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1.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 12): 3244-52, 2014 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-25478842

RESUMEN

The aspartate pathway is essential for the production of the amino acids required for protein synthesis and of the metabolites needed in bacterial development. This pathway also leads to the production of several classes of quorum-sensing molecules that can trigger virulence in certain microorganisms. The second enzyme in this pathway, aspartate ß-semialdehyde dehydrogenase (ASADH), is absolutely required for bacterial survival and has been targeted for the design of selective inhibitors. Fragment-library screening has identified a new set of inhibitors that, while they do not resemble the substrates for this reaction, have been shown to bind at the active site of ASADH. Structure-guided development of these lead compounds has produced moderate inhibitors of the target enzyme, with some selectivity observed between the Gram-negative and Gram-positive orthologs of ASADH. However, many of these inhibitor analogs and derivatives have not yet achieved the expected enhanced affinity. Structural characterization of these enzyme-inhibitor complexes has provided detailed explanations for the barriers that interfere with optimal binding. Despite binding in the same active-site region, significant changes are observed in the orientation of these bound inhibitors that are caused by relatively modest structural alterations. Taken together, these studies present a cautionary tale for issues that can arise in the systematic approach to the modification of lead compounds that are being used to develop potent inhibitors.


Asunto(s)
Aspartato-Semialdehído Deshidrogenasa/antagonistas & inhibidores , Aspartato-Semialdehído Deshidrogenasa/química , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Streptococcus pneumoniae/enzimología , Vibrio cholerae/enzimología , Aspartato-Semialdehído Deshidrogenasa/metabolismo , Ácido Aspártico/metabolismo , Vías Biosintéticas , Dominio Catalítico , Cólera/microbiología , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Infecciones Neumocócicas/microbiología , Conformación Proteica , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Streptococcus pneumoniae/química , Streptococcus pneumoniae/metabolismo , Vibrio cholerae/química , Vibrio cholerae/metabolismo
2.
Acta Crystallogr F Struct Biol Commun ; 70(Pt 10): 1340-5, 2014 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-25286936

RESUMEN

Homoserine O-acetyltransferase (HTA) catalyzes the formation of L-O-acetyl-homoserine from L-homoserine through the transfer of an acetyl group from acetyl-CoA. This is the first committed step required for the biosynthesis of methionine in many fungi, Gram-positive bacteria and some Gram-negative bacteria. The structure of HTA from Staphylococcus aureus (SaHTA) has been determined to a resolution of 2.45 Å. The structure belongs to the α/ß-hydrolase superfamily, consisting of two distinct domains: a core α/ß-domain containing the catalytic site and a lid domain assembled into a helical bundle. The active site consists of a classical catalytic triad located at the end of a deep tunnel. Structure analysis revealed some important differences for SaHTA compared with the few known structures of HTA.


Asunto(s)
Acetiltransferasas/química , Proteínas Bacterianas/química , Staphylococcus aureus/enzimología , Secuencia de Aminoácidos , Apoenzimas/química , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína
3.
Biochemistry ; 53(30): 4970-8, 2014 Aug 05.
Artículo en Inglés | MEDLINE | ID: mdl-25003821

RESUMEN

Canavan disease (CD) is a fatal, childhood neurological disorder caused by mutations in the ASPA gene, leading to catalytic deficiencies in the aspartoacylase (ASPA) enzyme and impaired N-acetyl-l-aspartic acid metabolism in the brain. To study the possible structural defects triggered by these mutations, four ASPA missense mutations associated with different disease severities have been structurally characterized. These mutant enzymes each have overall structures similar to that of the native ASPA enzyme, but with varying degrees of alterations that offer explanations for the respective loss of catalytic activity. The K213E mutant, a nonconservative mutant associated with a mild disease phenotype, has minimal structural differences compared to the native enzyme. In contrast, the loss of van der Waals contacts in the F295S mutant and the loss of hydrophobic and hydrogen bonding interactions in the Y231C mutant lead to a local collapse of the hydrophobic core structure in the carboxyl-terminal domain, contributing to a decrease in protein stability. The structure of the E285A mutant, the most common clinical mutant, reveals that the loss of hydrogen bonding interactions with the carboxylate side chain of Glu285 disturbs the active site architecture, leading to altered substrate binding and lower catalytic activity. Our improved understanding of the nature of these structural defects provides a basis for the development of treatment therapies for CD.


Asunto(s)
Amidohidrolasas/química , Amidohidrolasas/deficiencia , Enfermedad de Canavan/enzimología , Dominio Catalítico/genética , Mutación Missense/genética , Amidohidrolasas/genética , Enfermedad de Canavan/genética , Línea Celular , Cristalografía por Rayos X , Humanos , Relación Estructura-Actividad
4.
Acta Crystallogr D Biol Crystallogr ; 70(Pt 2): 442-50, 2014 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-24531478

RESUMEN

S-Adenosylmethionine (AdoMet) participates in a wide range of methylation and other group-transfer reactions and also serves as the precursor for two groups of quorum-sensing molecules that function as regulators of the production of virulence factors in Gram-negative bacteria. The synthesis of AdoMet is catalyzed by AdoMet synthetases (MATs), a ubiquitous family of enzymes found in species ranging from microorganisms to mammals. The AdoMet synthetase from the bacterium Campylobacter jejuni (cjMAT) is an outlier among this homologous enzyme family, with lower sequence identity, numerous insertions and substitutions, and higher catalytic activity compared with other bacterial MATs. Alterations in the structure of this enzyme provide an explanation for its unusual dimeric quaternary structure relative to the other MATs. Taken together with several active-site substitutions, this new structure provides insights into its improved kinetic properties with alternative substrates.


Asunto(s)
Proteínas Bacterianas/química , Campylobacter jejuni/química , Metionina Adenosiltransferasa/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Campylobacter jejuni/enzimología , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Metionina Adenosiltransferasa/genética , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido
5.
Chem Biol Drug Des ; 79(1): 128-36, 2012 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-22039970

RESUMEN

The aspartate biosynthetic pathway provides essential metabolites for many important biological functions, including the production of four essential amino acids. As this critical pathway is only present in plants and microbes, any disruptions will be fatal to these organisms. An early pathway enzyme, l-aspartate-ß-semialdehyde dehydrogenase, produces a key intermediate at the first branch point of this pathway. Developing potent and selective inhibitors against several orthologs in the l-aspartate-ß-semialdehyde dehydrogenase family can serve as lead compounds for antibiotic development. Kinetic studies of two small molecule fragment libraries have identified inhibitors that show good selectivity against l-aspartate-ß-semialdehyde dehydrogenases from two different bacterial species, Streptococcus pneumoniae and Vibrio cholerae, despite the presence of an identical constellation of active site amino acids in this homologous enzyme family. Structural characterization of enzyme-inhibitor complexes have elucidated different modes of binding between these structurally related enzymes. This information provides the basis for a structure-guided approach to the development of more potent and more selective inhibitors.


Asunto(s)
Antibacterianos/química , Aspartato-Semialdehído Deshidrogenasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Antibacterianos/síntesis química , Antibacterianos/farmacología , Aspartato-Semialdehído Deshidrogenasa/metabolismo , Sitios de Unión , Dominio Catalítico , Simulación por Computador , Cristalografía por Rayos X , Diseño de Fármacos , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Propionatos/química , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/enzimología , Especificidad por Sustrato , Vibrio cholerae/efectos de los fármacos , Vibrio cholerae/enzimología
6.
J Amino Acids ; 2011: 352538, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-22332000

RESUMEN

The aspartate pathway of amino acid biosynthesis is essential for all microbial life but is absent in mammals. Characterizing the enzyme-catalyzed reactions in this pathway can identify new protein targets for the development of antibiotics with unique modes of action. The enzyme aspartate ß-semialdehyde dehydrogenase (ASADH) catalyzes an early branch point reaction in the aspartate pathway. Kinetic, mutagenic, and structural studies of ASADH from various microbial species have been used to elucidate mechanistic details and to identify essential amino acids involved in substrate binding, catalysis, and enzyme regulation. Important structural and functional differences have been found between ASADHs isolated from these bacterial and fungal organisms, opening the possibility for developing species-specific antimicrobial agents that target this family of enzymes.

7.
Acta Crystallogr D Biol Crystallogr ; 66(Pt 2): 205-12, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20124701

RESUMEN

The enzyme aspartate semialdehyde dehydrogenase (ASADH) catalyzes a critical transformation that produces the first branch-point intermediate in an essential microbial amino-acid biosynthetic pathway. The first structure of an ASADH isolated from a fungal species (Candida albicans) has been determined as a complex with its pyridine nucleotide cofactor. This enzyme is a functional dimer, with a similar overall fold and domain organization to the structurally characterized bacterial ASADHs. However, there are differences in the secondary-structural elements and in cofactor binding that are likely to cause the lower catalytic efficiency of this fungal enzyme. Alterations in the dimer interface, through deletion of a helical subdomain and replacement of amino acids that participate in a hydrogen-bonding network, interrupt the intersubunit-communication channels required to support an alternating-site catalytic mechanism. The detailed functional information derived from this new structure will allow an assessment of ASADH as a possible target for antifungal drug development.


Asunto(s)
Aspartato-Semialdehído Deshidrogenasa/química , Candida albicans/enzimología , Secuencia de Aminoácidos , Aspartato-Semialdehído Deshidrogenasa/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Alineación de Secuencia
8.
Bioorg Med Chem Lett ; 20(2): 576-80, 2010 Jan 15.
Artículo en Inglés | MEDLINE | ID: mdl-20005097

RESUMEN

Potent, highly selective and orally-bioavailable MMP-13 inhibitors have been identified based upon a (pyridin-4-yl)-2H-tetrazole scaffold. Co-crystal structure analysis revealed that the inhibitors bind at the S(1)(') active site pocket and are not ligands for the catalytic zinc atom. Compound 29b demonstrated reduction of cartilage degradation biomarker (TIINE) levels associated with cartilage protection in a preclinical rat osteoarthritis model.


Asunto(s)
Inhibidores de la Metaloproteinasa de la Matriz , Osteoartritis/tratamiento farmacológico , Ácidos Picolínicos/química , Inhibidores de Proteasas/química , Tetrazoles/química , Administración Oral , Animales , Sitios de Unión , Cartílago/efectos de los fármacos , Cartílago/metabolismo , Dominio Catalítico , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Descubrimiento de Drogas , Metaloproteinasa 13 de la Matriz/metabolismo , Ácidos Picolínicos/síntesis química , Ácidos Picolínicos/farmacología , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/farmacología , Ratas , Tetrazoles/síntesis química , Tetrazoles/farmacología , Zinc/química
9.
J Biol Chem ; 283(23): 16216-25, 2008 Jun 06.
Artículo en Inglés | MEDLINE | ID: mdl-18334478

RESUMEN

The commitment step to the aspartate pathway of amino acid biosynthesis is the phosphorylation of aspartic acid catalyzed by aspartokinase (AK). Most microorganisms and plants have multiple forms of this enzyme, and many of these isofunctional enzymes are subject to feedback regulation by the end products of the pathway. However, the archeal species Methanococcus jannaschii has only a single, monofunctional form of AK. The substrate l-aspartate binds to this recombinant enzyme in two different orientations, providing the first structural evidence supporting the relaxed regiospecificity previously observed with several alternative substrates of Escherichia coli AK ( Angeles, T. S., Hunsley, J. R., and Viola, R. E. (1992) Biochemistry 31, 799-805 ). Binding of the nucleotide substrate triggers significant domain movements that result in a more compact quaternary structure. In contrast, the highly cooperative binding of the allosteric regulator l-threonine to multiple sites on this dimer of dimers leads to an open enzyme structure. A comparison of these structures supports a mechanism for allosteric regulation in which the domain movements induced by threonine binding causes displacement of the substrates from the enzyme, resulting in a relaxed, inactive conformation.


Asunto(s)
Proteínas Arqueales/química , Aspartato Quinasa/química , Methanococcus/enzimología , Treonina/química , Regulación Alostérica/fisiología , Proteínas Arqueales/genética , Aspartato Quinasa/genética , Dimerización , Methanococcus/genética , Estructura Cuaternaria de Proteína/fisiología , Estructura Terciaria de Proteína/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Relación Estructura-Actividad , Especificidad por Sustrato/fisiología
10.
J Med Chem ; 50(21): 5090-102, 2007 Oct 18.
Artículo en Inglés | MEDLINE | ID: mdl-17880056

RESUMEN

A new series of MEK1 inhibitors, the 4-anilino-5-carboxamido-2-pyridones, were designed and synthesized using a combination of medicinal chemistry, computational chemistry, and structural elucidation. The effect of variation in the carboxamide side chain, substitution on the pyridone nitrogen, and replacement of the 4'-iodide were all investigated. This study afforded several compounds which were either equipotent or more potent than the clinical candidate CI-1040 (1) in an isolated enzyme assay, as well as murine colon carcinoma (C26) cells, as measured by suppression of phosphorylated ERK substrate. Most notably, pyridone 27 was found to be more potent than 1 in vitro and produced a 100% response rate at a lower dose than 1, when tested for in vivo efficacy in animals bearing C26 tumors.


Asunto(s)
Amidas/síntesis química , Compuestos de Anilina/síntesis química , Antineoplásicos/síntesis química , MAP Quinasa Quinasa 1/antagonistas & inhibidores , MAP Quinasa Quinasa 2/antagonistas & inhibidores , Piridonas/síntesis química , Amidas/química , Amidas/farmacología , Compuestos de Anilina/química , Compuestos de Anilina/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Benzamidas/farmacología , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , MAP Quinasa Quinasa 1/química , MAP Quinasa Quinasa 2/química , Masculino , Ratones , Modelos Moleculares , Trasplante de Neoplasias , Fosforilación , Piridonas/química , Piridonas/farmacología , Ratas , Relación Estructura-Actividad
11.
J Biol Chem ; 282(38): 27781-91, 2007 Sep 21.
Artículo en Inglés | MEDLINE | ID: mdl-17623656

RESUMEN

Matrix metalloproteinase-13 (MMP13) is a Zn(2+)-dependent protease that catalyzes the cleavage of type II collagen, the main structural protein in articular cartilage. Excess MMP13 activity causes cartilage degradation in osteoarthritis, making this protease an attractive therapeutic target. However, clinically tested MMP inhibitors have been associated with a painful, joint-stiffening musculoskeletal side effect that may be due to their lack of selectivity. In our efforts to develop a disease-modifying osteoarthritis drug, we have discovered MMP13 inhibitors that differ greatly from previous MMP inhibitors; they do not bind to the catalytic zinc ion, they are noncompetitive with respect to substrate binding, and they show extreme selectivity for inhibiting MMP13. By structure-based drug design, we generated an orally active MMP13 inhibitor that effectively reduces cartilage damage in vivo and does not induce joint fibroplasias in a rat model of musculoskeletal syndrome side effects. Thus, highly selective inhibition of MMP13 in patients may overcome the major safety and efficacy challenges that have limited previously tested non-selective MMP inhibitors. MMP13 inhibitors such as the ones described here will help further define the role of this protease in arthritis and other diseases and may soon lead to drugs that safely halt cartilage damage in patients.


Asunto(s)
Cartílago/metabolismo , Artropatías/metabolismo , Metaloproteinasa 13 de la Matriz/fisiología , Animales , Colágeno/química , Colágeno/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Humanos , Iones , Metaloproteinasa 13 de la Matriz/química , Metaloproteinasa 13 de la Matriz/metabolismo , Modelos Biológicos , Modelos Químicos , Modelos Moleculares , Conejos , Ratas , Zinc/química
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