Selectivity analysis of 5-(arylthio)-2,4-diaminoquinazolines as inhibitors of Candida albicans dihydrofolate reductase by molecular dynamics simulations

A series of 5-(arylthio)-2,4-diaminoquinazolines are known as selective inhibitors of dihydrofolate reductase (DHFR) from Candida albicans. We have performed docking and molecular dynamics simulations of these inhibitors with C. albicans and human DHFR to understand the basis for selectivity of these agents. Study was performed on a selected set of 10 compounds with variation in structure and activity. Molecular dynamics simulations were performed at 300 K for 45 ps with equilibration for 10 ps. Trajectory data was analyzed on the basis of hydrogen bond interactions, energy of binding and conformational energy difference. The results indicate that hydrogen bonds formed between the compound and the active site residues are responsible for inhibition and higher potency. The selectivity index, i.e the ratio of I₅₀ against human DHFR to I₅₀ against fungal DHFR, is mainly determined by the conformation adapted by the compounds within the active site of two enzymes. Since the human DHFR active site is rigid, the compound is trapped in a higher energy conformation. This energy difference between the two conformations E mainly governs the selectivity against fungal DHFR. The information generated from this analysis of potency and selectivity should be useful for further work in the area of antifungal research.     

Further studies on the synthesis of quinazolines from 2-fluorobenzonitriles

Recently, we reported that appropriately substituted 2-fluorobenzonitriles undergo cyclization with guanidine carbonate to afford 2,4-diaminoquinazolines usually in good to excellent yield. This paper describes the preparation of a variety of new 2,4-diaminoquinazolines substituted at positions five or seven. In addition, the reactions of selected 2-fluorobenzonitriles with formamidine acetate or acetamidine acetate were examined. The results obtained demonstrate that the analogous 4-amino- and 2-methyl-4-aminoquinazolines can be prepared by this approach but that the yields are considerably lower than when guanidine carbonate is employed as the cyclization reagent.     

Synthesis of 5-(4-substituted benzyl)-2,4-diaminoquinazolines as inhibitors of candida albicans dihydrofolate reductase

Several 5-(4-substituted benzyl)-2,4-diaminoquinazolines were prepared as potentially selective inhibitors of Candida albicans dihydrofolate reductase. These compounds were synthesized by a novel route, which included as a key step the displacement of a fluoro group in 2,6-difluorobenzonitrile by the anions of ethyl or methyl 4-substituted phenylacetates. The resultant diarylacetates were saponified and decarboxylated to the 2-fluoro-6-(4-substituted phenyl)benzonitriles. Ring closure of these benzonitriles with guanidine carbonate gave the 5-(4-substituted benzyl)-2,4-diaminoquinazolines.     

A proposed model of Mycobacterium avium complex dihydrofolate reductase and its utility for drug design

A homology model of Mycobacterium avium complex dihydrofolate reductase (MAC DHFR) was constructed on the basis of the X-ray crystal structure of Mycobacterium tuberculosis (Mtb) DHFR. The homology searching of the MAC DHFR resulted in the identification of the Mtb DHFR structure (PDB 1DF7) as the template for the model building. The MAC enzyme sequence was aligned to that of the Mtb counterpart using a modified Needleman and Wunsch methodology. The initial geometry to be modeled was copied from the template, either fully or partially depending on whether the residues were conserved or not, respectively. Using a randomized modeling procedure, 10 independent models of the target protein were built. The cartesian average of all the model structures was then refined using molecular mechanics. The resulting model was assessed for stereochemical quality using a Ramachandran plot and by analyzing the consistency of the model with the experimental data. The structurally and functionally important residues were identified from the model. Further, 5-deazapteridines recently reported as inhibitors of MAC DHFR were docked into the active site of the developed model. All the seven inhibitors used in the docking study have a similar docking mode at the active site. The network of hydrogen bonds around the 2,4-diamino-5-deazapteridine ring was found to be crucial for the binding of the inhibitors with the active site residues. The 5-methyl group of the inhibitors was located in a narrow hydrophobic pocket at the bottom of the active site. The relative values of the three torsion angles of the inhibitors were found to be important for the proper orientation of the inhibitor functional groups into the active site.     

Synthesis, dihydrofolate reductase inhibition, anti-proliferative testing, and saturation transfer difference ¹H-NMR study of some new 2-substituted-4,6-diaminopyrimidine derivatives

A series of 2-substituted-4,6-diaminipyrimidine derivatives were synthesized and evaluated for their dihydrofolate reductase (DHFR) inhibitory activity. Saturation transfer difference (STD) (1)H-NMR experiments were used to probe the binding characteristics of the compounds with human DHFR enzyme. The most potent molecules, 12 and 15, in enzyme assay study showed the best results in STD experiments indicating their intimate interaction with the receptor. The docking studies were followed to explain the structural basis for the observed interaction between the ligands and DHFR. All the compounds were also assayed in vitro for their growth inhibitory activity on MCF-7, HepG2, SKHep1, and Hela tumor cell lines. Compounds 16, 17, and 22 demonstrated the most potent in vitro anti-proliferative activity among the others.     

Protein flexibility and species specificity in structure-based drug discovery: dihydrofolate reductase as a test system

In structure-based drug discovery, researchers would like to identify all possible scaffolds for a given target. However, techniques that push the boundaries of chemical space could lead to many false positives or inhibitors that lack specificity for the target. Is it possible to broadly identify the appropriate chemical space for the inhibitors and yet maintain target specificity? To address this question, we have turned to dihydrofolate reductase (DHFR), a well-studied metabolic enzyme of pharmacological relevance. We have extended our multiple protein structure (MPS) method for receptor-based pharmacophore models to use multiple X-ray crystallographic structures. Models were created for DHFR from human and Pneumocystis carinii. These models incorporate a fair degree of protein flexibility and are highly selective for known DHFR inhibitors over drug-like non-inhibitors. Despite sharing a highly conserved active site, the pharmacophore models reflect subtle differences between the human and P. carinii forms, which identify species-specific, high-affinity inhibitors. We also use structures of DHFR from Candida albicans as a counter example. The available crystal structures show little flexibility, and the resulting models give poorer performance in identifying species-specific inhibitors. Therapeutic success for this system may depend on achieving species specificity between the related human host and these key fungal targets. The MPS technique is a promising advance for structure-based drug discovery for DHFR and other proteins of biomedical interest.     

Further evaluation of 5-alkyl-5-deaza antifolates. 5-propyl- and 5-butyl-5-deaza analogues of aminopterin and methotrexate

5-Propyl-5-deaza and 5-butyl-5-deaza analogues of classical antifolates were synthesized by extensions of a previously reported general route which proceeds through 2,4-diamino-5-alkylpyrido[2,3-d]pyrimidine-6-carbonitrile intermediates followed by reductive condensation with diethyl N-4-(aminobenzoyl)-L-glutarnate to give diethyl esters of 5-alkyl-5-deazaaminopterin types. N¹⁰-Methyl derivatives, i.e., derivatives of 5-alkyl-5-deazamethotrexate, were also prepared by reductive methylation of the N¹⁰-H compounds. 5-Ethyl-5-deazamethotrexate was prepared using an alternative route through 6-(bromomethyl)-2,4-diamino-5-ethylpyrido[2,3-d]pyrimidine. These antifolates were evaluated for inhibition of dihydrofolate reductase (DHFR) from L1210 cells, their effect on L1210 and S180 tumor cell growth in culture, and carrier-mediated transport through L1210 cell membranes. Inhibitory effect on DHFR was lowered relative to methotrexate in 5-propyl-5-deazaaminopterin and 5-propyl-5-deazamethotrexate by 2- to 3-fold (K_i = 9.3 and 11.7 pM, respectively, vs. 4.3 pM for methotrexate) and by 17- to 18-fold in 5-butyl-5-deaza-aminopterin and 5-butyl-5-deazamethotrexate (K_i = 74 and 78 pM, respectively). Molecular modeling using graphics derived from human DHFR show the propyl and butyl compounds interacting with the enzyme in conformations that account for these slight decreases in binding.     

Structure-guided development of efficacious antifungal agents targeting Candida glabrata dihydrofolate reductase

Candida glabrata is a lethal fungal pathogen resistant to many antifungal agents and has emerged as a critical target for drug discovery. Over the past several years, we have been developing a class of propargyl-linked antifolates as antimicrobials and hypothesized that these compounds could be effective inhibitors of dihydrofolate reductase (DHFR) from C. glabrata. We initially screened a small collection of these inhibitors and found modest levels of potency. Subsequently, we determined the crystal structure of C. glabrata DHFR bound to a representative inhibitor with data to 1.6 A resolution. Using this structure, we designed and synthesized second-generation inhibitors. These inhibitors bind the C. glabrata DHFR enzyme with subnanomolar potency, display greater than 2000-fold levels of selectivity over the human enzyme, and inhibit the growth of C. glabrata at levels observed with clinically employed therapeutics.     

Modeling the inhibition of quadruple mutant <Emphasis Type="Italic">Plasmodium falciparum</Emphasis> dihydrofolate reductase by pyrimethamine derivatives

Modeling studies were performed on known inhibitors of the quadruple mutant Plasmodium falciparum dihydrofolate reductase (DHFR). GOLD was used to dock 32 pyrimethamine derivatives into the active site of DHFR obtained from the x-ray crystal structure 1J3K.pdb. Several scoring functions were evaluated and the Molegro Protein-Ligand Interaction Score was determined to have one of the best correlation to experimental pK _i . In conjunction with Protein-Ligand Interaction scores, predicted binding modes and key protein-ligand interactions were evaluated and analyzed in order to develop criteria for selecting compounds having a greater chance of activity versus resistant strains of Plasmodium falciparum. This methodology will be used in future studies for selection of compounds for focused screening libraries.     

Direct synthesis of 2,4-diaminoquinazolines from 2-fluorobenzonitriles

In a search for new methods for preparing 2,4-diaminoquinazolines having a diversity of substituents in the benzenoid ring, it was found that the reaction of 2,6-difluorobenzonitrile with guanidine carbonate gave 2,4-diamino-5-fluoroquinazoline in excellent yield. Extension of this approach to other 2-fluorobenzonitriles, some of which were elaborated for the first time, showed that this reaction possesses considerable generality. The cyclization was successful even when electron donating groups were present at position six. Only in two cases where a primary or secondary amino group was also present ortho to the cyano group was this transformation unsuccessful.     

Novel Sulfonamide Derivatives as Inhibitors of Histone Deacetylase

Inhibition of the enzyme histone deacetylase (HDAC) is emerging as a novel approach to the treatment of cancer. A series of novel sulfonamide derivatives were synthesized and evaluated for their ability to inhibit human HDAC. Compounds were identified which are potent enzyme inhibitors, with IC₅₀ values in the low nanomolar range against enzyme obtained from HeLa cell extracts, and with antiproliferative effects in cell culture. Extensive characterization of the structure–activity relationships of this series identified key requirements for activity. These include the direction of the sulfonamide bond and substitution patterns on the central phenyl ring. The alkyl spacer between the aromatic head group and the sulfonamide functionality also influenced the HDAC inhibitory activity. One of these compounds, m 11.1 , also designated PXD101, has entered clinical trials for solid tumors and haematological malignancies.     

5-(N-Arylnortropan-3-yl)- and 5-(N-Arylpiperidin-4-yl)-2,4-diaminopyrimidines. Novel Inhibitors of Dihydrofolate Reductase

Based on a computer-assisted analysis of the three-dimensional structure of the binary complex of E.coli dihydrofolate reductase (DHFR) with methotrexate, 5-(N-arylnortropan-3-yl)- and 5-(N-arylpiperidin-4-yl)-2,4-diaminopyrimidines 2 and 4 were designed as inhibitors of DHFR. Syntheses of the designed compounds have been carried out. The most potent compound 2a inhibited E. coli DHFR with K_i = 0.49.10^?9M. The activities within the series of compounds synthesized could be rationalized by molecular-modelling experiments which served as the basis of this work. Several compounds within the presented series exhibit antimalarial activities in vitro and in vivo.     

Incorporating dynamics in E. coli dihydrofolate reductase enhances structure-based drug discovery

Escherichia coli dihydrofolate reductase (DHFR) is a long-standing target for enzyme studies. The influence of protein motion on its catalytic cycle is significant, and the conformation of the M20 loop is of particular interest. We present receptor-based pharmacophore models-an equivalent of solvent-mapping of binding hotspots-based on ensembles of protein conformations from molecular dynamics simulations of DHFR.NADPH in both the closed and open conformation of the M20 loop. The optimal models identify DHFR inhibitors over druglike non-inhibitors; furthermore, high-affinity inhibitors of E. coli DHFR are preferentially identified over general DHFR inhibitors. As expected, models resulting from simulations with DHFR in the productive conformation with a closed M20 loop have better performance than those from the open-loop simulations. Model performance improves with increased dynamic sampling, indicating that including a greater degree of protein flexibility can enhance the quest for potent inhibitors. This was compared to the limited conformational sampling seen in crystal structures, which were suboptimal for this application.     

Evaluation of xanthine oxidase inhibitory activity of flavonoids by an online capillary electrophoresis-based immobilized enzyme microreactor

Xanthine oxidase (XOD) is a key enzyme in the human body to produce uric acid, and its inhibitor can be used for the treatment of hyperuricemia and gout. In this study, an online CE-based XOD immobilized enzyme microreactor (IMER) was developed for the enzyme kinetics assays and inhibitor screening. After 30 consecutive runs, the XOD activity remained about 95.6% of the initial immobilized activity. The Michaelis–Menten constant (K_m) of the immobilized XOD was determined as 0.39 mM using xanthine as substrate. The half-maximal inhibitory concentration and inhibition constant of the known inhibitor 4-aminopyrazolo[3,4-d]pyrimidine on XOD were determined as 11.9 and 5.2 μM, respectively. Then, the developed method was applied to evaluate the XOD inhibitory activity of 10 flavonoids, which indicated that dihydroquercetin, quercetin, biochanin A, and epicatechin had significant inhibitory effect on XOD. In addition, molecular docking results verified that the binding energy of the flavonoids with enzyme were in line with their inhibitory activity determined by XOD–IMER. Therefore, the developed XOD–IMER is a potential tool for the primary screening of XOD inhibitors from natural products.     

Solid-phase synthesis of 6-hydroxy-2,4-diaminoquinazolines

A new method for the solid-phase synthesis of 6-hydroxy-2,4-diaminoquinazolines has been developed. The synthesis utilizes solid-phase bound 6-hydroxy-2,4-dichloroquinazoline as a key intermediate. Sequential substitution of the two chlorines furnishes the title compounds regioselectively with high purity. A library of 18×22 compounds demonstrates the general utility of this approach.     

Application of flow microcalorimetry to the study of dehydrofolate reductase activities in crude tissue homogenates

Calorimetric methods are becoming important analytical tools in several areas of biochemical and biological research. In this work, a flow microcalorimetric method has been applied to the determination of dihydrofolate reductase (DHFR) activities in rat and human malignant tissue homogenates. In contrast to other commonly used DHFR analytical assays, the sensitivity of flow microcalorimetry allows direct measurements of this enzyme in crude tissue preparations. Our experimental data from rat tissue homogenates show that liver has the highest level of enzyme activity, while lung and brain have lower amounts of DHFR activity. The liver enzyme has a higher activity atpH 4.5, but the optimumpH for the lung and brain enzymes is 6.8. The substrate/cofactor molar ratio which gives the highest levels of DHFR activity is 1/1.5 for the liver and lung enzymes and 1/2.5 for brain DHFR. The DHFR in these rat tissue homogenates is activated by KCl or NaCl: in the presence of these salts (0.6M), the values of enzyme activity are 1.5–3 times higher than in their absence. Using flow microcalorimetry, very low levels of DHFR activity were also measured in human bone tumour homogenates, demonstrating the potential of the technique in the analysis of this enzyme in malignant tissues.     

Fluorinated phenylcyclopropylamines: Part 6. Effects of electron withdrawing or donating aryl substituents on the inhibition of tyramine oxidase from Arthrobacter sp. by diastereomeric 2-aryl-2-fluoro-cyclopropylamines

Diastereomeric arylcyclopropylamines substituted with fluorine in the 2-position and with electron donating or electron withdrawing groups at the aromatic ring were evaluated as inhibitors of microbial tyramine oxidase. The trans-isomers were consistently more potent inhibitors of the enzyme than the cis-isomers. Electron donating substituents increased the potency of tyramine oxidase inhibition, while electron withdrawing substituents decreased the activity. The results obtained are discussed in terms of pK_a and log D values of the inhibitors as well as the mechanism of action of tranylcypromines and the geometry of the active site of the enzyme.     

Synthesis and antifolate activity of new pyrrolo[2,3‐d]pyrimidine and thieno[2,3‐d]pyrimidine inhibitors of dihydrofolate reductase

Three previously undescribed dihydrofolate reductase (DHFR) inhibitors, N^α‐[4‐[N‐[(2,4‐diaminopyrrolo[2,3‐d]pyrimidin‐5‐yl)methyl]amino]benzoyl]‐N^δ‐hemiphthaloyl‐L‐ornithine (7) , N^α‐ [4‐ [N‐[(2,4‐diaminothieno[2,3‐d]pyrimidin‐5‐yl)methyl]amino]benzoyl]‐ N^δ‐hemiphthaloyl‐L‐ornithine (8) , and N‐[4‐[N‐[(2,4‐diaminothieno[2,3‐d]pyrimidin‐5‐yl)methyl]amino]benzoyl]‐L‐glutamic acid (12) , were synthesized and their antifolate activity was assessed. The ability of 7 and 8 to bind to DHFR and inhibit the growth of CCRF‐CEM human lymphoblastic leukemia cells in culture were dramatically reduced in comparison with the corresponding pteridine analogue, N^α‐(4‐amino‐4‐deoxypteroyl)‐N^δ‐hemiphmaloyl‐L‐ornithine ( 1 , PT523). In a similar manner, the antifolate activity of 12 was markedly reduced in comparison with that of the corresponding glutamate analogue, aminopterin ( 5 , AMT). In contrast, 7, 8 , and 12 all displayed excellent affinity for the reduced folate carrier (RFC) of CCRF‐CEM cells as measured by a standard competitive influx assay. Lack of a consistent correlation between the results of the growth inhibition assays and those of the DHFR and RFC binding assays results suggest that additional factors also play a role in the antifolate activity of these compounds.