Interactions between inhibitors of dihydrofolate reductase.
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ABSTRACT:
SUBMITTER: Bowden K
PROVIDER: S-EPMC1138366 | biostudies-other | 1989 Mar
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Dihydrofolate reductase (DHFR) reduces dihydrofolate (DHF) to tetrahydrofolate using NADPH as a cofactor. Due to its role in one carbon metabolism, chromosomal DHFR is the target of the antibacterial drug, trimethoprim. Resistance to trimethoprim has resulted in a type II DHFR that is not structurally related to the chromosomal enzyme target. Because of its metabolic significance, understanding DHFR kinetics and ligand binding behavior in more cell-like conditions, where the total macromolecule concentration can be as great as 300 mg/mL, is important. The progress-curve kinetics and ligand binding properties of the drug target (chromosomal E. coli DHFR) and the drug resistant (R67 DHFR) enzymes were studied in the presence of macromolecular cosolutes. There were varied effects on NADPH oxidation and binding to the two DHFRs, with some cosolutes increasing affinity and others weakening binding. However, DHF binding and reduction in both DHFRs decreased in the presence of all cosolutes. The decreased binding of ligands is mostly attributed to weak associations with the macromolecules, as opposed to crowder effects on the DHFRs. Computer simulations found weak, transient interactions for both ligands with several proteins. The net charge of protein cosolutes correlated with effects on NADP+ binding, with near neutral and positively charged proteins having more detrimental effects on binding. For DHF binding, effects correlated more with the size of binding pockets on the protein crowders. These nonspecific interactions between DHFR ligands and proteins predict that the in vivo efficiency of DHFRs may be much lower than expected from their in vitro rates.
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Project description:Streptococcus mutans is a major aetiological agent of dental caries. Formation of biofilms is a key virulence factor of S. mutans. Drugs that inhibit S. mutans biofilms may have therapeutic potential. Dihydrofolate reductase (DHFR) plays a critical role in regulating the metabolism of folate. DHFR inhibitors are thus potent drugs and have been explored as anticancer and antimicrobial agents. In this study, a library of analogues based on a DHFR inhibitor, trimetrexate (TMQ), an FDA-approved drug, was screened and three new analogues that selectively inhibited S. mutans were identified. The most potent inhibitor had a 50% inhibitory concentration (IC50) of 454.0±10.2nM for the biofilm and 8.7±1.9nM for DHFR of S. mutans. In contrast, the IC50 of this compound for human DHFR was ca. 1000nM, a >100-fold decrease in its potency, demonstrating the high selectivity of the analogue. An analogue that exhibited the least potency for the S. mutans biofilm also had the lowest activity towards inhibiting S. mutans DHFR, further indicating that inhibition of biofilms is related to reduced DHFR activity. These data, along with docking of the most potent analogue to the modelled DHFR structure, suggested that the TMQ analogues indeed selectively inhibited S. mutans through targeting DHFR. These potent and selective small molecules are thus promising lead compounds to develop new effective therapeutics to prevent and treat dental caries.
| S-EPMC4509821 | biostudies-literature
Project description:We report the first peptide based hDHFR inhibitors designed on the basis of structural analysis of dihydrofolate reductase (DHFR). A set of peptides were rationally designed and synthesized using solid phase peptide synthesis and characterized using nuclear magnetic resonance and enzyme immunoassays. The best candidate among them, a tetrapeptide, was chosen based on molecular mechanics calculations and evaluated in human lung adenocarcinoma cell line A549. It showed a significant reduction of cell proliferation and an IC50 of 82?µM was obtained. The interaction of the peptide with DHFR was supported by isothermal calorimetric experiments revealing a dissociation constant Kd of 0.7?µM and ?G of -34?±?1?kJ?mol-1. Conjugation with carboxylated polystyrene nanoparticles improved further its growth inhibitory effects. Taken together, this opens up new avenues to design, develop and deliver biocompatible peptide based anti-cancer agents.
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Project description:In vivo, as an advanced catalytic strategy, transient non-covalently bound multi-enzyme complexes can be formed to facilitate the relay of substrates, i. e. substrate channeling, between sequential enzymatic reactions and to enhance the throughput of multi-step enzymatic pathways. The human thymidylate synthase and dihydrofolate reductase catalyze two consecutive reactions in the folate metabolism pathway, and experiments have shown that they are very likely to bind in the same multi-enzyme complex in vivo. While reports on the protozoa thymidylate synthase-dihydrofolate reductase bifunctional enzyme give substantial evidences of substrate channeling along a surface "electrostatic highway," attention has not been paid to whether the human thymidylate synthase and dihydrofolate reductase, if they are in contact with each other in the multi-enzyme complex, are capable of substrate channeling employing surface electrostatics. This work utilizes protein-protein docking, electrostatics calculations, and Brownian dynamics to explore the existence and mechanism of the substrate channeling between the human thymidylate synthase and dihydrofolate reductase. The results show that the bound human thymidylate synthase and dihydrofolate reductase are capable of substrate channeling and the formation of the surface "electrostatic highway." The substrate channeling efficiency between the two can be reasonably high and comparable to that of the protozoa.
| S-EPMC4815301 | biostudies-literature
Project description:There is a pressing need for drugs effective against the opportunistic protozoan pathogen Cryptosporidium parvum. Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), have been widely exploited as chemotherapeutic targets. Although many DHFR inhibitors have been synthesized, only a few have been tested against C. parvum. To expedite and facilitate the discovery of effective anti-Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential inhibitors of C. parvum DHFR using the model eukaryote, Saccharomyces cerevisiae. We expressed the DHFR genes of C. parvum, Plasmodium falciparum, Toxoplasma gondii, Pneumocystis carinii, and humans in the same DHFR-deficient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the complementation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on the C. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinant C. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressing C. parvum DHFR. However, the inhibition was nonselective, as these compounds also strongly inhibited the growth of yeast dependent on the human enzyme. Conversely, the antibacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by the C. parvum enzyme. Future chemical refinement of the potent and selective lead compounds identified in this study may allow the design of an efficacious antifolate drug for the treatment of cryptosporidiosis.
| S-EPMC89807 | biostudies-literature
Project description:Resistance to trimethoprim (TMP) resulting from point mutations in the enzyme drug target dihydrofolate reductase (DHFR) drives the development of new antifolate inhibitors effective against methicillin-resistant Staphylococcus aureus (MRSA). For the past several years we have used structure-based design to create propargyl-linked antifolates that are highly potent antibacterial agents. In order to focus priority on the development of lead compounds with a low propensity to induce resistance, we prospectively evaluated resistance profiles for two of these inhibitors in an MRSA strain. By selection with the lead inhibitors, we generated resistant strains that contain single point mutations F98Y and H30N associated with TMP resistance and one novel mutation, F98I, in DHFR. Encouragingly, the pyridyl propargyl-linked inhibitor selects mutants at low frequency (6.85 × 10(-10) to 1.65 × 10(-9)) and maintains a low MIC (2.5 ?g/ml) and a low mutant prevention concentration (1.25 ?g/ml), strongly supporting its position as a lead compound. Results from this prospective screening method inform the continued design of antifolates effective against mutations at the Phe 98 position. Furthermore, the method can be used broadly to incorporate ideas for overcoming resistance early in the development process.
| S-EPMC3393473 | biostudies-literature
Project description:In this work, co-crystal screening was carried out for two important dihydrofolate reductase (DHFR) inhibitors, trimethoprim (TMP) and pyrimethamine (PMA), and for 2,4-diaminopyrimidine (DAP), which is the pharmacophore of these active pharmaceutical ingredients (API). The isomeric pyridinecarboxamides and two xanthines, theophylline (THEO) and caffeine (CAF), were used as co-formers in the same experimental conditions, in order to evaluate the potential for the pharmacophore to be used as a guide in the screening process. In silico co-crystal screening was carried out using BIOVIA COSMOquick and experimental screening was performed by mechanochemistry and supported by (solid + liquid) binary phase diagrams, infrared spectroscopy (FTIR) and X-ray powder diffraction (XRPD). The in silico prediction of low propensities for DAP, TMP and PMA to co-crystallize with pyridinecarboxamides was confirmed: a successful outcome was only observed for DAP + nicotinamide. Successful synthesis of multicomponent solid forms was achieved for all three target molecules with theophylline, with DAP co-crystals revealing a greater variety of stoichiometries. The crystalline structures of a (1:2) TMP:THEO co-crystal and of a (1:2:1) DAP:THEO:ethyl acetate solvate were solved. This work demonstrated the possible use of the pharmacophore of DHFR inhibitors as a guide for co-crystal screening, recognizing some similar trends in the outcome of association in the solid state and in the molecular aggregation in the co-crystals, characterized by the same supramolecular synthons.
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Project description:The lack of success in target-based screening approaches to the discovery of antibacterial agents has led to reemergence of phenotypic screening as a successful approach of identifying bioactive, antibacterial compounds. A challenge though with this route is then to identify the molecular target(s) and mechanism of action of the hits. This target identification, or deorphanization step, is often essential in further optimization and validation studies. Direct experimental identification of the molecular target of a screening hit is often complex, precisely because the properties and specificity of the hit are not yet optimized against that target, and so many false positives are often obtained. An alternative is to use computational, predictive, approaches to hypothesize a mechanism of action, which can then be validated in a more directed and efficient manner. Specifically here we present experimental validation of an in silico prediction from a large-scale screen performed against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis. The two potent anti-tubercular compounds studied in this case, belonging to the tetrahydro-1,3,5-triazin-2-amine (THT) family, were predicted and confirmed to be an inhibitor of dihydrofolate reductase (DHFR), a known essential Mtb gene, and already clinically validated as a drug target. Given the large number of similar screening data sets shared amongst the community, this in vitro validation of these target predictions gives weight to computational approaches to establish the mechanism of action (MoA) of novel screening hit.
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Project description:Homotetrameric R67 dihydrofolate reductase possesses 222 symmetry and a single active site pore. This situation results in a promiscuous binding site that accommodates either the substrate, dihydrofolate (DHF), or the cofactor, NADPH. NADPH interacts more directly with the protein as it is larger than the substrate. In contrast, the p-aminobenzoyl-glutamate tail of DHF, as monitored by nuclear magnetic resonance and crystallography, is disordered when bound. To explore whether smaller active site volumes (which should decrease the level of tail disorder by confinement effects) alter steady state rates, asymmetric mutations that decreased the half-pore volume by ?35% were constructed. Only minor effects on k(cat) were observed. To continue exploring the role of tail disorder in catalysis, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide-mediated cross-linking between R67 DHFR and folate was performed. A two-folate, one-tetramer complex results in the loss of enzyme activity where two symmetry-related K32 residues in the protein are cross-linked to the carboxylates of two bound folates. The tethered folate could be reduced, although with a ?30-fold decreased rate, suggesting decreased dynamics and/or suboptimal positioning of the cross-linked folate for catalysis. Computer simulations that restrain the dihydrofolate tail near K32 indicate that cross-linking still allows movement of the p-aminobenzoyl ring, which allows the reaction to occur. Finally, a bis-ethylene-diamine-?,?-amide folate adduct was synthesized; both negatively charged carboxylates in the glutamate tail were replaced with positively charged amines. The K(i) for this adduct was ?9-fold higher than for folate. These various results indicate a balance between folate tail disorder, which helps the enzyme bind substrate while dynamics facilitates catalysis.
| S-EPMC5147970 | biostudies-literature
Project description:For half a century, successful antifolate therapy against Plasmodium falciparum malaria has been attributed to host-parasite differences in drug binding to dihydrofolate reductase-thymidylate synthase (DHFR-TS). Selectivity may also arise through previously unappreciated differences in regulation of this drug target. The DHFR-TS of Plasmodium binds its cognate messenger RNA (mRNA) and inhibits its own translation. However, unlike translational regulation of DHFR or TS in humans, DHFR-TS mRNA binding is not coupled to enzyme active sites. Thus, antifolate treatment does not relieve translational inhibition and parasites cannot replenish dead enzyme.
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