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.     

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.     

Synthesis and biological activities of substituted dihydrobenzo[h]-pyrimido[4,5-b]quinolines as tetracyclic 5-deaza nonclassical folates

Novel tetracyclic compounds 1–4 have been synthesized via a regiospecific cyclocondensation reaction between substituted 6-aminopyrimidines 5– 7 and chlorovinyl aldehydes 13 and 14 . The linear structures of these compounds were established by ¹H nmr and ¹³C nmr spectral data and also by synthesis of the compounds via an unambiguous route. The growth of Manca human lymphoma cells was inhibited 50% by 1 and 4 at 4.5 × 10^?6 M and 1.2 × 10^?6 M respectively. These compounds also inhibited human dihydrofolate reductase (DHFR)by 50% at 4.4 × 10^?6 M and 1.4 × 10^?6 irrespectively and L. casei DHFR at 1.9 × 10^?5 M and 1.1 × 10^?5 M respectively. Compound 16 , a positional isomer of 1 , was the most potent of the compounds studied, it inhibited the growth of Manca human lymphoma cells by 50% at 9 × 10^?8 M. The IC₅₀ values of 16 for the inhibition of human DHFR and L. casei DHFR were 8 × 10^?8 M and 1.9 × 10^?5 M respectively.     

One site fits both: A model for the ternary complex of folate + NADPH in R67 dihydrofolate reductase, a D2 symmetric enzyme

R67 dihydrofolate reductase (DHFR) is a novel enzyme that confers resistance to the antibiotic trimethoprim. The crystal structure of R67 DHFR displays a toroidal structure with a central active-site pore. This homotetrameric protein exhibits 222 symmetry, with only a few residues from each chain contributing to the active site, so related sites must be used to bind both substrate (dihydrofolate) and cofactor (NADPH) in the productive R67 DHFR?NADPH?dihydrofolate complex. Whereas the site of folate binding has been partially resolved crystallographically, an interesting question remains: how can the highly symmetrical active site also bind and orient NADPH for catalysis? To model this ternary complex, we employed DOCK and SLIDE, two methods for docking flexible ligands into proteins using quite different algorithms. The bound pteridine ring of folate (Fol I) from the crystal structure of R67 DHFR was used as the basis for docking the nicotinamide-ribose-P_i (NMN) moiety of NADPH. NMN was positioned by both DOCK and SLIDE on the opposite side of the pore from Fol I, where it interacts with Fol I at the pore's center. Numerous residues serve dual roles in binding. For example, Gln 67 from both the B and D subunits has several contacts with the pteridine ring, while the same residue from the A and C subunits has several contacts with the nicotinamide ring. The residues involved in dual roles are generally amphipathic, allowing them to make both hydrophobic and hydrophilic contacts with the ligands. The result is a `hot spot' binding surface allowing the same residues to co-optimize the binding of two ligands, and orient them for catalysis.     

Very empirical treatment of solvation and entropy: a force field derived from Log Po/w

A non-covalent interaction force field model derived from the partition coefficient of 1-octanol/water solubility is described. This model, HINT for Hydropathic INTeractions, is shown to include, in very empirical and approximate terms, all components of biomolecular associations, including hydrogen bonding, Coulombic interactions, hydrophobic interactions, entropy and solvation/desolvation. Particular emphasis is placed on: (1) demonstrating the relationship between the total empirical HINT score and free energy of association, G _interaction; (2) showing that the HINT hydrophobic-polar interaction sub-score represents the energy cost of desolvation upon binding for interacting biomolecules; and (3) a new methodology for treating constrained water molecules as discrete independent small ligands. An example calculation is reported for dihydrofolate reductase (DHFR) bound with methotrexate (MTX). In that case the observed very tight binding, G _interaction–13.6 kcal mol^–1, is largely due to ten hydrogen bonds between the ligand and enzyme with estimated strength ranging between –0.4 and –2.3 kcal mol^–1. Four water molecules bridging between DHFR and MTX contribute an additional –1.7 kcal mol^–1 stability to the complex. The HINT estimate of the cost of desolvation is +13.9 kcal mol^–1.     

Towards understanding the origins of the different specificities of binding the reduced (NADPH) and oxidised (NADP+) forms of nicotinamide adenine dinucleotide phosphate coenzyme to dihydrofolate reductase

Lactobacillus casei dihydrofolate reductase (DHFR) binds more than a thousand times tighter to NADPH than to NADP⁺. The origins of the difference in binding affinity to DHFR between NADPH and NADP⁺ are investigated in the present study using experimental NMR data and hybrid density functional, B3LYP, calculations. Certain protein residues (Ala 6, Gln 7, Ile 13 and Gly 14) that are directly involved in hydrogen bonding with the nicotinamide carboxamide group show consistent differences in ¹H and ¹⁵N chemical shift between NADPH and NADP⁺ in a variety of ternary complexes. B3LYP calculations in model systems of protein-coenzyme interactions show differences in the H-bond geometry and differences in charge distribution between the oxidised and reduced forms of the nicotinamide ring. GIAO isotropic nuclear shieldings calculated for nuclei in these systems reproduce the experimentally observed trends in magnitudes and signs of the chemical shifts. The experimentally observed reduction in binding of NADP⁺ compared with NADPH results partly from NADP⁺ having to change its nicotinamide amide group from a cis- to a trans-conformation on binding and partly from the oxidised nicotinamide ring of NADP⁺ being unable to take up its optimal hydrogen bonding geometry in its interactions with protein residues.     

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.     

Molecular modeling of rice Rubisco

Molecular model of rice Rubisco has been constructed by molecular modeling method on a computer graphics workstation using graphics software TOM-FRODO and QUANTA/CHARMm,based on the three-dimensional structure of spinach Rubisco.The molecular modeling was carried out in the following procedure:amino-acid sequence substitution;manual adjustment of the initial model;and energy minimization.The three-dimensional structure of the quaternary complex Rubisco-CO2·Mg2+·CABP of rice Rubisco has also been modeled and optimized using the program package XPLOR.The main-chain structure of rice Rubisco does not show obvious difference from that of spinach Rubisco,and the side-chains are located in the most favorable positions and orientations.The active-site structures are basically the same for the two species.The mutant Leu335→Arg of rice Rubisco has been designed using the graphics software TURBO-FRODO and the program package XPLOR.Energy computation suggests that this mutation might increase the carboxylation     

Molecular simulations of DD-peptidase,a model ß-lactam-binding protein: Synergy between X-ray crystallography and computational chemistry

Using computer model building, the three-dimensional structure of an enzyme from Streptomyces R61 that is inhibited by ß-lactam antibiotics has been constructed starting from incomplete X-ray crystallographic data for this 37.4 kDa protein. The so-called DD-peptidase catalyzes transpeptidation and hydrolysis of peptides terminating in D-Ala-D-Ala and is a model for bacterial transpeptidases and carboxypeptidases essential in the biosynthesis of the peptidoglycan layer of the cell wall. The structure, which was completed with the SYBYL molecular modeling package, has been refined by energy minimization and molecular dynamics using Quanta/CHARMm software. A simulation of 105 ps was run with waters of solvation in the active site. From these computations, the interatomic distances between the active serine and key residues around the active site were determined. Inadequacies at reproducing geometric details of the ß-lactam ring of a cephalosporin are pointed out which are typical of most commercially available force fields.     

A molecular modelling study of the interaction of noradrenaline with theβ 2-adrenergic receptor

Summary A model of the ₂-adrenergic receptor binding site is built from the primary structure of the receptor, experimental evidence for key binding residues and analogy with a homologous protein of partially determined structure. It is suggested that residues Trp-109, Thr-110 and Asp-113 are involved in ligand binding. Noradrenaline is successfully docked into this model, and the results of an INDO molecular orbital calculation on the complex indicate that a charge transfer interaction between Trp-109 and noradrenaline is possible.     

Simultaneous saccharification and fermentation of cassava bagasse for l-(+)-lactic acid production using Lactobacilli

Saccharification and fermentation of cassava (Manihot esculenta) bagasse was carried out in a single step for the production of L-(+)-lactic acid by Lactobacillus casei and Lactobacillus delbrueckii. Using 15.5% w/v of cassava bagasse as the raw material, a maximum starch to lactic acid conversion of 96% was obtained with L. casei with a productivity rate of 1.40mg/mL·h and maximum yield of 83.8 mg/mL. It was 94% with L. delbrueckii with a productivity rate of 1.36 mg/mL·h. and maximum yeild of 81.9 mg/mL. Supplementation of bagasse with 0.01% w/v MnCl₂ showed positive influence on the lactic acid production by L. casei.     

Toward <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> DXR inhibitor design: homology modeling and molecular dynamics simulations

Mycobacterium tuberculosis 1-deoxy-d-xylulose-5-phosphate reductoisomerase (MtDXR) is a potential target for antitubercular chemotherapy. In the absence of its crystallographic structure, our aim was to develop a structural model of MtDXR. This will allow us to gain early insight into the structure and function of the enzyme and its likely binding to ligands and cofactors and thus, facilitate structure-based inhibitor design. To achieve this goal, initial models of MtDXR were generated using MODELER. The best quality model was refined using a series of minimizations and molecular dynamics simulations. A protein–ligand complex was also developed from the initial homology model of the target protein by including information about the known ligand as spatial restraints and optimizing the mutual interactions between the ligand and the binding site. The final model was evaluated on the basis of its ability to explain several site-directed mutagenesis data. Furthermore, a comparison of the homology model with the X-ray structure published in the final stages of the project shows excellent agreement and validates the approach. The knowledge gained from the current study should prove useful in the design and development of inhibitors as potential novel therapeutic agents against tuberculosis by either de novo drug design or virtual screening of large chemical databases. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Isotachophoretic analysis of the dihydrofolate reductase reaction in the presence of methotrexate and ascorbic acid

The antifolate methotrexate (MTX) is widely used in cancer chemotherapy. In this study, we show that MTX (MTX-Glu1) and MTX-polyglutamates (MTX-Glu2-5) strongly inhibited the growth of the leukemic cell line MOLT-4. This effect, however, was mitigated by ascorbic acid. We investigated whether ascorbic acid is able to reduce dihydrofolic acid (DHF) to tetrahydrofolic acid (THF) directly or by circumventing the MTX inhibition of dihydrofolate reductase (DHFR). The inhibition of this NADPH-dependent reduction of DHF by MTX-Glun in the absence or presence of ascorbate, was determined by analytical isotachophoresis. Using 0.01 M HCl/histidine, pH 6.0, as a leading electrolyte (L) and 0.005 M 2-(N-morpholino)ethanesulfonic acid (MES)/histidine, pH 6.0, as a terminating electrolyte (T), MTX-Glun derivatives including MTX-Glu1 could be easily separated, whereas the quantitative estimation of THF was not possible. A quantitative characterization of the DHFR reaction by measuring NADPH, NADP+ and ascorbate was achieved with another system (L: 0.01 M HCI/beta-alanine, pH 3.73; T: 0.01 M caproic acid, pH 3.27). Nanomolar concentrations of MTX-Glu1-5 inhibited consumption of NADPH and production of NADP+. Ascorbic acid was not able to reduce DHF, neither directly nor after inhibition of DHFR by MTX. However, ascorbic acid seemed to diminish the oxidation of THF and this may account for its capacity to reduce the inhibitory effect of MTX on MOLT-4 cells.     

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.     

Accelerated hydrolysis of esters catalyzed by receptors, I nonenzymatic cleavage ofp-nitrophenyl acetate by polypeptides

The hydrolysis kinetics ofp-nitrophenyl acetate by basic polypeptides with a variety of amino acid residues were analyzed. The results suggest that the tertiary structure of microenvironment brought about by random coil assembly is more important as a binding site than the number of basic residues (catalytic sites).     

基于真菌14α-去甲基化酶的非氮唑类抗真菌先导化合物的设计、合成及其体外抗真菌活性研究

基于真菌羊毛甾醇14α-去甲基化酶(CYP51)活性位点的三维结构设计并合成了新型四氢异喹啉类抗真菌先导化合物. 体外抗真菌活性研究显示: 设计的先导化合物具有较好的抗真菌活性. 其中化合物5f和5g对于5种测试菌的抗真菌活性强于或相当于对照药物氟康唑. 先导分子通过与靶酶活性腔氨基酸残基的非共价键结合产生抗真菌作用, 避免与血红素辅基Fe原子发生配位结合, 为一类具有新作用机理的非氮唑类抗真菌先导化合物. 本研究为抗真菌药物研究提供了新的结合方式及结构类型.     

A simple,rapid and reliable liquid chromatography–mass spectrometry method for determination of methotrexate in human plasma and its application to therapeutic drug monitoring

A simple, rapid and reliable liquid chromatography–electrospray ionization tandem mass spectrometry method was established and validated for the determination of methotrexate in human plasma. After a straightforward protein precipitation by acetonitrile–water (70:30, v/v), methotrexate (MTX) and p‐aminoacetophenone (used as internal standard, IS) were separated on a Column C₁₈ column (50 × 2.1 mm, 3 µm; Column Technology, Fremont, CA, USA) using a gradient elution with mobile phase of acetonitrile and 0.03% acetic acid aqueous solution at a flow rate of 0.5 mL/min. The total chromatographic runtime was 5 min for each injection. Quantification detection was performed in a triple‐quadruple tandem mass spectrometer under positive mode monitoring the following mass transitions: m/z 455.3 → 308.3 for MTX and m/z 136.1 → 94.4 for IS. The calibration curve was linear over the range of 0.05–25.0 µmol/L with a lower limit of quantification of 0.05 µmol/L. The intra‐ and interday precisions were <5.2%, the accuracy varied from ?4.1 to 4.5%. The recovery was >94%. The LC‐MS/MS method showed an excellent agreement with the existing HPLC‐UV method using Passing–Bablok regression and Bland–Altman difference plot analysis. The validated LC‐MS/MS can be successfully applied to the routine therapeutic drug monitoring of MTX in clinical laboratories. Copyright © 2015 John Wiley & Sons, Ltd.