Carinii Dihydrofolate Reductase Research Articles

Quantitative structure–activity relationships of dihydrofolatereductase inhibitors

Pneumocystis carinii is a nonpathogenic fungus found in respiratory tract of healthy humans. It is a major cause of death in AIDS patients. P. carinii dihydrofolatereductase inhibitors such as trimetrexate are currently used to treat P. carinii pneumonia. These drugs have several adverse side effects and, therefore, there is a critical need for the identification of novel dihydrofolatereductase inhibitors. In this work, quantitative structure – activity relationship models were derived for 395 stucturally diverse P. carinii dihydrofolatereductase inhibitors. A wide variety of molecular descriptors belonging to various structural properties were calculated for each molecule. Both linear (multiple linear regression) and non-linear (generalized regression neural network) models were developed to link the calculated descriptors as independent variables to their reported biological activity as dependent variable. Different variable transformation was performed on the both dependent and independent variables to obtain better multiple linear regression models. At first glance, it seemed applying variable transformation on the dependent variable was necessary to improve quality of the quantitative structure–activity relationships models because it made the model more predictive. Upon further inspection, the results were not necessarily surprising. In the other words, back-transforming predictions did not give unbiased estimations of pIC50. At the final trial, generalized neural network was employed to develop predictive quantitative structure–activity relationships model.

Structure-activity correlations for three pyrido[2,3-d]pyrimidine antifolates binding to human and Pneumocystis carinii dihydrofolate reductase.

To further define the interactions that enhance the selectivity of binding and to directly compare the binding of the most potent analogue {N(6)-methyl-N(6)-(3,4,5-trifluorophenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine; compound 26} in the series of bicyclic pyrido[2,3-d]pyrimidine analogues of piritrexim (PTX) with native human (h), Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) dihydrofolate reductase (DHFR) enzymes, the crystal structures of hDHFR complexed with N(6)-methyl-N(6)-(4-isopropylphenyl)pyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 22), of hDHFR complexed with compound 26 and of pcDHFR complexed with N(6)-methyl-N(6)-1-naphthylpyrido[2,3-d]pyrimidine-2,4,6-triamine (compound 24) are reported as ternary complexes with NADPH. This series of bicyclic pyrido[2,3-d]pyrimidines were designed in which there was a transposition of the 5-methyl group of PTX to the N9 position of the pyrido[2,3-d]pyrimidine. It was hypothesized that the N9-methyl group would preferentially interact with Ile123 of pcDHFR (and Ile123 of pjDHFR), but not with the shorter Val115 in hDHFR. Structure-activity data for this series of antifolates revealed that a trifluoro derivative (26) was the most selective against pjDHFR compared with mammalian DHFR (h/pj = 35.7). Structural data for the hDHFR-26 complex revealed that 26 binds in a different conformation from that observed in the pcDHFR-26 complex. In the hDHFR-26 complex the trifluorophenyl ring of 26 occupies a position near the cofactor-binding site, with close intermolecular contacts with Asp21, Ser59 and Ile60, whereas this ring in the pcDHFR-26 complex is positioned away from the cofactor site and near Ile65, with weaker contacts with Ile65, Phe69 and Ile123. Comparison of the intermolecular contacts between the N9-methyl group with Val115/Ile123 validates the hypothesis that the N9-methyl substituent preferentially interacts with Ile123 compared with Val115 of hDHFR, as the weaker contact with Val115 in the hDHFR structure is consistent with its weaker binding affinity compared with pcDHFR. The results for the structures of hDHFR-22 and pcDHFR-24 show that their inhibitor-binding orientation is similar to that observed in pcDHFR-26 and the pcDHFR variant (F69N) reported previously. The naphthyl moiety of 24 makes several intermolecular contacts with the active-site residues in pcDHFR that help to stabilize the binding, resulting in a more potent inhibitor.

In silico ligand-based pharmacophore model generation for the identification of novel Pneumocystis carinii DHFR inhibitors

The ubiquitous enzyme, dihydrofolate reductase (DHFR), is a well-studied metabolic enzyme of significant pharmacological relevance. The past research studies have shown that DHFR has emerged as an important therapeutic target for the treatment of various diseases including cancers, bacterial and protozoal infections, and the opportunistic infections that are associated with AIDS. DHFR has been successfully used and selected as a target for the purpose of devising a cure for various diseases by discovering the antimicrobial drugs against a range of pathogenic microorganisms including the opportunistic microorganisms Pneumocystis carinii (pc), Toxoplasma gondii and Mycobacterium avium complex. However, it has been reported that blockage of the enzymatic activity of DHFR is a crucial and most significant element in terms of the treatment of a wide range of diseases. DHFR is also a key enzyme in the treatment of Pneumocystosis. The discovery and development of type-specific pcDHFR inhibitors is of both research and clinical interests. Ligand-based pharmacophore modeling is playing a vital role for the identification of ligand features for the particular targets. In this study, we present a model for the design of ligand-based pharmacophore onto the set of 10 compounds of eight different classes along with the standard drug trimethoprim. The ligand-based pharmacophore model has been identified to facilitate the discovery of type-specific pcDHFR inhibitors. A pharmacophore model was generated using Ligand Scout 3.02 with diverse classes of pcDHFR inhibitors. The proposed pharmacophore model generated in this study revealed that the model contains two HBAs, two HBDs, and one HY/AR volume. Ligand Scout 3.02 has been used to predict the pharmacophore features for pcDHFR inhibitors, and the distances between pharmacophore features have been computed by the effective use of the software VMD. The results indicate that the in silico methods are valuable for the prediction of the biological activity of the compound or compound library by screening it against a predicted pharmacophore. Thus, the results obtained in this study can be considered to be useful and reliable tools for the identification of structurally diverse compounds with the desired biological activity. The model has also been validated, firstly by mapping of five test compounds on to our model and secondly by docking these test compounds into the active site of pcDHFR.

CoMFA/CoMSIA 3D-QSAR of pyrimidine inhibitors of Pneumocystis carinii dihydrofolate reductase

Pneumocystis carinii is typically a non-pathogenic fungus found in the respiratory tract of healthy humans. However, it may cause P. carinii pneumonia (PCP) in people with immune deficiency, affecting mainly premature babies, cancer patients and transplant recipients, and people with acquired immunodeficiency syndrome (AIDS). In the latter group, PCP occurs in approximately 80% of patients, a major cause of death. Currently, there are many available therapies to treat PCP patients, including P. carinii dihydrofolate reductase (PcDHFR) inhibitors, such as trimetrexate (TMX), piritrexim (PTX), trimethoprim (TMP), and pyrimethamine (PMT). Nevertheless, the high percentage of adverse side effects and the limited therapeutic success of the current drug therapy justify the search for new drugs rationally planned against PCP. This work focuses on the study of pyrimidine inhibitors of PcDHFR, using both CoMFA and CoMSIA 3D-QSAR methods.

Structural analysis of Pneumocystis carinii dihydrofolate reductase complexed with NADPH and 2,4-diamino-6-[2-(5-carboxypent-1-yn-1-yl)-5-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine.

Structural data are reported for 2,4-diamino-6-[2-(5-carboxypent-1-yn-1-yl)-5-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine (PY1014) complexed with Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to 1.8 Å resolution. These data reveal that the carboxylate of the ω-carboxyalkynyl side chain of PY1014, the most pcDHFR-selective analog in this series, forms ionic interactions with the conserved Arg75 in the substrate-binding pocket of pcDHFR. The reversal of the 2',5'-substitution pattern of this analog compared with the highly selective diaminopyrimidine analog PY1011 (i.e. the 5'-pentynylcarboxy-5'-methoxy pattern of PY1014 versus the 3',4'-dimethoxy-5'-pentynylcarboxy pattern of PY1011) is necessary to achieve optimal interaction with Arg75 as observed in other structures. The larger diaminopyrido[2,3-d]pyrimidine ring of PY1014 places the 5'-methoxy group closer to Leu25 and Ser64 than does the diaminopyrimidine ring of PY1011. The 5'-methoxy O atom forms a hydrogen bond to the amide of Leu25 (O···N, 2.7 Å) and the 5'-methoxy methyl group makes a hydrophobic contact of 3.1 Å with C(β) of Ser64. Although the IC(50) values of PY1014 and PY1011 are similar, inhibition data show that the selectivity of PY1011 for pcDHFR is significantly greater. The greater selectivity for pcDHFR compared with mammalian DHFR of these inhibitors is also influenced by the enhanced hydrophobic interactions of the side-chain methylene atoms with Phe69 of pcDHFR compared with Asn64 of mammalian DHFR.

Structural analysis of Pneumocystis carinii and human DHFR complexes with NADPH and a series of five potent 6-[5'-(ω-carboxyalkoxy)benzyl]pyrido[2,3-d]pyrimidine derivatives.

Structural data are reported for five antifolates, namely 2,4-diamino-6-[5'-(5-carboxypentyloxy)-2'-methoxybenzyl]-5-methylpyrido[2,3-d]pyrimidine, (1), and the 5'-[3-(ethoxycarbonyl)propoxy]-, (2), 5'-[3-(ethoxycarbonyl)butoxy]-, (3), 5'-[3-(ethoxycarbonyl)pentyloxy]-, (4), and 5'-benzyloxy-, (5), derivatives, which are potent and selective for Pneumocystis carinii dihydrofolate reductase (pcDHFR). Crystal structures are reported for their ternary complexes with NADPH and pcDHFR refined to between 1.4 and 2.0 Å resolution and for that of 3 with human DHFR (hDHFR) to 1.8 Å resolution. These data reveal that the carboxylate of the ω-carboxyalkoxy side chain of 1, the most potent inhibitor in this series, forms ionic interactions with the conserved Arg75 in the substrate-binding pocket of pcDHFR, whereas the less potent ethyl esters of 2-4 bind with variable side-chain conformations. The benzyloxy side chain of 5 makes no contact with Arg75 and is the least active inhibitor in this series. These structural results suggest that the weaker binding of this series compared with that of their pyrimidine homologs in part arises from the flexibility observed in their side-chain conformations, which do not optimize intermolecular contact to Arg75. Structural data for the binding of 3 to both hDHFR and pcDHFR reveals that the inhibitor binds in two different conformations, one similar to each of the two conformations observed for the parent pyrido[2,3-d]pyrimidine, piritrexim (PTX), bound to hDHFR. The structure of the pcDHFR complex of 4 reveals disorder in the side-chain orientation; one orientation has the ω-carboxyalkoxy side chain positioned in the folate-binding pocket similar to the others in this series, while the second orientation occupies a new site near the nicotinamide ring of NADPH. This alternate binding site has not been observed in other DHFR structures. Structural data for the pcDHFR complex of 5 show that its benzyl side chain forms intermolecular van der Waals interactions with Phe69 in the binding pocket that could account for its enhanced binding selectivity compared with the other analogs in this series.

Abstract 2676: Unusual binding of TMP in two different sites in the binary complex with the Q35S/N64F double mutant of human dihydrofolate reductase

Abstract In an effort to understand the role of specific active site residues in modulating the binding selectivity of inhibitors against dihydrofolate reductase (DHFR) from different species, a series of single and double mutants of DHFR were made at positions 35 and 64 of human (h) DHFR. To explore sequence differences between human and the pathogenic organisms Pneumocystis carinii (pc) and Pneumocystis jirovecii (pj) DHFR, the causative agent of Pneumocystis pneumonia (PcP) in patients with AIDS, kinetic data were measured for trimethoprim, used for treatment of AIDS-related pneumonia. Crystal structure data are reported for the Q35S/N64F double mutant of hDHFR as a binary complex with trimethoprim (TMP). These substitutions make the human enzyme resemble a cross between pjDHFR which has Ser/Ser at these positions and pcDHFR which has Lys/Phe at these sites. Kinetic data show enhanced affinity for the binding of TMP to this double mutant (Ki 604 nM) compared to wild type hDHFR (Ki 1757 nM). The Ki of TMP is 43 nM for pjDHFR and 222 nM for pcDHFR. Structural data for the binary complex of TMP with this double mutant of hDHFR revealed the unexpected binding of two copies of TMP; one bound in the normal binding site while the other occupies a shallow pocket different from that involved in binding the γ-glutamate of folate substrates or inhibitors. The trimethoxyphenyl ring of the second TMP makes a series of hydrophobic contacts with the trimethoxyphenyl ring of the first TMP and with the phenyl ring of residue F31 and the mutated F64 in this structure. Structural data for the binary complex of wild type hDHFR with TMP does not indicate such binding. The conformation of the two TMP in the double mutant complex differ significantly from each other and from that observed for the wild type hDHFR binary complex. Analysis of all DHFR TMP complexes reveals the bridge torsion angles fall into two broad ranges and the two TMP in the mutant hDHFR complex fall into the less populated group. These data suggest that the combination of hydrophobic contacts from the normal F31 in hDFHR and the mutant F64 provide a unique environment to create sufficient van der Waals contact energy to stabilize binding in this unusual site. Supported in part by NIH GM51670 (VC). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2676.

2,4-Diamino-5-methyl-6-substituted arylthio-furo[2,3- d]pyrimidines as novel classical and nonclassical antifolates as potential dual thymidylate synthase and dihydrofolate reductase inhibitors

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.

Using Molecular Docking, 3D-QSAR, and Cluster Analysis for Screening Structurally Diverse Data Sets of Pharmacological Interest

In this study, we propose a drug design approach which includes docking, molecular fingerprints based cluster analysis, and 'induced' descriptors based receptor-dependent 3D-QSAR. The method was shown to be very useful for screening and modeling structurally diverse data sets of pharmacological interest. Different from other receptor-dependent 3D-QSAR, no ambiguous alignments are required for the construction of the models, and the computational cost is relatively lower. Moreover, 'induced' descriptors were shown to be very powerful in "capturing" ligand-receptor intermolecular interactions. The methodology was validated for eight data sets sampled from the literature and from public databases: human sex hormone-binding globulin, human corticosteroid-binding globulin, anthrax lethal factor, HIV-1 reverse transcriptase, neuraminidase A, thrombin, trypsin, and Pneumocystis carinii dihydrofolate reductase data sets. The resulting models were interpretable; the constructed QSAR equations have high statistical significance and predictive strength; and the drug design solutions were shown to be useful for guiding ligand modification for the development of new inhibitors for a broad range of molecular targets.

Structural analysis of a holoenzyme complex of mouse dihydrofolate reductase with NADPH and a ternary complex with the potent and selective inhibitor 2,4-diamino-6-(2'-hydroxydibenz[b,f]azepin-5-yl)methylpteridine.

It has been shown that 2,4-diamino-6-arylmethylpteridines and 2,4-diamino-5-arylmethylpyrimidines containing an O-carboxylalkyloxy group in the aryl moiety are potent and selective inhibitors of the dihydrofolate reductase (DHFR) from opportunistic pathogens such as Pneumocystis carinii, the causative agent of Pneumocystis pneumonia in HIV/AIDS patients. In order to understand the structure-activity profile observed for a series of substituted dibenz[b,f]azepine antifolates, the crystal structures of mouse DHFR (mDHFR; a mammalian homologue) holo and ternary complexes with NADPH and the inhibitor 2,4-diamino-6-(2'-hydroxydibenz[b,f]azepin-5-yl)methylpteridine were determined to 1.9 and 1.4 A resolution, respectively. Structural data for the ternary complex with the potent O-(3-carboxypropyl) inhibitor PT684 revealed no electron density for the O-carboxylalkyloxy side chain. The side chain was either cleaved or completely disordered. The electron density fitted the less potent hydroxyl compound PT684a. Additionally, cocrystallization of mDHFR with NADPH and the less potent 2'-(4-carboxybenzyl) inhibitor PT682 showed no electron density for the inhibitor and resulted in the first report of a holoenzyme complex despite several attempts at crystallization of a ternary complex. Modeling data of PT682 in the active site of mDHFR and P. carinii DHFR (pcDHFR) indicate that binding would require ligand-induced conformational changes to the enzyme for the inhibitor to fit into the active site or that the inhibitor side chain would have to adopt an alternative binding mode to that observed for other carboxyalkyloxy inhibitors. These data also show that the mDHFR complexes have a decreased active-site volume as reflected in the relative shift of helix C (residues 59-64) by 0.6 A compared with pcDHFR ternary complexes. These data are consistent with the greater inhibitory potency against pcDHFR.

New insights into DHFR interactions: Analysis of Pneumocystis carinii and mouse DHFR complexes with NADPH and two highly potent 5‐(ω‐carboxy(alkyloxy) trimethoprim derivatives reveals conformational correlations with activity and novel parallel ring stacking interactions

Structural data are reported for two highly potent antifolates, 2,4-diamino-5-[3',4'-dimethoxy-5'-(5-carboxy-1-pentynyl)]benzylpyrimidine (PY1011), with 5000-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR), relative to rat liver DHFR, and 2,4-diamino-5-[2-methoxy-5-(4-carboxybutyloxy)benzyl]pyrimidine (PY957), that has 80-fold selectivity for pcDHFR. Crystal structures are reported for NADPH ternary complexes with PY957 and pcDHFR, refined to 2.2 A resolution; with PY1011 and pcDHFR, refined to 2.0 A resolution; and with PY1011 and mouse DHFR (mDHFR), refined to 2.2 A resolution. These results reveal that the carboxylate of the omega-carboxyalkyloxy side chain of these inhibitors form ionic interactions with the conserved Arg in the substrate binding pocket of DHFR. These data suggest that the enhanced inhibitory activity of PY1011 compared with PY957 is, in part, due to the favorable contacts with Phe69 of pcDHFR by the methylene carbons of the inhibitor side chain that are oriented by the triple bond of the 1-pentynyl side chain. These contacts are not present in the PY957 pcDHFR complex, or in the PY1011 mDHFR complex. In the structure of mDHFR the site of Phe69 in pcDHFR is occupied by Asn64. These data also revealed a preference for an unusual parallel ring stacking interaction between Tyr35 of the active site helix and Phe199 of the C-terminal beta sheet in pcDHFR and by Tyr33 and Phe179 in mDHFR that is independent of bound ligand. A unique His174-His187 parallel ring stacking interaction was also observed only in the structure of pcDHFR. These ring stacking interactions are rarely found in any other protein families and may serve to enhance protein stability.

Design, Synthesis, and Antifolate Activity of New Analogues of Piritrexim and Other Diaminopyrimidine Dihydrofolate Reductase Inhibitors with ω-Carboxyalkoxy or ω-Carboxy-1-alkynyl Substitution in the Side Chain

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.

CoMFA and CoMSIA Analyses of Pneumocystis carinii Dihydrofolate Reductase, Toxoplasma gondii Dihydrofolate Reductase, and Rat Liver Dihydrofolate Reductase

In a continuing effort to develop potent and selective dihydrofolate reductase (DHFR) inhibitors against opportunistic pathogens, we developed three-dimensional quantitative structure-activity relationship (3D QSAR) models for the inhibitory activity against Pneumocystis carinii (pc) DHFR, Toxoplasma gondii (tg) DHFR, and rat liver DHFR, using a data set of 179 structurally diverse compounds. To ensure a balanced distribution of more potent and less potent drugs in the training set, three different 90-compound training sets taken from the main data set were used, one for each enzyme, while the remaining 89 compounds in the main data set in each case were used as the test set. Three methods, namely, conventional CoMFA, all orientation search (AOS) CoMFA, and CoMSIA were applied to the training sets. While the AOS CoMFA models gave the best internal predictions (cross-validated r(2) values from the training sets), which are satisfactory, CoMSIA models gave the best external predictions (predictive r(2) values from the test sets). Both AOS CoMFA and CoMSIA analyses were used to construct stdev*coefficient contour maps which can be used to design new compounds in an interactive fashion.

Structure determination of tetrahydroquinazoline antifolates in complex with human and Pneumocystis carinii dihydrofolate reductase: correlations between enzyme selectivity and stereochemistry.

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.

Primary Structure of Dihydrofolate Reductase and Mitochondrial Ribosomal Protein L36 Genes from the BasidiomyceteCoprinus cinereus

We amplified and sequenced the dihydrofolate reductase (DHFR) gene of the basidiomycete Coprinus cinereus. Downstream of the DHFR coding region, a mitochondrial (mt) ribosomal protein L36 (RPL36) gene was discovered in the opposite orientation to DHFR gene. Putative polyadenylation signals of the two genes overlapped, both containing the 8-bp palindrome 5'-aatatatt-3'. The finding that C. cinereus DHFR gene is closely clustered with a mt protein gene strongly suggests that C. cinereus DHFR is closely related to mt function and evolution. The amino acid sequence of C. cinereus DHFR is most homologous to eukaryotic proteins such as Cryptococcus neoformans and Pneumocystis carinii DHFRs. However, the sequence of C. cinereus mt RPL36 closely resembles RPL36 of bacteria and cyanobacteria such as Synechocystis sp. and Escherichia coli. This result strongly supports the serial endosymbiotic theory of the development of ancestral eukaryotes, and suggests that C. cinereus mt RPL36 gene originated from the ancestral eubacterial genome.

Interaction Energy Analysis of Nonclassical Antifolates with Pneumocystis carinii Dihydrofolate Reductase

The x-ray structure of the Pneumocystis carinii dihydrofolate reductase (DHFR):trimethoprim:NADPH ternary complex obtained from the Protein Databank was used as a structural template to generate models for the following complexes: P. carinii DHFR:piritrexim:NADPH, P. carinii DHFR:epiroprim:NADPH, and P. carinii DHFR:trimetrexate:NADPH. Each of these complexes, including the original trimethoprim complex was then modeled in 60 angstrom cubes of explicit water and minimized to a rms gradient between 1.0 to 3.0 x 10-5 kcal/angstrom. Subsequently, each antifolate structure was subdivided into distinct substructural regions. The minimized complexes were used to calculate interaction energies for each intact antifolate and its corresponding substructural regions with the P. carinii DHFR binding site residues, the DHFR protein, the solvated complex ( which consists of P. carinii DHFR, NADPH, and solvent water), solvent water alone, and NADPH. Antifolate substructural regions which contained nitrogen and carbon atoms in an aromatic environment (i. e. the pteridyl, pyridopyrimidinyl, and diaminopyrimidinyl subregions) contributed most to the stability of antifolate interactions, while interaction energies for the hydrocarbon aromatic rings, methoxy, and ethoxy groups were much less stable. Additionally, interaction energy analyses were calculated for carbon and nitrogen atoms of the pteridyl, pyridopyrimidinyl, and diaminopyrimidinyl subregions and for the carbon and oxygen atoms of methoxy and ethoxy subregions. The contributions of hydrogen atoms were included with those of the carbon, nitrogen and oxygen atoms to which they are attached. These analyses revealed that the carbon atoms of the pteridyl, pyridopyrimidinyl, and diaminopyrimidinyl subregions generally contributed most to the stability of those regions. Carbon atoms also contributed favorably to the stability of the methoxy group interactions. Those substructural regions which exhibit relatively unfavorable interaction energies may constitute important modification targets in the design of improved P. carinii DHFR inhibitors. Interaction energies for different groups of atoms within the substructural regions suggest strategies for modification of the substructural regions.

Development of a yeast assay for rapid screening of inhibitors of human-derived Pneumocystis carinii dihydrofolate reductase.

Human-derived Pneumocystis carinii dihydrofolate reductase (DHFR) was expressed in a Saccharomyces cerevisiae strain whose growth depends on complementation by this enzyme. We utilized a quantitative assay to measure the sensitivity of this yeast strain to DHFR inhibitors. This assay should be useful for identifying new inhibitors of human-derived P. carinii DHFR.

Analysis of quinazoline and pyrido[2,3-d]pyrimidine N9-C10 reversed-bridge antifolates in complex with NADP+ and Pneumocystis carinii dihydrofolate reductase.

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.

Structure-based enzyme inhibitor design: modeling studies and crystal structure analysis ofPneumocystis cariniidihydrofolate reductase ternary complex with PT653 and NADPH

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.

2,4-Diamino-6,7-dihydro-5H-cyclopenta[d]pyrimidine Analogues of Trimethoprim as Inhibitors of Pneumocystis carinii and Toxoplasma gondii Dihydrofolate Reductase.

2,4-Diamino-6,7-dihydro-5<i>H</i>-cyclopenta[<i>d</i>]pyrimidine Analogues of Trimethoprim as Inhibitors of <i>Pneumocystis </i><i>c</i><i>arinii </i>and <i>Toxoplasma </i><i>g</i><i>ondii </i>Dihydrofolate Reductase.