,4-diamino-5-methyl-6-[2'-(5-carboxy-1-butynyl)-5'-methoxy]benzyl]pyrimidine (13), with an IC(50) value of 0.65 nM against P. carinii DHFR, 0.57 nM against M. avium DHFR, and 55 nM against rat DHFR. The potency of 13 against P. carinii DHFR was 20-fold greater than that of PTX (IC(50) = 13 nM), and its selectivity index (SI) relative to rat DHFR was 85, whereas PTX was nonselective. The activity of 13 against P. carinii DHFR was 20 000 times greater than that of TMP, with an SI of 96, whereas that of TMP was only 14. However 13 was no more potent than PTX against M. avium DHFR, and its SI was no better than that of TMP. Molecular modeling dynamics studies using compounds 10 and 13 indicated a slight binding preference for the latter, in qualitative agreement with the IC(50) data. Among the pteridines, the most potent against P. carinii DHFR and M. avium DHFR was the 2'-(5-carboxy-1-butynyl)dibenz[b,f]azepinyl derivative 20 (IC(50) = 2.9 nM), whereas the most selective was the 2'-(5-carboxy-1-pentynyl) analogue 21, with SI values of >100 against both P. carinii and M. avium DHFR relative to rat DHFR. The final compound, 2,4-diamino-5-[3'-(4-carboxy-1-butynyl)-4'-bromo-5'-methoxybenzyl]pyrimidine (22), was both potent and selective against M. avium DHFR (IC(50) = 0.47 nM, SI = 1300) but was not potent or selective against either P. carinii or T. gondii DHFR.

The opportunistic apicomplexan parasite Toxoplasma gondii damages fetuses in utero and threatens immunocompromised individuals. The toxicity associated with standard antitoxoplasmal therapies, which target the folate pathway, underscores the importance of examining alternative pharmacological strategies. Parasitic protozoa cannot synthesize purines de novo; consequently, targeting purine salvage enzymes is a plausible pharmacological strategy. Several enzymes critical to purine metabolism have been studied in T. gondii, but IMP dehydrogenase (IMPDH), which catalyzes the conversion of IMP to XMP, has yet to be characterized. Thus, we have cloned the gene encoding this enzyme in T. gondii. Northern blot analysis shows that two IMPDH transcripts are present in T. gondii tachyzoites. The larger transcript contains an open reading frame of 1,656 nucleotides whose deduced protein sequence consists of 551 amino acids (TgIMPDH). The shorter transcript is an alternative splice product that generates a 371-amino-acid protein lacking the active-site flap (TgIMPDH-S). When TgIMPDH is expressed as a recombinant protein fused to a FLAG tag, the fusion protein localizes to the parasite cytoplasm. Immunoprecipitation with anti-FLAG was employed to purify recombinant TgIMPDH, which converts IMP to XMP as expected. Mycophenolic acid is an uncompetitive inhibitor relative to NAD+, with a intercept inhibition constant (Kii) of 0.03+/-0.004 microM. Tiazofurin and its seleno analog were not inhibitory to the purified enzyme, but adenine dinucleotide analogs such as TAD and the nonhydrolyzable beta-methylene derivatives of TAD or SAD were inhibitory, with Kii values 13- to 60-fold higher than that of mycophenolic acid.

Pneumonia caused by Pneumocystis jirovecii is still a major opportunistic infection among patients with AIDS. This opportunitistic pathogen is susceptible to therapy with inhibitors of the enzyme dihydrofolate reductase (DHFR) that target cell growth. Recent studies have shown that recombinant human-derived Pneumocystis DHFR (pDHFR) differs from rat-derived pDHFR by 38% in amino acid sequence. However, characterization of drug susceptibility, kinetics, and the three-dimensional structure of human-derived pDHFR has been hampered by the limited availability of purified material. The present study was undertaken to develop procedures to prepare sufficient enzyme for structure/function studies. Protein yield was limited when human-derived pDHFR was expressed in Escherichia coli using a pET28a(+) vector with an N-terminal His-tag for the 25 kDa protein. Therefore, the protein was expressed in Sf21 insect cells by baculovirus infection. The soluble enzyme was purified from cell lysates via Ni-chelated chromatographic columns, yielding about 5.1 mg of human-derived pDHFR fusion protein per liter of Sf21 culture. The purified protein had the expected mass as determined from Western blots with antibody for the N-terminal His-tag. This His-tagged recombinant DHFR from human-derived Pneumocystis was catalytically active and demonstrated kinetics similar to the recombinant enzyme from rat-derived Pneumocystis. The present studies for production of soluble human-derived pDHFR indicated that the baculovirus expression system supported production of significantly purer catalytically active enzyme in higher yields than that expressed in bacterial cultures. These protocols now make it possible to facilitate screening of antifolates with selectivity for human-derived pDHFR and to determine its three-dimensional structure.

2,4-diamino-5-methyl-6-(substituted-phenyl)thiopyrrolo[2,3-d]pyrimidines 4-11 were synthesized as dihydrofolate reductase (DHFR) inhibitors against opportunistic pathogens that afflict patients with AIDS. Synthesis was achieved from 2,4-diamino-5-methypyrrolo[2,3-d]pyrimidine and substituted phenylthiols under modified conditions reported by Gangjee et al. Some of these compounds were potent and selective against DHFR from both Toxoplasma gondii and Mycobacterium avium compared to mammalian DHFR. Compound 11 with a 1-naphthyl substituent is 16-fold more potent and equally selective against Toxoplasma gondii DHFR as the clinically used trimethoprim.

Six previously undescribed N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepines with water-solubilizing O-carboxyalkyloxy or O-carboxybenzyloxy side chains at the 2'-position were synthesized and compared with trimethoprim (TMP) and piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three of the opportunistic organisms known to cause significant morbidity and mortality in patients with AIDS and other disorders of the immune system. The ability of the new analogues to inhibit reduction of dihydrofolate to tetrahydrofolate by Pc, Tg, Ma, and rat DHFR was determined, and the selectivity index (SI) was calculated from the ratio IC(50)(rat DHFR)/IC(50)(Pc, Tg, or Ma DHFR). The IC(50) values of the 2'-O-carboxypropyl analogue (10), with SI values in parentheses, were 1.1 nM (1300) against Pc DHFR, 9.9 nM (120) against Tg DHFR, and 2.0 nM (600) against Ma DHFR. The corresponding values for the 2'-O-(4-carboxybenzyloxy) analogue (12) were 1.0 nM (560), 22 nM (21), and 0.75 nM (630). By comparison, the IC(50) and SI values for TMP were Pc, 13 000 nM (14); Tg, 2800 nM (65); and Ma, 300 nM (610). For the prototypical potent but nonselective inhibitors PTX and TMX, respectively, these values were Pc, 13 nM (0.26) and 47 nM (0.17); Tg, 4.3 nM (0.76) and 16 nM (0.50); Ma, 0.61 nM (5.4) and 1.5 nM (5.3). Thus 10 and 12 met the criterion for DHFR inhibitors that combine the high selectivity of TMP with the high potency of PTX and TMX.

A series of dihydrofolate reductase (DHFR) inhibitors, where the methylenamino-bridge of non-classical inhibitors was replaced with an ester function, have been prepared as potential soft drugs intended for inhalation against Pneumocystis carinii pneumonia (PCP). Several of the new ester-based inhibitors that should serve as good substrates for the ubiquitous esterases and possibly constitute safer alternatives to metabolically stable DHFR inhibitors administered orally, were found to be potent inhibitors of P. carinii DHFR (pcDHFR). Although the objectives of the present program is to achieve a favorable toxicity profile by applying the soft drug concept, a high preference for inhibition of the fungal DHFR versus the mammalian DHFR is still desirable to suppress host toxicity at the site of administration. Compounds with a slight preference for the fungal enzyme were identified. The selection of the target compounds for synthesis was partly guided by an automated docking and scoring procedure as well as molecular dynamics simulations. The modest selectivity of the synthesized inhibitors was reasonably well predicted, although a correct ranking of the relative affinities was not successful in all cases.

2,4-Diamino-5-[3',4'-dimethoxy-5'-(5-carboxy-1-pentynyl)]benzylpyrimidine (6) and 2,4-diamino-5-[3',4'-dimethoxy-5'-(4-carboxyphenylethynyl)benzylpyrimidine (7) were synthesized from 2,4-diamino-5-(5'-iodo-3',4'-dimethoxybenzyl)pyrimidine (9) via a Sonogashira reaction with appropriate acetylenic esters followed by saponification, and were tested as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat in comparison with the widely used antibacterial agent 2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine (trimethoprim, TMP). The selectivity index (SI) for each compound was calculated by dividing its 50% inhibitory concentration (IC(50)) against rat DHFR by its IC(50) against Pc, Tg, or Ma DHFR. The IC(50) of 6 against Pc DHFR was 1.0 nM, with an SI of 5000. Compound 7 had an IC(50) of 8.2 nM against Ma DHFR, with an SI of 11000. By comparison, the IC(50) of TMP was 12000 nM against Pc, 300 nM against Ma, and 180000 against rat DHFR. The potency and selectivity values of 6 and 7 were not as high against Tg as they were against Pc or Ma DHFR, but nonetheless exceeded those of TMP. Because of the outstanding selectivity of 6 against Pc and of 7 against Ma DHFR, these novel analogues may be viewed as promising leads for further structure-activity optimization.

Structural data are reported for the first examples of the tetrahydroquinazoline antifolate (6R,6S)-2,4-diamino-6-(1-indolinomethyl)-5,6,7,8-tetrahydroquinazoline (1) and its trimethoxy analogue (6R,6S)-2,4-diamino-6-(3',4',5'-trimethoxybenzyl)-5,6,7,8-tetrahydroquinazoline (2) as inhibitor complexes with dihydrofolate reductase (DHFR) from human (hDHFR) and Pneumocystis carinii (pcDHFR) sources. The indoline analogue (1) was crystallized as ternary complexes with NADPH and hDHFR (1.9 A resolution) and pcDHFR (2.3 A resolution), while the trimethoxy quinazoline analogue (2) was crystallized as a binary complex with hDHFR in two polymorphic rhombohedral R3 lattices: R3(1) to 1.8 A resolution and R3(2) to 2.0 A resolution. Structural analysis of these potent and selective DHFR-inhibitor complexes revealed preferential binding of the 6S-equatorial isomer in each structure. This configuration is similar to that of the natural tetrahydrofolate substrate; that is, 6S. These data also show that in both the hDHFR and pcDHFR ternary complexes with (1) the indoline ring is partially disordered, with two static conformations that differ between structures. These conformers also differ from that observed for the trimethoxybenzyl ring of tetrahydroquinazoline (2). There is also a correlation between the disorder of the flexible loop 23 and the disorder of the cofactor nicotinamide ribose ring in the pcDHFR-NADPH-(1) ternary complex. Comparison of the Toxoplasma gondii DHFR (tgDHFR) sequence with those of other DHFRs provides insight into the role of sequence and conformation in inhibitor-binding preferences which may aid in the design of novel antifolates with specific DHFR selectivity.

In a continuing effort to design small-molecule inhibitors of dihydrofolate reductase (DHFR) that combine the enzyme-binding selectivity of 2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine (trimethoprim, TMP) with the potency of 2,4-diamino-5-methyl-6-(2',5'-dimethoxybenzyl)pyrido[2,3-d]pyrimidine (piritrexim, PTX), seven previously undescribed 2,4-diamino-5-[2'-methoxy-5'-(substituted benzyl)]pyrimidines were synthesized in which the substituent at the 5'-position was a carboxyphenyl group linked to the benzyl moiety by a bridge of two or four atoms in length. The new analogues were all obtained from 2,4-diamino-5-(5'-iodo-2'-methoxybenzyl)pyrimidine via a Sonogashira reaction, followed, where appropriate, by catalytic hydrogenation. The new analogues were tested as inhibitors of DHFR from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three life-threatening pathogens often found in AIDS patients and individuals whose immune system is impaired as a result of treatment with immunosuppressive chemotherapy or radiation. The selectivity index (SI) of each compound was obtained by dividing its 50% inhibitory concentration (IC(50)) against Pc, Tg, or Ma DHFR by its IC(50) against rat DHFR. 2,4-Diamino-[2'-methoxy-5'-(3-carboxyphenyl)ethynylbenzyl]pyrimidine (28), with an IC(50) of 23 nM and an SI of 28 in the Pc DHFR assay, had about the same potency as PTX and was 520 times more potent than TMP. As an inhibitor of Tg DHFR, 28 had an IC(50) of 5.5 nM (510-fold lower than that of TMP and similar to that of PTX) and an SI value of 120 (2-fold better than TMP and vastly superior to PTX). Against Ma DHFR, 28 had IC(50) and SI values of 1.5 nM and 430, respectively, compared with 300 nM and 610 for TMP. Although it had 2.5-fold lower potency than 28 against Ma DHFR (IC(50) = 3.7 nM) and was substantially weaker against Pc and Tg DHFR, 2,4-diamino-[2'-methoxy-5'-(4-carboxyphenyl)ethynylbenzyl]pyrimidine (29), with the carboxy group at the para rather than the meta position, displayed 2200-fold selectivity against the Ma enzyme and was the most selective 2,4-diamino-5-(5'-substituted benzyl)pyrimidine inhibitor of this enzyme we have encountered to date. Additional bioassay data for these compounds are also reported.

A series of 2,4-diamino-6-(arylaminomethyl)pyrido[2,3-d]pyrimidines were synthesized and evaluated as inhibitors of Pneumocystis carinii (pc), Toxoplasma gondii (tg), and rat liver (rl) dihydrofolate reductase (DHFR) and as inhibitors of the growth of tumor cell lines in culture. Compounds 4-15 were designed as part of a continuing effort to examine the effects of substitutions at the 5-position, in the two-atom bridge, and in the side chain phenyl ring on structure-activity/selectivity relationships of 2,4-diaminopyrido[2,3-d]pyrimidines against a variety of DHFRs. Reductive amination of the common intermediate 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile 16 with the appropriate anilines afforded the target compounds 4-12. Nucleophilic substitution or reductive methylation afforded the N10-methyl target compounds 13-15. As predicted, compounds 4-15 were, in general, less potent against all three DHFRs compared to the corresponding 2,4-diamino-5-methyl analogues previously reported; however, the greater decrease in potency against rlDHFR compared to pcDHFR and tgDHFR resulted in appreciable selectivity toward pathogenic DHFRs from different pathogens. The 2',5'-dichloro analogue 8 showed selectivity ratios (IC(50) against rlDHFR/IC(50) against pcDHFR or tgDHFR) of 15.7 and 23 for pcDHFR and tgDHFR, respectively. Thus, the selectivity of 8 for pcDHFR is higher than the first line clinical agent trimethoprim (TMP). In a P. carinii cell culture study, analogue 8 exhibited 88% cell growth inhibition at a concentration of 10 muM and afforded marginal effects in an in vivo study in the T. gondii mouse model. Selected compounds were evaluated in the National Cancer Institute (NCI) in vitro preclinical antitumor screening program and inhibited the growth of tumor cells in culture at micromolar to submicromolar concentrations and were selected for evaluation in a NCI in vivo hollow fiber assay.

A series of 6-aralkyl substituted 2,4-diaminothieno[2,3-d]pyrimidines in which the 6-aryl group is separated from the thieno[2,3-d]pyrimidine ring by two to five methylene groups were synthesized and studied as inhibitors of dihydrofolate reductase from Pneumocystis carinii, Toxoplasma gondii, Mycobacterium avium, and rat liver. Compounds in which the thieno[2,3-d]pyrimidine ring is separated from the 6-aryl substituent by three methylene groups were the most potent inhibitors of the series (with IC(50) values ranging from 0.24 and 11.0 microM) but those with two methylene groups between the aromatic rings were the most selective agents.

We have identified and mapped a gene in Toxoplasma gondii that encodes a homologue of SRCAP (Snf2-related CBP activator protein), a member of the SNF/SWI family of chromatin remodeling factors. The genomic locus (TgSRCAP) is present as a single copy and contains 16 introns. The predicted cDNA contains an open reading frame of 8,775 bp and encodes a protein of 2,924 amino acids. We have identified additional SRCAP-like sequences in Apicomplexa for comparison by screening genomic databases. An analysis of SRCAP homologues between species reveals signature features that may be indicative of SRCAP members. Expression of mRNA encoding TgSRCAP is upregulated when tachyzoite (invasive form) parasites are induced to differentiate into bradyzoites (encysted form) in vitro. Recombinant TgSRCAP protein is functionally equivalent to the human homologue, being capable of increasing transcription mediated by CREB.

As part of an ongoing effort to discover novel small-molecule antifolates combining the enzyme-binding species selectivity of trimethoprim (TMP) with the potency of piritrexim (PTX), 10 previously unreported 2,4-diamino-5-(2'-methoxy-5'-substituted)benzylpyrimidines (2-11) containing a carboxyl group at the distal end of the 5'-substituent were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three of the opportunistic pathogens frequently responsible for life-threatening illness in people with impaired immune systems as a result of HIV infection or immunosuppressive chemotherapy. The selectivity index of DHFR inhibition was evaluated by comparing the potency of each compound against the parasite enzymes with its potency against rat liver DHFR. 2,4-Diamino-5-[5'-(5-carboxy-1-pentynyl)-2'-methoxybenzyl]pyrimidine (3) inhibited Pc DHFR with a selectivity index of 79 and was 430 times more potent than TMP. 2,4-Diamino-5-[5'-(4-carboxy-1-butynyl)-2'-methoxybenzyl]pyrimidine (2), with one less carbon than 3 in the side chain, had a selectivity index of 910 against Ma DHFR and was 43 times more potent than TMP. 2,4-Diamino-5-[5'-(5-carboxypentyl)-2'-methoxybenzyl]pyrimidine (6) had a selectivity index of 490 against Tg DHFR and was 320 times more potent than TMP. 2,4-Diamino-5-[5'-(6-carboxy-1-hexynyl)-2'-methoxybenzyl]pyrimidine (4), with one more carbon than 3, was less potent against all three of the parasite enzymes than either 3 or 6 and also had a lower selectivity index than 3 against the Pc enzyme. However, 4 was the only member of the series with a selectivity index of >300 against both Tg and Ma DHFR. Given that PTX is at least 10 times more potent against rat DHFR than against P. carinii or T. gondii DHFR and that the selectivity index of several of the compounds matches or exceeds that of TMP as well as PTX, our results suggest that it may be possible to develop clinically useful nonclassical antifolates that are both potent and selective against the major opportunistic pathogens of AIDS.

A concise new route allowing easy access to five previously unreported 2,4-diamino-6-(substituted benzyl)pyrido[2,3-d]pyrimidines (2a-e) was developed, involving condensation of 2,4-dipivaloylamino-5-bromopyrido[2,3-d]pyrimidine (6) with an organozinc halide in the presence of a catalytic amount of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II).CH(2)Cl(2), followed by removal of the pivaloyl groups with base. Also prepared via a scheme based on the Taylor ring expansion/ring annulation synthesis were three heretofore undescribed 2,4-diamino-5-(substituted benzyl)-7H-pyrrolo[2,3-d]pyrimidines (3b-c). Standard spectrophotometric assays were used to compare the ability of 2a-e and 3b-c to inhibit dihydrofolate reductase (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium, three examples of opportunistic pathogens to which AIDS patients are highly vulnerable because of their immunocompromised state. For comparison, 13 previously untested 2,4-diamino-6-(substituted benzyl)quinazolines (17a-m) were also evaluated as inhibitors of these enzymes, as well as the enzyme from rat liver. None of the quinazolines or pyridopyrimidines tested was more potent against the P. carinii enzyme than the structurally related reference compound piritrexim (1), and none showed selectivity for the P. carinii enzyme over the rat enzyme. One of the pyridopyrimidines (2c) showed 10-fold selectivity for T. gondii versus rat DHFR, and two of them (2b, 2c) showed selectivity for the M. avium enzyme. However, this gain in species selectivity was achieved at the cost of decreased in potency, as has been noted with many other lipophilic DHFR inhibitors.

As part of a research effort to improve the quality of current chemotherapy of Pneumocystis carinii pneumonia, we report a structure-based design project to optimise activity, species selectivity and pharmaceutical properties of the triazenyl-pyrimethamine TAB (4) (IC(50)=0.17 microM; rat liver DHFR IC(50)/P. carinii DHFR IC(50)=114). This has led us to design, synthesise and evaluate four new series of pyrimethamine derivatives bearing triazole, triazolium, triazinium and amino moieties at the 3'-position of the p-chlorophenyl ring. Such stabilised 'triazene' derivatives address the potentially compromised pharmaceutical profile of TAB and the 3'-amine substituted agents afford conformationally flexible substitutes. The benzylamino-pyrimethamine derivative (24a) (IC(50)=0.12 microM, rat liver DHFR IC(50)/P. carinii DHFR IC(50): 5.26) was the most potent and the only P. carinii-selective antifolate of the new series.

Structural studies of two ternary complexes of Pneumocystis carinii dihydrofolate reductase (pcDHFR) with the cofactor NADP(+) and potent antifolates, the N9-C10 reversed-bridge inhibitor 2,4-diamino-6-[N-(2',5'-dimethoxybenzyl)-N-methylamino]quinazoline (1) and its 3',5'-dimethoxypyrido[2,3-d]pyrimidine analog (2), were carried out. Data for the monoclinic crystals were refined to 1.90 A resolution for the complex with (1) (R = 0.178) and to 2.1 A resolution for the complex with (2) (R = 0.193). The effect of the N9-C10 reversed-bridge geometry is to distort the bridge from coplanarity with the pyrido[2,3-d]pyrimidine or quinazoline ring system and to twist the C10 methylene conformation toward a gauche conformation. This change also influences the conformation of the methoxybenzyl ring, moving it away from a trans position. This change places the 5'-methoxy group deeper within the hydrophobic pocket made by Ile65, Pro66 and Phe69 of the pcDHFR active site. These results also revealed the first observation of an unusual conformation for the reversed-bridge geometry (C5-C6-N9-C10 torsion angle) in antifolate (2). The electron density is consistent with the presence of two models (conformers 2-1 and 2-2) that result from inversion of the geometry at N9. The four examples of N9-C10 reversed-bridge antifolates cluster in two conformations, with the structure of quinazoline (1) similar to that previously reported for its 2',5'-dimethoxypyrido[2,3-d]pyrimidine analog (3). The two conformers of (2) differ from these and each other by a twisted-bridge geometry that results in the dimethoxybenzyl ring occupying the same conformational space. Conformer 2-2 also has the N9-C10 reversed bridge perpendicular to the pyrido[2,3-d]pyrimidine plane, in contrast to the gauche-trans conformation normally observed. As a result of these changes, the N9 methyl probes conformational space in the active site not normally occupied by antifolate structures. The N9 methyl of conformer 2-2 makes close contacts to the conserved Leu25 as well as the hydroxyl O atoms of the nicotinamide ribose and Ser64, whereas the other three reversed-bridge conformers make weak hydrophobic contacts with Ile123, Thr61 and Ile65. These antifolates are ten times more selective for pcDHFR than the C9-N10 bridge parent trimetrexate. However, pyrido[2,3-d]pyrimidines (2) and (3) are three times more selective for pcDHFR than quinazoline (1) is for rat liver DHFR. These data suggest that the loss of hydrogen-bonding interactions with N8 is more important to potency than the interactions of the methoxybenzyl substituents.

Structural data are reported for N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepine (PT653), an example of structure-based inhibitor design with 21-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR) relative to rat liver dihydrofolate reductase (rlDHFR). These data test the hypothesis that 2,4-diaminopteridines with a bulky N,N-diarylaminomethyl side chain at the 6-position could fit better into the larger active site of pcDHFR than into that of mammalian DHFR. The crystal structure of the ternary complex of NADPH, PT653 and pcDHFR, refined to 2.4 A resolution, reveals that PT653 binds in a different orientation than predicted from modeling studies reported previously [Rosowsky et al. (1999), J. Med. Chem. 42, 4853-4860]. These crystal data show that the pteridine-ring plane is tilted compared with that observed in the crystal structure of the pcDHFR methotrexate (MTX) NADPH ternary complex used as a template to model PT653 binding. Also, as a result of this tilt, the dibenzoazepine ring is bound deeper into the p-aminobenzoyl folate binding pocket of pcDHFR, thereby relieving close intermolecular contacts predicted from the modeling data. By far the most significant structural change, but more subtle in magnitude, is the ligand-induced conformational shift of 1.2 A away from the inhibitor of residues 61-66 in helix C. The other major effect is the unwinding of the short helical segment involving loop 47 which has a different conformation to that observed in other pcDHFR complexes [Cody et al. (1999), Biochemistry, 38, 4303-4312]. The favorable pcDHFR selectivity of PT653 could be a result of ligand-induced fit of the large hydrophobic dibenzazepine ring which occupies regions of the enzyme active site not probed by other antifolates and which take advantage of sequence and conformational differences between the structures of human and pcDHFR. These data suggest that such hydrophobic analogs could be used as lead compounds in the design of more pcDHFR-selective antifolates. Enzyme inhibition data also show that PT653 is 102-fold selective for Toxoplasma gondii (tg) DHFR relative to rlDHFR. Homology-modeling studies of the tgDHFR structure suggest that differences in ligand-binding orientation and enzyme sequence could influence the enhanced selectivity of PT653 for tgDHFR.

A series of previously undescribed 2,4-diamino-5-[2-methoxy-5-alkoxybenzyl]pyrimidines (3a-e) and 2,4-diamino-5-[2-methoxy-5-(omega-carboxyalkyloxy)benzyl]pyrimidines (3f-k) with up to eight CH2 groups in the alkoxy or omega-carboxyalkyloxy side chain were synthesized and tested for the ability to inhibit partially purified dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat liver in comparison with two standard inhibitors, trimethoprim (1) and piritrexim (2). The latter drug is known to be extremely potent but shows a marked preference for binding to mammalian DHFR, whereas the former is very selective for the parasite enzymes but is a much weaker inhibitor. The underlying strategy for the synthesis of compounds 3a-k was that a hybrid structure embodying some features of both 1 and 2 might possess a more favorable combination of potency and selectivity than either parent drug. The choice of analogues 3f-k was based on the idea that the acidic omega-carboxyl group might interact preferentially with a basic center in the active site of DHFR from any of the parasite species relative to the active site of mammalian DHFR. In addition, the omega-carboxyl group was expected to improve water solubility relative to 1 or 2. In standardized spectrophotometric assays with dihydrofolate as the substrate and NADPH as the cofactor, 2,4-diamino-5-[(2-methoxy-4-carboxybutyloxy)benzyl]pyrimidine (3g) inhibited Pc DHFR with an IC(50) of 0.049 microM and rat DHFR with IC(50) of 3.9 microM. Its potency against Pc DHFR was 140-fold greater than that of 1 and close to that of 2, and its selectivity index, defined as the ratio IC(50)(rat liver)/IC(50)(P. carinii), was 8-fold higher than that of 1 and >10(4)-fold higher than that of 2. Although it was less potent and less selective against Tg than Pc DHFR, it was very potent as well as highly selective against Ma DHFR, with an IC(50) of 0.0058 microM and an IC(50)(rat liver)/IC(50)(M. avium) ratio of >600. Because of this favorable combination of potency and selectivity relative to 1 and 2, compound 3g may be viewed as a promising lead in the search for new antifolates with potential clinical activity against P. carinii and other opportunistic pathogens in patients with AIDS.

Six 2,4-diaminopyrido[2,3-d]pyrimidines with a 6-methylthio bridge to an aryl group were synthesized and biologically evaluated as inhibitors of Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase (DHFR). The syntheses of analogues 3-8 were achieved by nucleophilic displacement of 2,4-diamino-6-bromomethylpyrido[2,3-d]pyrimidine 14 with various arylthiols. The alpha-naphthyl analogue 4 showed the highest selectivity ratios of 3.6 and 8.7 against pcDHFR and tgDHFR, respectively, versus rat liver (rl) DHFR. The beta-naphthyl analogue 5 exhibited the highest potency within the series with an IC(50) value against pcDHFR and tgDHFR of 0.17 and 0.09 microM, respectively. Analogue 4 was evaluated for in vitro antimycobacterium activity and was shown to inhibit the growth of Mycobacterium tuberculosis H(37)Rv cells by 58% at a concentration of 6.25 microg/mL.

The synthesis, anti-Pneumocystis carinii activity and DNA binding properties of eight new N,N'-bis[4-(N-alkylamidino)phenyl]homopiperazines are reported. Compounds 2 and 8 were the most potent and caused about 70% inhibition of Pneumocystis carinii growth in a cell culture model at 1 microM concentrations.

While assays of many antifolate inhibitors for dihydrofolate reductase (DHFR) have been performed using rat DHFR as a target, neither the sequence nor the structure of rat DHFR is known. Here, we report the isolation of the rat DHFR gene through screening of a rat liver cDNA library. The rat liver DHFR gene has an open reading frame of 561 bp encoding a protein of 187 amino acids. Comparisons of the rat enzyme with those from other species indicate a high level of conservation at the primary sequence level and more so for the amino acid residues comprising the active site of the enzyme. Expression of the rat DHFR gene in bacteria produced a recombinant protein with high enzymatic activity. The recombinant protein also paralleled the human enzyme with respect to the inhibition by most of the antifolates tested with PT652 and PT653 showing a reversal in their patterns. Our results indicated that rat DHFR can be used as a model to study antifolate compounds as potential drug candidates. However, variations between rat and human DHFR enzymes, coupled with unique features in the inhibitors, could lead to the observed differences in enzyme sensitivity and selectivity.

The triazenyl-pyrimethamine derivative 3a (TAB), a potent and selective inhibitor of Pneumocystis carinii DHFR, was selected as the starting point for a lead optimization study. Molecular modeling studies, corroborated by a recent crystal structure determination of the ternary complex of P. carinii DHFR--NADPH bound to TAB, predicted that modifications to the acetoxy residue of the lead inhibitor could exploit binding opportunities in the vicinity of an active site pocket bounded by residues Ile33, Lys37, and Leu72. Substitutions in the benzyl moiety with electron-donating and electron-withdrawing groups were predicted to probe face-edge interactions with amino acid Phe69 unique to the P. carinii enzyme. New triazenes 10a--v and 12a--f were prepared by coupling the diazonium tetrafluoroborate salt 6b of aminopyrimethamine with substituted benzylamines or phenethylamines. The most potent of the new inhibitors against P. carinii DHFR was the naphthylmethyl-substituted triazene 10t (IC(50): 0.053 microM), but a more substantial increase in potency against the rat liver DHFR led to a reduction in selectivity (ratio rat liver DHFR IC(50)/P. carinii DHFR IC(50): 5.36) compared to the original lead structure 3a (ratio rat liver DHFR IC(50)/P. carinii DHFR IC(50): 114).

A series of lipophilic soft drugs structurally related to the nonclassical dihydrofolate reductase (DHFR) inhibitors trimetrexate and piritrexim have been designed, synthesized, and evaluated in DHFR assays, with special emphasis on the inhibition of P. carinii DHFR. The best inhibitors, encompassing an ester bond in the bridge connecting the two aromatic systems, were approximately 10 times less potent than trimetrexate and piritrexim. The metabolites were designed to be poor inhibitors. Furthermore, molecular dynamics simulations of three ligands in complex with DHFR from Pneumocystis carinii and from the human enzyme were conducted in order to better understand the factors determining the selectivity. A correct ranking of the relative inhibition of DHFR was achieved utilizing the linear interaction energy method. The soft drugs are intended for local administration. One representative ester was selected for a pharmacokinetic study in rats where it was found to undergo fast metabolic degradation to the predicted inactive metabolites.

Classical and nonclassical isosteric C8-N9 bridged analogues of the multitargeted antifolate LY231514 were synthesized as inhibitors of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and as antitumor and antiopportunistic infection agents. The syntheses of the analogues were accomplished by reductive amination of the appropriate anilines with 2-amino-4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine (28) followed by saponification of the ethyl esters, for the classical analogue 6. The N9-methyl analogues were obtained from the N9-H precursors by reductive methylation. In general, the nonclassical compounds 7-17 were similar in potency to TMP against Toxoplasma gondii DHFR, with selectivity ratios greater than 38 and 21 for 11 and 16, respectively. These compounds were poor inhibitors of Pneumocystis carinii DHFR and rat liver DHFR. The nonclassical analogues were also inactive against TS. The classical analogue 6 was a marginal inhibitor of isolated human TS (IC50 = 46 microM) and of human DHFR (IC50 = 10 microM), however, it was a potent inhibitor of the growth of two human head and neck squamous cell carcinoma cell lines and of CCRF-CEM human lymphoblastic leukemia cells in culture and was similar to LY231514 against ZR-75-1 human breast carcinoma cell line. Evaluation of 6 against MTX-resistant sublines indicated that DHFR is not the major target of 6. Metabolite protection studies of the growth inhibitory activity of 6 suggest that TS is a major target of this drug and that polyglutamyl forms of 6 may serve as the intracellular TS inhibitors. These studies also suggest that 6 has a site of action in addition to sites in the folate pathway.