RESUMEN
Folate enzymes, namely, dihydrofolate reductase (DHFR) and pteridine reductase (PTR1) are acknowledged targets for the development of antiparasitic agents against Trypanosomiasis and Leishmaniasis. Based on the amino dihydrotriazine motif of the drug Cycloguanil (Cyc), a known inhibitor of both folate enzymes, we have identified two novel series of inhibitors, the 2-amino triazino benzimidazoles (1) and 2-guanidino benzimidazoles (2), as their open ring analogues. Enzymatic screening was carried out against PTR1, DHFR, and thymidylate synthase (TS). The crystal structures of TbDHFR and TbPTR1 in complex with selected compounds experienced in both cases a substrate-like binding mode and allowed the rationalization of the main chemical features supporting the inhibitor ability to target folate enzymes. Biological evaluation of both series was performed against T. brucei and L. infantum and the toxicity against THP-1 human macrophages. Notably, the 5,6-dimethyl-2-guanidinobenzimidazole 2g resulted to be the most potent (Ki = 9 nM) and highly selective TbDHFR inhibitor, 6000-fold over TbPTR1 and 394-fold over hDHFR. The 5,6-dimethyl tricyclic analogue 1g, despite showing a lower potency and selectivity profile than 2g, shared a comparable antiparasitic activity against T. brucei in the low micromolar domain. The dichloro-substituted 2-guanidino benzimidazoles 2c and 2d revealed their potent and broad-spectrum antitrypanosomatid activity affecting the growth of T. brucei and L. infantum parasites. Therefore, both chemotypes could represent promising templates that could be valorized for further drug development.
Asunto(s)
Antagonistas del Ácido Fólico , Tetrahidrofolato Deshidrogenasa , Triazinas , Trypanosoma brucei brucei , Trypanosoma brucei brucei/efectos de los fármacos , Trypanosoma brucei brucei/enzimología , Humanos , Tetrahidrofolato Deshidrogenasa/metabolismo , Tetrahidrofolato Deshidrogenasa/química , Antagonistas del Ácido Fólico/farmacología , Antagonistas del Ácido Fólico/química , Triazinas/farmacología , Triazinas/química , Tripanocidas/farmacología , Tripanocidas/química , Proguanil/farmacología , Proguanil/química , Timidilato Sintasa/antagonistas & inhibidores , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Leishmania infantum/efectos de los fármacos , Leishmania infantum/enzimología , Bencimidazoles/farmacología , Bencimidazoles/química , Relación Estructura-Actividad , Antiprotozoarios/farmacología , Antiprotozoarios/química , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/metabolismo , Proteínas Protozoarias/química , OxidorreductasasRESUMEN
Human thymidylate synthase (hTS) is a validated drug target for chemotherapy. A virtual screening experiment was used to prioritize a list of compounds from African Natural Products Databases docked against the orthosteric binding pocket of hTS. Consensus scores of binding affinities from ensemble-based virtual screening, hydrated docking and MM-PBSA calculations ranked compounds NEA4433 and NEA4434 as the best candidates owing to binding affinity scores in the picomolar order, their excellent ADMET profiles and the good stability of the protein-ligand complexes formed. The current study demonstrates the role of water in small molecule binding to hTS in mediating protein-ligand interactions. Similarly, the robust ensemble docking (relaxed scheme complex) ranked NEA4433 and NEA4434 as the best candidates. Furthermore, the best candidates prioritized were shown to strongly interact with the same residues that interacted with hTS substrate and cofactor.
Asunto(s)
Timidilato Sintasa , Humanos , Timidilato Sintasa/química , Simulación del Acoplamiento Molecular , Ligandos , Unión ProteicaRESUMEN
Drugs that target human thymidylate synthase (hTS), a dimeric enzyme, are widely used in anticancer therapy. However, treatment with classical substrate-site-directed TS inhibitors induces over-expression of this protein and development of drug resistance. We thus pursued an alternative strategy that led us to the discovery of TS-dimer destabilizers. These compounds bind at the monomer-monomer interface and shift the dimerization equilibrium of both the recombinant and the intracellular protein toward the inactive monomers. A structural, spectroscopic, and kinetic investigation has provided evidence and quantitative information on the effects of the interaction of these small molecules with hTS. Focusing on the best among them, E7, we have shown that it inhibits hTS in cancer cells and accelerates its proteasomal degradation, thus causing a decrease in the enzyme intracellular level. E7 also showed a superior anticancer profile to fluorouracil in a mouse model of human pancreatic and ovarian cancer. Thus, over sixty years after the discovery of the first TS prodrug inhibitor, fluorouracil, E7 breaks the link between TS inhibition and enhanced expression in response, providing a strategy to fight drug-resistant cancers.
Asunto(s)
Neoplasias Ováricas , Timidilato Sintasa , Femenino , Animales , Ratones , Humanos , Sitios de Unión , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Fluorouracilo/farmacología , Neoplasias Ováricas/tratamiento farmacológico , Inhibidores Enzimáticos/farmacologíaRESUMEN
Human thymidylate synthase (hTS) is essential for DNA replication and therefore a therapeutic target for cancer. Effective targeting requires knowledge of the mechanism(s) of regulation of this 72 kDa homodimeric enzyme. Here, we investigate the mechanism of binding cooperativity of the nucleotide substrate. We have employed exquisitely sensitive methyl-based CPMG and CEST NMR experiments enabling us to identify residues undergoing bifurcated linear 3-state exchange, including concerted switching between active and inactive conformations in the apo enzyme. The inactive state is populated to only ~1.3%, indicating that conformational selection contributes negligibly to the cooperativity. Instead, methyl rotation axis order parameters, determined by 2H transverse relaxation rates, suggest that rigidification of the enzyme upon substrate binding is responsible for the entropically-driven cooperativity. Lack of the rigidification in product binding and substrate binding to an N-terminally truncated enzyme, both non-cooperative, support this idea. In addition, the lack of this rigidification in the N-terminal truncation indicates that interactions between the flexible N-terminus and the rest of the protein, which are perturbed by substrate binding, play a significant role in the cooperativity-a novel mechanism of dynamic allostery. Together, these findings yield a rare depth of insight into the substrate binding cooperativity of an essential enzyme.
Asunto(s)
Nucleótidos , Timidilato Sintasa , Humanos , Conformación Molecular , Nucleótidos/metabolismo , Unión Proteica , Conformación Proteica , Timidilato Sintasa/química , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismoRESUMEN
Breast cancer is a major cause of death in women worldwide. In this study, 60 female rats were classified into 6 groups; negative control, α-aminophosphonates, arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one, DMBA, DMBA & α-aminophosphonates, and DMBA & arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one. New α-aminophosphonates and arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one were synthesized and elucidated by different spectroscopic and elemental analysis. Histopathological examination showed marked proliferation of cancer cells in the DMBA group. Treatment with α-aminophosphonates mainly decreased tumor mass. Bcl2 expression increased in DMBA-administered rats and then declined in the treated groups, mostly with α-aminophosphonates. The level of CA15-3 markedly declined in DMBA groups treated with α-aminophosphonates and arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one. Gene expression of GST-P, PCNA, PDK, and PIK3CA decreased in the DMBA group treated with α-aminophosphonates and arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one, whereas PIK3R1 and BAX increased in the DMBA group treated with α-aminophosphonates and arylidine derivatives of 3-acetyl-1-aminoquinolin-2(1H)-one. The molecular docking postulated that the investigated compounds can inhibt the Thymidylate synthase TM due to high hydrophobicity charachter.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias Mamarias Experimentales/tratamiento farmacológico , Timidilato Sintasa/antagonistas & inhibidores , 9,10-Dimetil-1,2-benzantraceno , Animales , Antineoplásicos/farmacología , Células CACO-2 , Simulación por Computador , Evaluación Preclínica de Medicamentos , Femenino , Peces , Humanos , Neoplasias Mamarias Experimentales/inducido químicamente , Neoplasias Mamarias Experimentales/patología , Modelos Moleculares , Simulación del Acoplamiento Molecular , Terapia Molecular Dirigida/métodos , Organofosfonatos/síntesis química , Organofosfonatos/química , Organofosfonatos/farmacología , Organofosfonatos/uso terapéutico , Extractos Vegetales , Quinolinas/síntesis química , Quinolinas/química , Quinolinas/farmacología , Quinolinas/uso terapéutico , Ratas , Timidilato Sintasa/químicaRESUMEN
Thymidylate is a vital DNA precursor synthesized by thymidylate synthases. ThyX is a flavin-dependent thymidylate synthase found in several human pathogens and absent in humans, which makes it a potential target for antimicrobial drugs. This enzyme methylates the 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate (dTMP) using a reduced flavin adenine dinucleotide (FADH-) as prosthetic group and (6R)-N5,N10-methylene-5,6,7,8-tetrahydrofolate (CH2THF) as a methylene donor. Recently, it was shown that ThyX-catalyzed reaction is a complex process wherein FADH- promotes both methylene transfer and reduction of the transferred methylene into a methyl group. Here, we studied the dynamic and photophysics of FADH- bound to ThyX, in several substrate-binding states (no substrate, in the presence of dUMP or folate or both) by femtosecond transient absorption spectroscopy. This methodology provides valuable information about the ground-state configuration of the isoalloxazine moiety of FADH- and the rigidity of its local environment, through spectra shape and excited-state lifetime parameters. In the absence of substrate, the environment of FADH- in ThyX is only mildly more constrained than that of free FADH- in solution. The addition of dUMP however narrows the distribution of ground-state configurations and increases the constraints on the butterfly bending motion in the excited state. Folate binding results in the selection of new ground-state configurations, presumably located at a greater distance from the conical intersection where excited-state decay occurs. When both substrates are present, the ground-state configuration appears on the contrary rather limited to a geometry close to the conical intersection, which explains the relatively fast excited-state decay (100 ps on the average), even if the environment of the isoalloxazine is densely packed. Hence, although the environment of the flavin is dramatically constrained, FADH- retains a dynamic necessary to shuttle carbon from folate to dUMP. Our study demonstrates the high sensitivity of FADH- photophysics to the constraints exerted by its immediate surroundings.
Asunto(s)
Dinitrocresoles/metabolismo , Simulación de Dinámica Molecular , Timidilato Sintasa/metabolismo , Biocatálisis , Dinitrocresoles/química , Estructura Molecular , Oxidación-Reducción , Thermotoga maritima/enzimología , Timidilato Sintasa/químicaRESUMEN
Folate enzyme cofactors and their derivatives have the unique ability to provide a single carbon unit at different oxidation levels for the de novo synthesis of amino-acids, purines, or thymidylate, an essential DNA nucleotide. How these cofactors mediate methylene transfer is not fully settled yet, particularly with regard to how the methylene is transferred to the methylene acceptor. Here, we uncovered that the bacterial thymidylate synthase ThyX, which relies on both folate and flavin for activity, can also use a formaldehyde-shunt to directly synthesize thymidylate. Combining biochemical, spectroscopic and anaerobic crystallographic analyses, we showed that formaldehyde reacts with the reduced flavin coenzyme to form a carbinolamine intermediate used by ThyX for dUMP methylation. The crystallographic structure of this intermediate reveals how ThyX activates formaldehyde and uses it, with the assistance of active site residues, to methylate dUMP. Our results reveal that carbinolamine species promote methylene transfer and suggest that the use of a CH2O-shunt may be relevant in several other important folate-dependent reactions.
Asunto(s)
Formaldehído/metabolismo , Nucleótidos/metabolismo , Thermotoga maritima/enzimología , Timidilato Sintasa/metabolismo , Biocatálisis , Espectroscopía de Resonancia Magnética con Carbono-13 , Dominio Catalítico , Activación Enzimática , Flavinas/metabolismo , Metilación , Electricidad Estática , Timidilato Sintasa/químicaRESUMEN
Novel evidence is presented allowing further clarification of the mechanism of the slow-binding thymidylate synthase (TS) inhibition by N4-hydroxy-dCMP (N4-OH-dCMP). Spectrophotometric monitoring documented time- and temperature-, and N4-OH-dCMP-dependent TS-catalyzed dihydrofolate production, accompanying the mouse enzyme incubation with N4-OH-dCMP and N5,10-methylenetetrahydrofolate, known to inactivate the enzyme by the covalent binding of the inhibitor, suggesting the demonstrated reaction to be uncoupled from the pyrimidine C(5) methylation. The latter was in accord with the hypothesis based on the previously presented structure of mouse TS (cf. PDB ID: 4EZ8), and with conclusions based on the present structure of the parasitic nematode Trichinella spiralis, both co-crystallized with N4-OH-dCMP and N5,10-methylenetetrahdrofolate. The crystal structure of the mouse TS-N4-OH-dCMP complex soaked with N5,10-methylenetetrahydrofolate revealed the reaction to run via a unique imidazolidine ring opening, leaving the one-carbon group bound to the N(10) atom, thus too distant from the pyrimidine C(5) atom to enable the electrophilic attack and methylene group transfer.
Asunto(s)
Desoxicitidina Monofosfato/análogos & derivados , Inhibidores Enzimáticos/farmacología , Timidilato Sintasa/antagonistas & inhibidores , Trichinella/enzimología , Animales , Cristalografía por Rayos X , Desoxicitidina Monofosfato/química , Desoxicitidina Monofosfato/farmacología , Inhibidores Enzimáticos/química , Humanos , Ratones , Simulación del Acoplamiento Molecular , Espectrofotometría , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Triquinelosis/parasitologíaRESUMEN
Tetrahydrofolic acid and formaldehyde are key human metabolites but their physiologically relevant chemistry is undefined. Our NMR studies confirm formaldehyde as a product of tetrahydrofolic acid degradation but also reveal their reaction regulates the stability of tetrahydrofolic acid. These observations identify a novel non-enzymatic feedback mechanism regulating formaldehyde and folate metabolism that has important implications for folate-targeting chemotherapy in cancer and other diseases.
Asunto(s)
Formaldehído/metabolismo , Tetrahidrofolatos/metabolismo , Timidilato Sintasa/metabolismo , Biocatálisis , Estabilidad de Enzimas , Formaldehído/química , Humanos , Estructura Molecular , Tetrahidrofolatos/química , Timidilato Sintasa/químicaRESUMEN
Methylation of 2-deoxyuridine-5'-monophosphate (dUMP) at the C5 position by the obligate dimeric thymidylate synthase (TSase) in the sole de novo biosynthetic pathway to thymidine 5'-monophosphate (dTMP) proceeds by forming a covalent ternary complex with dUMP and cosubstrate 5,10-methylenetetrahydrofolate. The crystal structure of an analog of this intermediate gives important mechanistic insights but does not explain the half-of-the-sites activity of the enzyme. Recent experiments showed that the C5 proton and the catalytic Cys are eliminated in a concerted manner from the covalent ternary complex to produce a noncovalent bisubstrate intermediate. Here, we report the crystal structure of TSase with a close synthetic analog of this intermediate in which it has partially reacted with the enzyme but in only one protomer, consistent with the half-of-the-sites activity of this enzyme. Quantum mechanics/molecular mechanics simulations confirmed that the analog could undergo catalysis. The crystal structure shows a new water 2.9 Å from the critical C5 of the dUMP moiety, which in conjunction with other residues in the network, may be the elusive general base that abstracts the C5 proton of dUMP during the reaction.
Asunto(s)
Timidilato Sintasa/química , Dominio Catalítico , Cristalografía por Rayos X , Cinética , Modelos Moleculares , Timidilato Sintasa/metabolismoRESUMEN
A homo-dimeric enzyme, thymidylate synthase (TS), has been a long-standing molecular target in chemotherapy. To further elucidate properties and interactions with ligands of wild-type mouse thymidylate synthase (mTS) and its two single mutants, H190A and W103G, spectroscopic and theoretical investigations have been employed. In these mutants, histidine at position 190 and tryptophan at position 103 are substituted with alanine and glycine, respectively. Several emission-based spectroscopy methods used in the paper demonstrate an especially important role for Trp 103 in TS ligands binding. In addition, the Advanced Poisson-Boltzmann Solver (APBS) results show considerable differences in the distribution of electrostatic potential around Trp 103, as compared to distributions observed for all remaining Trp residues in the mTS family of structures. Together, spectroscopic and APBS results reveal a possible interplay between Trp 103 and His190, which contributes to a reduction in enzymatic activity in the case of H190A mutation. Comparison of electrostatic potential for mTS complexes, and their mutants, with the substrate, dUMP, and inhibitors, FdUMP and N4-OH-dCMP, suggests its weaker influence on the enzyme-ligand interactions in N4OH-dCMP-mTS compared to dUMP-mTS and FdUMP-mTS complexes. This difference may be crucial for the explanation of the "abortive reaction" inhibitory mechanism of N4OH-dCMP towards TS. In addition, based on structural analyses and the H190A mutant capacity to form a denaturation-resistant complex with N4-OH-dCMP in the mTHF-dependent reaction, His190 is apparently responsible for a strong preference of the enzyme active center for the anti rotamer of the imino inhibitor form.
Asunto(s)
Nucleótidos de Desoxiuracil/metabolismo , Modelos Teóricos , Espectrometría de Fluorescencia/métodos , Electricidad Estática , Timidilato Sintasa/metabolismo , Sustitución de Aminoácidos , Animales , Desoxicitidina Monofosfato/análogos & derivados , Desoxicitidina Monofosfato/metabolismo , Nucleótidos de Desoxiuracil/química , Fluorodesoxiuridilato/metabolismo , Ratones , Modelos Moleculares , Análisis Multivariante , Conformación Proteica , Timidilato Sintasa/químicaRESUMEN
The observables associated with protein intrinsic fluorescence - spectra, time decays, anisotropies - offer opportunities to monitor in real time and non-invasively a protein's functional form and its interchange with other forms with different functions. We employed these observables to sketch the fluorometric profiles of two functional forms of human thymidylate synthase (hTS), a homodimeric enzyme crucial for cell proliferation and thus targeted by anticancer drugs. The protein takes an active and an inactive form. Stabilization of the latter by peptides that, unlike classical hTS inhibitors, bind it at the monomer/monomer interface offers an alternative inhibition mechanism that promises to avoid the onset of drug resistance in anticancer therapy. The fluorescence features depicted herein can be used as tools to identify and quantify each of the two protein forms in solution, thus making it possible to investigate the kinetic and thermodynamic aspects of the active/inactive conformational interchange. Two examples of fluorometrically monitored interconversion kinetics are provided.
Asunto(s)
Polarización de Fluorescencia , Timidilato Sintasa/química , Nucleótidos de Desoxiuracil/química , Nucleótidos de Desoxiuracil/metabolismo , Humanos , Cinética , Simulación de Dinámica Molecular , Mutagénesis Sitio-Dirigida , Estructura Cuaternaria de Proteína , Timidilato Sintasa/genética , Timidilato Sintasa/metabolismoRESUMEN
White spot syndrome virus (WSSV), the causative agent of white spot disease (WSD) severely affecting crustacean life forms, is highly contagious and forms the principal cause of massive economic losses in the shrimp aquaculture industry. Previous studies have demonstrated thymidylate synthase as a successful anti-cancer therapeutic drug target, leading to various anti-cancer drugs. The differential utilization of nucleotide precursors between white spot syndrome virus and shrimp encouraged us to analyze WSSV-thymidylate synthase (wTS). Here, we report the crystal structures of wTS in its apo-form and as a ternary complex with deoxyuridine monophosphate (dUMP) and methotrexate at a resolution of 2.35 Å and 2.6 Å, respectively. wTS possesses a fold characteristic to known thymidylate synthase (TS) structures. Like other TS structures, the apo-form of wTS displays an open conformation, whereas the wTS ternary complex attains a closed conformation. While the C-terminal loop maintains a typical distance from methotrexate, the Sγ atom of the catalytic Cys is positioned farther from the C6 atom of dUMP. Altogether, we report the first TS structure from a crustacean virus and highlight its distinction from shrimp and other TS structures.
Asunto(s)
Nucleótidos de Desoxiuracil/química , Metotrexato/química , Penaeidae/virología , Timidilato Sintasa/química , Virus del Síndrome de la Mancha Blanca 1/química , Animales , Crustáceos/virología , Escherichia coli/química , Humanos , Enlace de Hidrógeno , Ligandos , Ratones , Modelos Moleculares , Conformación Molecular , Penaeidae/química , Dominios Proteicos , Proteínas RecombinantesRESUMEN
Folates are essential biomolecules required to carry out many crucial processes in leishmania parasite. Dihydrofolate reductase-thymidylate synthase (DHFR-TS) and pteridine reductase 1 (PTR1) involved in folate biosynthesis in leishmania have been established as suitable targets for development of chemotherapy against leishmaniasis. In the present study, various computational tools such as homology modelling, pharmacophore modelling, docking, molecular dynamics and molecular mechanics have been employed to design dual DHFR-TS and PTR1 inhibitors. Two designed molecules, i.e. 2-(4-((4-nitrobenzyl)oxy)phenyl)-1H-benzo[d]imidazole and 2-(4-((2,4-dichlorobenzyl)oxy)phenyl)-1H-benzo[d]oxazolemolecules were synthesized. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was performed to evaluate in vitro activity of molecules against promastigote form of Leishmania donovani using Miltefosine as standard. 2-(4-((4-nitrobenzyl)oxy)phenyl)-1H-benzo[d]imidazole and 2-(4-((2,4-dichlorobenzyl)oxy)phenyl)-1H-benzo[d]oxazolemolecules were found to be moderately active with showed IC50 = 68 ± 2.8 µM and 57 ± 4.2 µM, respectively.
Asunto(s)
Antiprotozoarios/farmacología , Leishmania donovani/efectos de los fármacos , Complejos Multienzimáticos/química , Oxidorreductasas/química , Proteínas Protozoarias/química , Tetrahidrofolato Deshidrogenasa/química , Timidilato Sintasa/química , Antiprotozoarios/síntesis química , Antiprotozoarios/química , Bencimidazoles/síntesis química , Bencimidazoles/química , Bencimidazoles/farmacología , Benzoxazoles/síntesis química , Benzoxazoles/química , Benzoxazoles/farmacología , Descubrimiento de Drogas , Concentración 50 Inhibidora , Leishmania donovani/metabolismo , Modelos Moleculares , Fosforilcolina/análogos & derivados , Fosforilcolina/farmacología , Relación Estructura-ActividadRESUMEN
Thymidylate synthase (TS) catalyzes the production of the nucleotide dTMP from deoxyuridine monophosphate (dUMP), making the enzyme necessary for DNA replication and consequently a target for cancer therapeutics. TSs are homodimers with active sites separated by â¼30 Å. Reports of half-the-sites activity in TSs from multiple species demonstrate the presence of allosteric communication between the active sites of this enzyme. A simple explanation for the negative allosteric regulation occurring in half-the-sites activity would be that the two substrates bind with negative cooperativity. However, previous work on Escherichia coli TS revealed that dUMP substrate binds without cooperativity. To gain further insight into TS allosteric function, binding cooperativity in human TS is examined here. Isothermal titration calorimetry and two-dimensional lineshape analysis of NMR titration spectra are used to characterize the thermodynamics of dUMP binding, with a focus on quantification of cooperativity between the two substrate binding events. We find that human TS binds dUMP with â¼9-fold entropically driven positive cooperativity (ρITC = 9 ± 1, ρNMR = 7 ± 1), in contrast to the apparent strong negative cooperativity reported previously. Our work further demonstrates the necessity of globally fitting isotherms collected under various conditions, as well as accurate determination of binding competent protein concentration, for calorimetric characterization of homotropic cooperative binding. Notably, an initial curvature of the isotherm is found to be indicative of positively cooperative binding. Two-dimensional lineshape analysis NMR is also found to be an informative tool for quantifying binding cooperativity, particularly in cases in which bound intermediates yield unique resonances.
Asunto(s)
Timidilato Sintasa/metabolismo , Escherichia coli/enzimología , Humanos , Espectroscopía de Resonancia Magnética , Modelos Biológicos , Especificidad por Sustrato , Temperatura , Timidilato Sintasa/química , Uridina Monofosfato/metabolismoRESUMEN
Cryptosporidiosis is a human gastrointestinal disease caused by protozoans of the genus Cryptosporidium, which can be fatal in immunocompromised individuals. The essential enzyme, thymidylate synthase (TS), is responsible for de novo synthesis of deoxythymidine monophosphate. The TS active site is relatively conserved between Cryptosporidium and human enzymes. In previous work, we identified compound 1, (2-amino-4-oxo-4,7-dihydro-pyrrolo[2,3-d]pyrimidin-methyl-phenyl-l-glutamic acid), as a promising selective Cryptosporidium hominis TS (ChTS) inhibitor. In the present study, we explore the structure-activity relationship around 1 glutamate moiety by synthesizing and biochemically evaluating the inhibitory activity of analogues against ChTS and human TS (hTS). X-Ray crystal structures were obtained for compounds bound to both ChTS and hTS. We establish the importance of the 2-phenylacetic acid moiety methylene linker in optimally positioning compounds 23, 24, and 25 within the active site. Moreover, through the comparison of structural data for 5, 14, 15, and 23 bound in both ChTS and hTS identified that active site rigidity is a driving force in determining inhibitor selectivity.
Asunto(s)
Cryptosporidium/enzimología , Glutamatos/química , Fenilacetatos/química , Pirimidinas/química , Pirroles/química , Timidilato Sintasa/antagonistas & inhibidores , Dominio Catalítico , Diseño de Fármacos , Humanos , Modelos Moleculares , Estructura Molecular , Unión Proteica , Timidilato Sintasa/químicaRESUMEN
Thymidylate synthase (TS) is a dimeric enzyme conserved in all life forms that exhibits the allosteric feature of half-the-sites activity. Neither the reason for nor the mechanism of this phenomenon is understood. We used a combined nuclear magnetic resonance (NMR) and molecular dynamics approach to study a stable intermediate preceding hydride transfer, which is the rate-limiting and half-the-sites step. In NMR titrations with ligands leading to this intermediate, we measured chemical shifts of the apoenzyme (lig0), the saturated holoenzyme (lig2), and the typically elusive singly bound (lig1) states. Approximately 40 amides showed quartet patterns providing direct NMR evidence of coupling between the active site and probes >30 Å away in the distal subunit. Quartet peak patterns have symmetrical character, indicating reciprocity in communicating the first and second binding events to the distal protomer. Quartets include key catalytic residues and map to the dimer interface ß-sheet, which also represents the shortest path between the two active sites. Simulations corroborate the coupling observed in solution in that there is excellent overlap between quartet residues and main-chain atoms having intersubunit cross-correlated motions. Simulations identify five hot spot residues, three of which lie at the kink in the unique ß-bulge abutting the active sites on either end of the sheet. Interstrand cross-correlated motions become more organized and pronounced as the enzyme progresses from lig0 to lig1 and ultimately lig2. Coupling in the apparently symmetrical complex has implications for half-the-sites reactivity and potentially resolves the paradox of inequivalent TS active sites despite the vast majority of X-ray structures appearing to be symmetrical.
Asunto(s)
Multimerización de Proteína/fisiología , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Dominio Catalítico/fisiología , Conformación Proteica en Lámina beta/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Thymidylate synthase (TS), found in all organisms, is an essential enzyme responsible for the de novo synthesis of deoxythymidine monophosphate. The TS active sites of the protozoal parasite Cryptosporidium hominis and human are relatively conserved. Evaluation of antifolate compound 1 and its R-enantiomer 2 against both enzymes reveals divergent inhibitor selectivity and enzyme stereospecificity. To establish how C. hominis and human TS (ChTS and hTS) selectively discriminate 1 and 2, respectively, we determined crystal structures of ChTS complexed with 2 and hTS complexed with 1 or 2. Coupled with the previously determined structure of ChTS complexed with 1, we discuss a possible mechanism for enzyme stereospecificity and inhibitor selectivity.
Asunto(s)
Cryptosporidium/enzimología , Antagonistas del Ácido Fólico/farmacología , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Antagonistas del Ácido Fólico/química , Humanos , Modelos Moleculares , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Especificidad de la Especie , Relación Estructura-Actividad , Timidilato Sintasa/antagonistas & inhibidoresRESUMEN
The thymidylate synthases ThyA and Thy1 are enzymes that catalyse the formation of thymidine monophosphate from 2'-deoxyuridine monophosphate. Thy1 (or ThyX) requires flavin for catalytic reactions, while ThyA does not. In the present study, the crystal structure of the flavin-dependent thymidylate synthase Thy1 from Thermus thermophilus HB8 (TtThy1, TTHA1096) was determined in complex with FAD and phosphate at 2.5â Å resolution. TtThy1 is a tetrameric molecule like other Thy1 proteins, to which four FAD molecules are bound. In the crystal of TtThy1, two phosphate ions were bound to each dUMP-binding site. The characteristic feature of TtThy1 is the existence of an extra C-terminal domain (CTD) consisting of three α-helices and a ß-strand. The function of the CTD is unknown and database analysis showed that this CTD is only shared by part of the Deinococcus-Thermus phylum.
Asunto(s)
Flavina-Adenina Dinucleótido/metabolismo , Thermus thermophilus/enzimología , Timidilato Sintasa/química , Timidilato Sintasa/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Flavina-Adenina Dinucleótido/química , Modelos Moleculares , Conformación Proteica , Dominios Proteicos , Homología de SecuenciaRESUMEN
Chemical quenching offers a complementary approach to studying the mechanism of a flavoenzyme, supplementing the information learned from spectroscopic, structural, and computational methods. Generally, in a chemical quench experiment, an enzymatic turnover is quickly stopped at various stages with a chemical agent, and the individual reaction mixtures at each time point are analyzed for the reactants, products and any intermediates. The order by which bonds are made and broken in the reaction is indicated by the identities of the captured intermediates, and the rates of individual steps in the mechanism are determined from the amounts of various chemical species at different time points. This chapter outlines general considerations in selecting a chemical quencher of a particular enzyme-catalyzed reaction and methods for analyzing captured reaction intermediates, with a focus on flavoenzymes. The investigation of flavin-dependent thymidylate synthase is used as a case study to illustrate the concepts and workflow of quenching, isolating, and characterizing quencher-modified reaction intermediates and drawing mechanistic conclusions from the identities of these molecules.