RESUMEN
Several 2-amino-4-carboxypyridine, 4- and 5-carboxypyridone-based compounds were prepared and tested against three members of the chorismate-utilising enzyme family, anthranilate synthase, isochorismate synthase and salicylate synthase. Most compounds exhibited low micromolar inhibition of these three enzymes. The most potent inhibitor was a 4-carboxypyridone analogue bearing a lactate side chain on the pyridyl nitrogen which exhibited inhibition constants of 5, 91 and 54 muM against anthranilate synthase, isochorismate synthase and salicylate synthase respectively.
Asunto(s)
Aminopiridinas/química , Aminopiridinas/farmacología , Ácido Corísmico/metabolismo , Ácidos Isonicotínicos/química , Ácidos Isonicotínicos/farmacología , Piridinas/química , Piridinas/farmacología , Piridonas/química , Piridonas/farmacología , Aminopiridinas/síntesis química , Aminopiridinas/metabolismo , Antranilato Sintasa/antagonistas & inhibidores , Antranilato Sintasa/química , Antranilato Sintasa/metabolismo , Bacterias/enzimología , Dominio Catalítico , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Transferasas Intramoleculares/antagonistas & inhibidores , Transferasas Intramoleculares/química , Transferasas Intramoleculares/metabolismo , Ácidos Isonicotínicos/síntesis química , Ácidos Isonicotínicos/metabolismo , Liasas/antagonistas & inhibidores , Liasas/química , Liasas/metabolismo , Modelos Moleculares , Piridinas/síntesis química , Piridinas/metabolismo , Piridonas/síntesis química , Piridonas/metabolismoRESUMEN
EntC, one of two isochorismate synthases in Escherichia coli, is specific to the biosynthesis of the siderophore enterobactin. Here, we report the crystal structure of EntC in complex with isochorismate and Mg(2+)at 2.3 A resolution, the first structure of a chorismate-utilizing enzyme with a non-aromatic reaction product. EntC exhibits a complex alpha+beta fold like the other chorismate-utilizing enzymes, such as salicylate synthase and anthranilate synthase. Comparison of active site structures allowed the identification of several residues, not discussed previously, that might be important for the isochorismate activity of the EntC. Although EntC, MenF and Irp9 all convert chorismate to isochorismate, only Irp9 subsequently exhibits isochorismate pyruvate lyase activity resulting in the formation of salicylate and pyruvate as the reaction products. With a view to understanding the roles of these amino acid residues in the conversion of chorismate to isochorismate and to obtaining clues about the pyruvate lyase activity of Irp9, several mutants of EntC were generated in which the selected residues in EntC were substituted for those of Irp9: these included A303T, L304A, F327Y, I346L and F359Q mutations. Biochemical analysis of these mutants indicated that the side chain of A303 in EntC may be crucial in the orientation of the carbonyl to allow formation of a hydrogen bond with isochorismate. Some mutations, such as L304A and F359Q, give rise to a loss of catalytic activity, whereas others, such as F327Y and I346L, show that subtle changes in the otherwise closely similar active sites influence activity. We did not find a combination of these residues that conferred pyruvate lyase activity.
Asunto(s)
Ácido Corísmico/metabolismo , Enterobactina/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimología , Transferasas Intramoleculares/química , Transferasas Intramoleculares/metabolismo , Secuencia de Aminoácidos , Biocatálisis , Dominio Catalítico , Ácido Corísmico/química , Cristalografía por Rayos X , Pruebas de Enzimas , Datos de Secuencia Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Estructura Secundaria de Proteína , Alineación de Secuencia , Especificidad por SustratoRESUMEN
Enfuvirtide (also known as Fuzeon, T-20, or DP-178) is an antiretroviral fusion inhibitor which prevents human immunodeficiency virus type 1 (HIV-1) from entering host cells. This linear 36-mer synthetic peptide is indicated, in combination with other antiretroviral agents, for the treatment of HIV-1-infected individuals and AIDS patients with multidrug-resistant HIV infections. Although enfuvirtide is an efficient anti-HIV-1 drug, its clinical use is limited by a short plasma half-life, i.e., approximately 2 h, which requires twice-daily subcutaneous injections, often resulting in skin sensitivity reaction side effects at the injection sites. Ultimately, 80% of patients stop enfuvirtide treatment within 6 months because of these side effects. We report on the development of long-lasting enfuvirtide conjugates by the use of the site-specific conjugation of enfuvirtide to an antithrombin-binding carrier pentasaccharide (CP) through polyethylene glycol (PEG) linkers of various lengths. These conjugates showed consistent and broad anti-HIV-1 activity in the nanomolar range. The coupling of the CP to enfuvirtide only moderately affected the in vitro anti-HIV-1 activity in the presence of antithrombin. Most importantly, one of these conjugates, enfuvirtide-PEG(12)-CP (EP40111), exhibited a prolonged elimination half-life of more than 10 h in rat plasma compared to the half-life of native enfuvirtide, which was 2.8 h. On the basis of the pharmacokinetic properties of antithrombin-binding pentasaccharides, the anticipated half-life of EP40111 in humans would putatively be about 120 h, which would allow subcutaneous injection once a week instead of twice daily. In conclusion, EP40111 is a promising compound with strong potency as a novel long-lasting anti-HIV-1 drug.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Fármacos Anti-VIH/farmacología , Proteína gp41 de Envoltorio del VIH/administración & dosificación , Proteína gp41 de Envoltorio del VIH/farmacología , VIH-1/efectos de los fármacos , Fragmentos de Péptidos/administración & dosificación , Fragmentos de Péptidos/farmacología , Secuencia de Aminoácidos , Animales , Fármacos Anti-VIH/síntesis química , Antitrombinas/metabolismo , Línea Celular , Supervivencia Celular , Cromatografía Líquida de Alta Presión , Preparaciones de Acción Retardada , Portadores de Fármacos , Enfuvirtida , Inhibidores del Factor Xa , Femenino , Proteína gp41 de Envoltorio del VIH/síntesis química , Semivida , Humanos , Datos de Secuencia Molecular , Neutrófilos/efectos de los fármacos , Neutrófilos/virología , Fragmentos de Péptidos/síntesis química , Polietilenglicoles/química , Polisacáridos/química , Ratas , Ratas Wistar , Espectrometría de Masas en TándemRESUMEN
A library of 2,5-dihydrochorismate analogues were designed as inhibitors of the chorismate-utilising enzymes including anthranilate synthase, isochorismate synthase, salicylate synthase and 4-amino-4-deoxychorismate synthase. The inhibitors were synthesised in seven or eight steps from shikimic acid, sourced from star anise. The compounds exhibited moderate but differential inhibition against the four chorismate-utilising enzymes.
Asunto(s)
Antranilato Sintasa/antagonistas & inhibidores , Proteínas Bacterianas/metabolismo , Ligasas de Carbono-Nitrógeno/antagonistas & inhibidores , Ácido Corísmico/análogos & derivados , Transferasas Intramoleculares/antagonistas & inhibidores , Liasas/antagonistas & inhibidores , Antranilato Sintasa/metabolismo , Ligasas de Carbono-Nitrógeno/metabolismo , Ácido Corísmico/síntesis química , Ácido Corísmico/metabolismo , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/metabolismo , Escherichia coli/enzimología , Transferasas Intramoleculares/metabolismo , Liasas/metabolismo , Serratia marcescens/enzimología , TransaminasasAsunto(s)
Compuestos Bicíclicos con Puentes/química , Inhibidores Enzimáticos/química , Sitios de Unión , Compuestos Bicíclicos con Puentes/síntesis química , Compuestos Bicíclicos con Puentes/farmacología , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Helicobacter pylori/enzimología , Hidroliasas , Modelos Moleculares , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Streptomyces coelicolor/enzimologíaRESUMEN
The in silico design, synthesis, and biological evaluation of ten potent type II dehydroquinase inhibitors are described. These compounds contain an anhydroquinate core, incorporated as a mimic of the enolate reaction intermediate. This substructure is attached by a variety of linking units to a terminal phenyl group that binds in an adjacent pocket. Inhibitors were synthesised from (-)-quinic acid using palladium-catalysed Stille and carboamidation chemistry. Several inhibitors exhibited nanomolar inhibition constants against type II dehydroquinases from Streptomyces coelicolor and Mycobacterium tuberculosis. These are among the most potent inhibitors of these enzymes reported to date.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hidroliasas/antagonistas & inhibidores , Evaluación Preclínica de Medicamentos , Inhibidores Enzimáticos/síntesis química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Modelos Moleculares , Conformación MolecularRESUMEN
We describe the rational design of a novel, highly potent inhibitor of type II dehydroquinase, the dicarboxylate 6. The incorporation of a carboxylate at the 3-position mimics the putative enolate intermediate in the reaction mechanism, and allows a potential electrostatic binding interaction with the arginine on the active site flap. This results in a 1000-fold increase in potency, making the dicarboxylate 6 the most potent inhibitor of type II dehydroquinase reported to date, with a high ligand efficiency of -0.68 kcal mol(-1) per nonhydrogen atom. The systematic dissection of 6 in compounds 7-12, all of which show a drop in potency, confirm the synergistic importance of the two carboxylates, the C3 and C4 hydroxyl groups, and the anhydroquinate ring structure for the potency of 6.
Asunto(s)
Alcoholes/química , Ácidos Carboxílicos/química , Inhibidores Enzimáticos/farmacología , Hidroliasas/antagonistas & inhibidores , Mycobacterium tuberculosis/efectos de los fármacos , Streptomyces coelicolor/efectos de los fármacos , Antibacterianos/síntesis química , Antibacterianos/farmacología , Sitios de Unión , Diseño de Fármacos , Inhibidores Enzimáticos/síntesis química , Cetonas/química , Cinética , Ligandos , Mycobacterium tuberculosis/enzimología , Streptomyces coelicolor/enzimología , Relación Estructura-Actividad , TermodinámicaRESUMEN
The irreversible inhibition of 8-amino-7-oxononanoate synthase by trifluoroalanine involves decarboxylative defluorination of the inhibitor-PLP aldimine followed by attack of the conjugated imine by the amino group of the active site lysine to afford a covalently bound difluorinated intermediate which can subsequently undergo further HF losses and hydrolysis to afford a 2-(pyridoximine phosphate) acetoyl protein adduct.
Asunto(s)
Aciltransferasas/química , Aciltransferasas/metabolismo , Alanina/análogos & derivados , Alanina/metabolismo , Ligasas/antagonistas & inhibidores , Aciltransferasas/antagonistas & inhibidores , Sitios de Unión , Catálisis , Hidrólisis , Lisina/química , Modelos Moleculares , Conformación ProteicaRESUMEN
The salicylate synthase, Irp9, from Yersinia enterocolitica is involved in the biosynthesis of the siderophore yersiniabactin. It is a bifunctional enzyme that forms salicylate and pyruvate from chorismate and water via the intermediate isochorismate. Here we report the first crystal structure of Irp9 and also of its complex with the reaction products salicylate and pyruvate at 1.85 A and 2.1 A resolution, respectively. Like other members of the chorismate-utilizing enzyme family, e.g. the TrpE subunit of anthranilate synthase and the PabB subunit of 4-amino-4-deoxychorismate synthase, Irp9 has a complex alpha/beta fold. The crystal structure of Irp9 contains one molecule each of phosphate and acetate derived from the crystallization buffer. The Irp9-products complex structure was obtained by soaking chorismate into Irp9, demonstrating that the enzyme is still catalytically active in the crystal. Both structures contain Mg(2+) in the active site. There is no evidence of the allosteric tryptophan binding site found in TrpE and PabB. Mutagenesis of Glu240, His321 and Tyr372 provided some insight into the mechanism of the two transformations catalyzed by Irp9. Knowledge of the structure of Irp9 will guide the search for potent inhibitors of salicylate formation, and hence of bacterial iron uptake, which is directly related to the virulence of Yersinia.
Asunto(s)
Liasas/química , Estructura Cuaternaria de Proteína , Ácido Pirúvico/química , Salicilatos/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Liasas/genética , Liasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutación , Ácido Pirúvico/metabolismo , Salicilatos/metabolismo , Alineación de Secuencia , Yersinia enterocolitica/enzimologíaRESUMEN
The reactive beta-ketoacid pyridoxal-5'-phosphate aldimine formed in the condensation step of the 8-amino-7-oxononanoate synthase reaction was 'trapped' in the enzyme-bound form by carrying out the reaction with l-alanine methyl ester and pimeloyl-CoA affording the more stable methyl ester of the putative intermediate, the characterisation of which provides the first definitive evidence for a beta-ketoacid intermediate in an alpha-oxamine synthase mechanism.
Asunto(s)
Aciltransferasas/metabolismo , Aciltransferasas/química , Modelos Moleculares , Estructura Molecular , Fosfato de Piridoxal/metabolismoRESUMEN
In some bacteria, salicylate is synthesized using the enzymes isochorismate synthase and isochorismate pyruvate lyase. In contrast, gene inactivation and complementation experiments with Yersinia enterocolitica suggest the synthesis of salicylate in the biosynthesis of the siderophore yersiniabactin involves a single protein, Irp9, which converts chorismate directly into salicylate. In the present study, Irp9 was for the first time heterologously expressed in Escherichia coli as a hexahistidine fusion protein, purified to near homogeneity, and characterized biochemically. The recombinant protein was found to be a dimer, each subunit of which has a molecular mass of 50 kDa. Enzyme assays, reverse-phase high-pressure liquid chromatography and 1H nuclear magnetic resonance (NMR) spectroscopic analyses confirmed that Irp9 is a salicylate synthase and converts chorismate to salicylate with a K(m) for chorismate of 4.2 microM and a k(cat) of 8 min(-1). The reaction was shown to proceed through the intermediate isochorismate, which was detected directly using 1H NMR spectroscopy.
Asunto(s)
Liasas/metabolismo , Salicilatos/metabolismo , Yersinia enterocolitica/enzimología , Ácido Corísmico/análogos & derivados , Ácido Corísmico/metabolismo , Cromatografía Líquida de Alta Presión , Clonación Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Liasas/biosíntesis , Liasas/genética , Espectroscopía de Resonancia Magnética , Peso Molecular , Proteínas Recombinantes/metabolismo , Sideróforos/metabolismoRESUMEN
Analogues of chorismate and isochorismate were designed and tested as potential inhibitors in the first inhibition study against a salicylate synthase.