Virtual screening,docking, and dynamics of potential new inhibitors of dihydrofolate reductase from Yersinia pestis

In the present work, we propose to design drugs that target the enzyme dihydrofolate redutase (DHFR) as a means of a novel drug therapy against plague. Potential inhibitors of DHFR from Yersinia pestis (YpDHFR) were selected by virtual screening and subjected to docking, molecular dynamics (MD) simulations, and Poisson–Boltzmann surface area method, in order to evaluate their interactions in the active sites of YpDHFR and human DHFR (HssDHFR). The results suggested selectivity for three compounds that were further used to propose the structures of six new potential selective inhibitors for YpDHFR.     

Analysis of Coxiela burnetti dihydrofolate reductase via in silico docking with inhibitors and molecular dynamics simulation

Coxiella burnetii is a gram-negative bacterium able to infect several eukaryotic cells, mainly monocytes and macrophages. It is found widely in nature with ticks, birds, and mammals as major hosts. C. burnetii is also the biological warfare agent that causes Q fever, a disease that has no vaccine or proven chemotherapy available. Considering the current geopolitical context, this fact reinforces the need for discovering new treatments and molecular targets for drug design against C. burnetii. Among the main molecular targets against bacterial diseases reported, the enzyme dihydrofolate reductase (DHFR) has been investigated for several infectious diseases. In the present work, we applied molecular modeling techniques to evaluate the interactions of known DHFR inhibitors in the active sites of human and C. burnetii DHFR (HssDHFR and CbDHFR) in order to investigate their potential as selective inhibitors of CbDHFR. Results showed that most of the ligands studied compete for the binding site of the substrate more effectively than the reference drug trimethoprim. Also the most promising compounds were proposed as leads for the drug design of potential CbDHFR inhibitors.     

Comparative mapping of on-targets and off-targets for the discovery of anti-trypanosomatid folate pathway inhibitors

BackgroundMulti-target approaches are necessary to properly analyze or modify the function of a biochemical pathway or a protein family. An example of such a problem is the repurposing of the known human anti-cancer drugs, antifolates, as selective anti-parasitic agents. This requires considering a set of experimentally validated protein targets in the folate pathway of major pathogenic trypanosomatid parasites and humans: (i) the primary parasite on-targets: pteridine reductase 1 (PTR1) (absent in humans) and bifunctional dihydrofolate reductase-thymidylate synthase (DHFR–TS), (ii) the primary off-targets: human DHFR and TS, and (iii) the secondary on-target: human folate receptor β, a folate/antifolate transporter.MethodsWe computationally compared the structural, dynamic and physico-chemical properties of the targets. We based our analysis on available inhibitory activity and crystallographic data, including a crystal structure of the bifunctional T. cruzi DHFR–TS with tetrahydrofolate bound determined in this work. Due to the low sequence and structural similarity of the targets analyzed, we employed a mapping of binding pockets based on the known common ligands, folate and methotrexate.ResultsOur analysis provides a set of practical strategies for the design of selective trypanosomatid folate pathway inhibitors, which are supported by enzyme inhibition measurements and crystallographic structures.ConclusionsThe ligand-based comparative computational mapping of protein binding pockets provides a basis for repurposing of anti-folates and the design of new anti-trypanosmatid agents.General significanceApart from the target-based discovery of selective compounds, our approach may be also applied for protein engineering or analyzing evolutionary relationships in protein families.     

Specificity in structure-based drug design: Identification of a novel,selective inhibitor of Pneumocystis carinii dihydrofolate reductase

Specificity is an important aspect of structure-based drug design. Distinguishing between related targets in different organisms is often the key to therapeutic success. Pneumocystis carinii is a fungal opportunist which causes a crippling pneumonia in immunocompromised individuals. We report the identification of novel inhibitors of P. cariniidihydrofolate reductase (DHFR) that are selective versus inhibition of human DHFR using computational molecular docking techniques. The Fine Chemicals Directory, a database of commercially available compounds, was screened with the DOCK program suite to produce a list of potential P. carinii DHFR inhibitors. We then used a postdocking refinement directed at discerning subtle structural and chemical features that might reflect species specificity. Of 40 compounds predicted to exhibit anti-PneumocystisDHFR activity, each of novel chemical framework, 13 (33%) show IC₅₀ values better than 150 μM in an enzyme assay. These inhibitors were further assayed against human DHFR: 10 of the 13 (77%) bind preferentially to the fungal enzyme. The most potent compound identified is a 7 μM inhibitor of P. carinii DHFR with 25-fold selectivity. The ability of molecular docking methods to locate selective inhibitors reinforces our view of structure-based drug discovery as a valuable strategy, not only for identifying lead compounds, but also for addressing receptor specificity. Proteins 29:59–67, 1997. © 1997 Wiley-Liss, Inc.     

The influence of glycerol on the binding of methotrexate to Mycobacterium tuberculosis dihydrofolate reductase: a molecular modelling study

The enzyme, dihydrofolate reductase (DHFR), from Mycobacterium tuberculosis (mt-DHFR) is believed to be a potential drug target for the treatment of tuberculosis. The co-crystal structure of mt-DHFR bound with glycerol (GOL), NAPDH and methotrexate (MTX) reveals a GOL binding site on the enzyme. This GOL binding site could be very important for the design of novel, selective mt-DHFR inhibitors, because this binding site is absent on human DHFR (h-DHFR). We have performed molecular dynamic simulations and free energy calculations to evaluate the binding affinity of GOL and its free energy contribution to the binding of MTX to mt-DHFR. The results showed that GOL does not bind tightly to mt-DHFR. Although GOL itself contributed free energy on MTX binding to mt-DHFR, GOL also increased the flexibilities of MTX, so that MTX cannot maintain strong electronic interactions with ARG32 and ARG60, which caused the total binding free energy to decrease. These data suggest that GOL binding is weak and it could be expelled from the binding site, to allow inhibitors containing appropriate side chains to bind. This observation can be used to inform future drug design studies, especially those aimed at improving drug selectivity against h-DHFR.     

Three-dimensional structure of M. tuberculosis dihydrofolate reductase reveals opportunities for the design of novel tuberculosis drugs

Dihydrofolate reductase (DHFR) catalyzes the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate and is essential for the synthesis of thymidylate, purines and several amino acids. Inhibition of the enzyme's activity leads to arrest of DNA synthesis and cell death. The enzyme has been studied extensively as a drug target for bacterial, protozoal and fungal infections, and also for neoplastic and autoimmune diseases. Here, we report the crystal structure of dihydrofolate reductase from Mycobacterium tuberculosis, a human pathogen responsible for the death of millions of human beings per year. Three crystal structures of ternary complexes of M. tuberculosis DHFR with NADP and different inhibitors have been determined, as well as the binary complex with NADP, with resolutions ranging from 1.7 to 2.0 A. The three DHFR inhibitors are the anticancer drug methotrexate, the antimicrobial trimethoprim and Br-WR99210, an analogue of the antimalarial agent WR99210. Structural comparison of these complexes with human dihydrofolate reductase indicates that the overall protein folds are similar, despite only 26 % sequence identity, but that the environments of both NADP and of the inhibitors contain interesting differences between the enzymes from host and pathogen. Specifically, residues Ala101 and Leu102 near the N6 of NADP are distinctly more hydrophobic in the M. tuberculosis than in the human enzyme. Another striking difference occurs in a region near atoms N1 and N8 of methotrexate, which is also near atom N1 of trimethoprim, and near the N1 and two methyl groups of Br-WR99210. A glycerol molecule binds here in a pocket of the M. tuberculosis DHFR:MTX complex, while this pocket is essentially filled with hydrophobic side-chains in the human enzyme. These differences between the enzymes from pathogen and host provide opportunities for designing new selective inhibitors of M. tuberculosis DHFR.     

Comparative protein modeling of spermidine synthase from Plasmodium falciparum: A potential target for anti-malarial drug therapy

Malaria, caused by protozoan parasites of the genus Plasmodium, affects up to 500 million individuals and kills over 1 million people every year. The increasing resistance of the malaria parasites has enforced strategies for finding new drug targets. In recent years, enzymes associated with the polyamine metabolism have attracted attention as drug targets. Cytosolic Plasmodium falciparum spermidine synthase (PfPAPT) is a potential target for antimalarial chemotherapy. Contrasting with the other enzymes involved in the parasite polyamine amine biosynthesis, little information is available about this enzyme, and its crystallographic structure is unknown yet. In this paper I propose a theoretical low-resolution 3D model for PfPAPT based on crystal structure of the Arabidopsis thaliana, by multiple alignment followed by intensive optimization; validation and dynamic simulations in water. Comparison between the active sites of PfPAPT and human PAPT revealed key differences that could be useful for the design of new selective inhibitors of Plasmodium PAPT.     

Synthesis and characterization of potent inhibitors of Trypanosoma cruzi dihydrofolate reductase

Dihydrofolate reductase (DHFR) of the parasite Trypanosoma cruzi (T. cruzi) is a potential target for developing drugs to treat Chagas’ disease. We have undertaken a detailed structure–activity study of this enzyme. We report here synthesis and characterization of six potent inhibitors of the parasitic enzyme. Inhibitory activity of each compound was determined against T. cruzi and human DHFR. One of these compounds, ethyl 4-(5-[(2,4-diamino-6-quinazolinyl)methyl]amino-2-methoxyphenoxy)butanoate (6b) was co-crystallized with the bifunctional dihydrofolate reductase-thymidylate synthase enzyme of T. cruzi and the crystal structure of the ternary enzyme:cofactor:inhibitor complex was determined. Molecular docking was used to analyze the potential interactions of all inhibitors with T. cruzi DHFR and human DHFR. Inhibitory activities of these compounds are discussed in the light of enzyme–ligand interactions. Binding affinities of each inhibitor for the respective enzymes were calculated based on the experimental or docked binding mode. An estimated 60–70% of the total binding energy is contributed by the 2,4-diaminoquinazoline scaffold.     

A three-dimensional structure of Plasmodium falciparum serine hydroxymethyltransferase in complex with glycine and 5-formyl-tetrahydrofolate. Homology modeling and molecular dynamics

Cytosolic Plasmodium falciparum serine hydroxymethyltransferase (pfSHMT) is a potential target for antimalarial chemotherapy. Contrasting with the other enzymes involved in the parasite folate cycle, little information is available about this enzyme, and its crystallographic structure is unknown yet. In this paper, we propose a theoretical low-resolution 3D model for pfSHMT in complex with glycine and 5-formyl tetrahydrofolate (5-FTHF) based on homology modeling by multiple alignment followed by intensive optimization, validation and dynamics simulations in water. Comparison between the active sites of our model and that of crystallographic Human SHMT (hSHMT) revealed key differences that could be useful for the design of new selective inhibitors of pfSHMT.     

Crystal structure of the C183S/C217S mutant of human CA VII in complex with acetazolamide

Human carbonic anhydrase VII (hCA VII) is a cytosolic member of the α-CA family. This enzyme is mainly localized in a number of brain tissues such as the cortex, hippocampus and thalamus and has been noted for its contribution in generating neuronal excitation and seizures. Recently, it has been also proposed that hCA VII may be involved in the control of neuropathic pain, thus its inhibition may offer a new approach in designing pain killers useful for combating neuropathic pain. We report here the X-ray crystallographic structure of a mutated form of human CA VII in complex with acetazolamide, a classical sulfonamide inhibitor. These crystallographic studies provide important implications for the rational drug design of selective CA inhibitors with clinical applications.     

Mobility of the spin-labeled side chains of some novel antifolate inhibitors in their complexes with dihydrofolate reductase

Four spin-labeled inhibitors of dihydrofolate reductase (DHFR) have been synthesized, each of which has the 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO) reporting group at a different distance from the 2,4-diaminopyrimidine moiety by which the inhibitors are anchored and oriented in the active site. Inhibitors in which the TEMPO group is attached by a short side chain are weakly bound to DHFR from bacteria (Streptococcus faecium and Lactobacillus casei), to the bovine enzyme and to recombinant human DHFR. However, binding is sufficiently tight, especially in the ternary complexes with NADPH, for recording of the EPR spectra of the bound ligands. The spectra indicate that when these inhibitors are bound to the enzyme the TEMPO group is highly immobilized with correlation time, tau c, 4-20ns. Inhibitors that have the reporter group attached to the glutamate moiety of methotrexate bind to all four DHFRs more tightly than the inhibitors with shorter side chains by factors of up to 10(6). However, in most complexes formed by the inhibitors with longer side chains immobilization of the TEMPO group is slight (tau c 0.2-4 ns). These results are in general agreement with predictions from X-ray crystallographic results including thermal factors but there are some unanticipated differences between some results for bacterial and eukaryotic enzymes. Three of the splin-labeled inhibitors would provide good probes for distance measurements in and around the active site of mammalian DHFR.     

Human dihydrofolate reductase and thymidylate synthase form a complex in vitro and co-localize in normal and cancer cells

Enzymes involved in thymidylate biosynthesis, thymidylate synthase (TS), and dihydrofolate reductase (DHFR) are well-known targets in cancer chemotherapy. In this study, we demonstrated for the first time, that human TS and DHFR form a strong complex in vitro and co-localize in human normal and colon cancer cell cytoplasm and nucleus. Treatment of cancer cells with methotrexate or 5-fluorouracil did not affect the distribution of either enzyme within the cells. However, 5-FU, but not MTX, lowered the presence of DHFR-TS complex in the nucleus by 2.5-fold. The results may suggest the sequestering of TS by FdUMP in the cytoplasm and thereby affecting the translocation of DHFR-TS complex to the nucleus. Providing a strong likelihood of DHFR-TS complex formation in vivo, the latter complex is a potential new drug target in cancer therapy. In this paper, known 3D structures of human TS and human DHFR, and some protozoan bifunctional DHFR-TS structures as templates, are used to build an in silico model of human DHFR–TS complex structure, consisting of one TS dimer and two DHFR monomers. This complex structure may serve as an initial 3D drug target model for prospective inhibitors targeting interfaces between the DHFR and TS enzymes.     

Structure-based approach to pharmacophore identification, in silico screening, and three-dimensional quantitative structure-activity relationship studies for inhibitors of Trypanosoma cruzi dihydrofolate reductase function

We have employed a structure-based three-dimensional quantitative structure-activity relationship (3D-QSAR) approach to predict the biochemical activity for inhibitors of T. cruzi dihydrofolate reductase-thymidylate synthase (DHFR-TS). Crystal structures of complexes of the enzyme with eight different inhibitors of the DHFR activity together with the structure in the substrate-free state (DHFR domain) were used to validate and refine docking poses of ligands that constitute likely active conformations. Structural information from these complexes formed the basis for the structure-based alignment used as input for the QSAR study. Contrary to indirect ligand-based approaches the strategy described here employs a direct receptor-based approach. The goal is to generate a library of selective lead inhibitors for further development as antiparasitic agents. 3D-QSAR models were obtained for T. cruzi DHFR-TS (30 inhibitors in learning set) and human DHFR (36 inhibitors in learning set) that show a very good agreement between experimental and predicted enzyme inhibition data. For crossvalidation of the QSAR model(s), we have used the 10% leave-one-out method. The derived 3D-QSAR models were tested against a few selected compounds (a small test set of six inhibitors for each enzyme) with known activity, which were not part of the learning set, and the quality of prediction of the initial 3D-QSAR models demonstrated that such studies are feasible. Further refinement of the models through integration of additional activity data and optimization of reliable docking poses is expected to lead to an improved predictive ability.     

Molecular docking studies on DMDP derivatives as human DHFR inhibitors

Molecular docking is routinely used for understanding drug‐receptor interaction in modern drug design. Here, we describe the docking of 2, 4-diamino-5-methyl-5-deazapteridine (DMDP) derivatives as inhibitors to human dihydrofolate reductase (DHFR). We docked 78 DMDP derivates collected from literature to DHFR and studied their specific interactions with DHFR. A new shape-based method, LigandFit, was used for docking DMDP derivatives into DHFR active sites. The result indicates that the molecular docking approach is reliable and produces a good correlation coefficient (r² = 0.499) for the 73 compounds between docking score and IC₅₀ values (Inhibitory Activity). The chloro substituted naphthyl ring of compound 63 makes significant hydrophobic contact with Leu 22, Phe 31 and Pro 61 of the DHFR active site leading to enhanced inhibition of the enzyme. The docked complexes provide better insights to design more potent DHFR inhibitors prior to their synthesis.     

Design,docking studies and molecular dynamics of new potential selective inhibitors of Plasmodium falciparum serine hydroxymethyltransferase

In this paper, former studies on the interactions of the natural substrate and potential inhibitors of Plasmodium falciparum serine hydroxymethyltransferase (PfSHMT) were used to design five new potential selective inhibitors to this enzyme. Results of the docking energies calculations of these structures inside the active sites of PfSHMT and human SHMT were used to select a more suitable structure as a potential selective inhibitor to PfSHMT. Further molecular dynamics studies of this molecule and 5-formyl-6-hydrofolic acid (natural substrate) docked inside these enzymes' active sites revealed important features for additional refinements of this structure and also additional residues in the PfSHMT active site to be considered further for designing selective inhibitors.     

Cloning,recombinant expression and inhibitor profiles of dihydrofolate reductase from the Australian sheep blow fly,Lucilia cuprina

While dihydrofolate reductase (DHFR) is an important drug target in mammals, bacteria and protozoa, no inhibitors of this enzyme have been developed as commercial insecticides. We therefore examined the potential of this enzyme as a drug target in an important ectoparasite of livestock, the Australian sheep blow fly, Lucilia cuprina (Diptera: Calliphoridae) (Wiedemann). The non‐specific DHFR inhibitors aminopterin and methotrexate significantly inhibited the growth of L. cuprina larvae, with IC₅₀ values at µg levels. Trimethoprim and pyrimethamine were 5–30‐fold less active. Relative IC₅₀ values for the inhibition of recombinant L. cuprina DHFR by various inhibitors were in accordance with their relative effects on larval growth. The active‐site amino acid residues of L. cuprina DHFR differed by between 34% and 50% when compared with two mammalian species, as well as two bacteria and two protozoa. There were significant charge and size differences in specific residues between the blow fly and human DHFR enzymes, notably the L. cuprina Asn21, Lys31 and Lys63 residues. This study provides bioassay evidence to highlight the potential of blow fly DHFR as an insecticide target, and describes differences in active site residues between blow flies and other organisms which could be exploited in the design of blow fly control chemicals.     

Structural studies provide clues for analog design of specific inhibitors of Cryptosporidium hominis thymidylate synthase–dihydrofolate reductase

Cryptosporidium is the causative agent of a gastrointestinal disease, cryptosporidiosis, which is often fatal in immunocompromised individuals and children. Thymidylate synthase (TS) and dihydrofolate reductase (DHFR) are essential enzymes in the folate biosynthesis pathway and are well established as drug targets in cancer, bacterial infections, and malaria. Cryptosporidium hominis has a bifunctional thymidylate synthase and dihydrofolate reductase enzyme, compared to separate enzymes in the host. We evaluated lead compound 1 from a novel series of antifolates, 2-amino-4-oxo-5-substituted pyrrolo[2,3-d]pyrimidines as an inhibitor of Cryptosporidium hominis thymidylate synthase with selectivity over the human enzyme. Complementing the enzyme inhibition compound 1 also has anti-cryptosporidial activity in cell culture. A crystal structure with compound 1 bound to the TS active site is discussed in terms of several van der Waals, hydrophobic and hydrogen bond interactions with the protein residues and the substrate analog 5-fluorodeoxyuridine monophosphate (TS), cofactor NADPH and inhibitor methotrexate (DHFR). Another crystal structure in complex with compound 1 bound in both the TS and DHFR active sites is also reported here. The crystal structures provide clues for analog design and for the design of ChTS–DHFR specific inhibitors.     

Bifunctional thymidylate synthase-dihydrofolate reductase in protozoa

Protozoa contain thymidylate synthase (TS) and dihydrofolate reductase (DHFR) on the same polypeptide. In the bifunctional protein, the DHFR domain is on the amino terminus, TS is on the carboxyl terminus, and the two domains are separated by a junction peptide of varying size depending on the source. The native protein is composed of a dimer of two such subunits and is 110-140 kDa. Most studies of the bifunctional TS-DHFR have been performed with the protein from anti-folate resistant strains of Leishmania major, which show amplification of the TS-DHFR gene and overproduction of the bifunctional protein. The Leishmania TS-DHFR has also been highly expressed in heterologous systems. There appears to be extensive communication among domains and channeling of the H2folate product of TS to DHFR. Anti-folates commonly used to treat microbial infections are poor inhibitors of L. major DHFR. However, selective inhibition of L. major vs. human DHFR does not appear difficult to achieve, and selective inhibitors are known. The TS-DHFR from Plasmodium falciparum has also been cloned and has recently been expressed in Escherichia coli, albeit in small amounts. Interestingly, pyrimethamine-resistant strains of P. falciparum all have a common point mutation in the DHFR coding sequence (Thr/Ser 108 to Asn), which causes decreased binding of the folate analog. It is suggested that if an appropriate inhibitor of the pyrimethamine-resistant P. falciparum DHFRs can be found, it may serve in combination with pyrimethamine as an antimalarial regimen with low propensity for the development of resistance. In the future, we project that we will have a detailed knowledge of the structure and function of TS-DHFRs, and have the essential tools necessary for a molecular-based approach to drug design.     

Determining the structural basis for specificity of ligands using crystallographic screening

Crystallographic screening has been used to identify new inhibitors for potential target for drug development. Here, we describe the application of the crystallographic screening to assess the structural basis of specificity of ligands against a protein target. The method is efficient and results in detailed crystallographic information. The utility of the method is demonstrated in the study of the structural basis for specificity of ligands for human purine nucleoside phosphorylase (PNP). Purine nucleoside phosphorylase catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleotides and deoxynucleosides. This enzyme is a target for inhibitor development aiming at T-cell immune response modulation and has been submitted to extensive structure-based drug design. This methodology may help in the future development of a new generation of PNP inhibitors.     

Novel non-active site inhibitor of Cryptosporidium hominis TS-DHFR identified by a virtual screen

The essential enzyme thymidylate synthase-dihydrofolate reductase (TS-DHFR) is a validated drug target for many pathogens, but has been elusive in Cryptosporidium hominis, as active site inhibitors of the enzymes from related parasitic protozoa show decreased potency and lack of species specificity over the human enzymes. As a rational approach to discover novel inhibitors, we conducted a virtual screen of a non-active site pocket in the DHFR linker region. From this screen, we have identified and characterized a noncompetitive inhibitor, flavin mononucleotide (FMN), with micromolar potency that is selective for ChTS-DHFR versus the human enzymes. These results describe a novel allosteric pocket amenable to inhibitor targeting, and a lead compound with which to move towards potent, selective inhibitors of ChTS-DHFR.