RESUMEN
The emergence of antibiotic-resistant strains of pathogenic bacteria is an increasing threat to global health that underscores an urgent need for an expanded antibacterial armamentarium. Gram-negative bacteria, such as Escherichia coli, have become increasingly important clinical pathogens with limited treatment options. This is due in part to their lipopolysaccharide (LPS) outer membrane components, which dually serve as endotoxins while also protecting Gram-negative bacteria from antibiotic entry. The LpxC enzyme catalyzes the committed step of LPS biosynthesis, making LpxC a promising target for new antibacterials. Here, we present the first structure of an LpxC enzyme in complex with the deacetylation reaction product, UDP-(3-O-(R-3-hydroxymyristoyl))-glucosamine. These studies provide valuable insight into recognition of substrates and products by LpxC and a platform for structure-guided drug discovery of broad spectrum Gram-negative antibiotics.
Asunto(s)
Amidohidrolasas/química , Escherichia coli/enzimología , Ácidos Mirísticos/química , Protones , Uridina Difosfato N-Acetilglucosamina/análogos & derivados , Amidohidrolasas/metabolismo , Cristalografía por Rayos X , Lipopolisacáridos/biosíntesis , Lipopolisacáridos/química , Ácidos Mirísticos/metabolismo , Estructura Terciaria de Proteína , Uridina Difosfato N-Acetilglucosamina/química , Uridina Difosfato N-Acetilglucosamina/metabolismoRESUMEN
The JAK-STAT pathway mediates signaling by cytokines, which control survival, proliferation, and differentiation of a variety of cells. In recent years, a single point mutation (V617F) in the tyrosine kinase JAK2 was found to be present with a high incidence in myeloproliferative disorders (MPDs). This mutation led to hyperactivation of JAK2, cytokine-independent signaling, and subsequent activation of downstream signaling networks. The genetic, biological, and physiological evidence suggests that JAK2 inhibitors could be effective in treating MPDs. De novo design efforts of new scaffolds identified 1-amino-5H-pyrido[4,3-b]indol-4-carboxamides as a new viable lead series. Subsequent optimization of cell potency, metabolic stability, and off-target activities of the leads led to the discovery of 7-(2-aminopyrimidin-5-yl)-1-{[(1R)-1-cyclopropyl-2,2,2-trifluoroethyl]amino}-5H-pyrido[4,3-b]indole-4-carboxamide (65). Compound 65 is a potent, orally active inhibitor of JAK2 with excellent selectivity, PK profile, and in vivo efficacy in animal models.
Asunto(s)
Carbolinas/síntesis química , Indoles/síntesis química , Janus Quinasa 2/antagonistas & inhibidores , Trastornos Mieloproliferativos/tratamiento farmacológico , Piridinas/síntesis química , Pirimidinas/síntesis química , Administración Oral , Animales , Carbolinas/farmacocinética , Carbolinas/farmacología , Cristalografía por Rayos X , Perros , Haplorrinos , Hepatocitos/metabolismo , Indoles/farmacocinética , Indoles/farmacología , Janus Quinasa 2/metabolismo , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Estructura Molecular , Fosforilación , Policitemia Vera/tratamiento farmacológico , Piridinas/farmacocinética , Piridinas/farmacología , Pirimidinas/farmacocinética , Pirimidinas/farmacología , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
p70 ribosomal S6 kinase (p70S6K) is a downstream effector of the mTOR signaling pathway involved in cell proliferation, cell growth, cell-cycle progression, and glucose homeostasis. Multiple phosphorylation events within the catalytic, autoinhibitory, and hydrophobic motif domains contribute to the regulation of p70S6K. We report the crystal structures of the kinase domain of p70S6K1 bound to staurosporine in both the unphosphorylated state and in the 3'-phosphoinositide-dependent kinase-1-phosphorylated state in which Thr-252 of the activation loop is phosphorylated. Unphosphorylated p70S6K1 exists in two crystal forms, one in which the p70S6K1 kinase domain exists as a monomer and the other as a domain-swapped dimer. The crystal structure of the partially activated kinase domain that is phosphorylated within the activation loop reveals conformational ordering of the activation loop that is consistent with a role in activation. The structures offer insights into the structural basis of the 3'-phosphoinositide-dependent kinase-1-induced activation of p70S6K and provide a platform for the rational structure-guided design of specific p70S6K inhibitors.
Asunto(s)
Proteínas Quinasas S6 Ribosómicas 70-kDa/química , Cromatografía en Gel , Cristalografía por Rayos X , Humanos , Fosforilación , Reacción en Cadena de la Polimerasa , Unión Proteica , Multimerización de Proteína , Estructura Secundaria de Proteína , Proteínas Quinasas S6 Ribosómicas 70-kDa/genética , Proteínas Quinasas S6 Ribosómicas 70-kDa/metabolismo , Estaurosporina/metabolismo , UltracentrifugaciónRESUMEN
A high throughput screening campaign was designed to identify allosteric inhibitors of Chk1 kinase by testing compounds at high concentration. Activity was then observed at K(m) for ATP and at near-physiological concentrations of ATP. This strategy led to the discovery of a non-ATP competitive thioquinazolinone series which was optimized for potency and stability. An X-ray crystal structure for the complex of our best inhibitor bound to Chk1 was solved, indicating that it binds to an allosteric site approximately 13A from the ATP binding site. Preliminary data is presented for several of these compounds.
Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Quinasas/efectos de los fármacos , Quinazolinas/síntesis química , Quinazolinas/farmacología , Sitios de Unión , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Técnicas Químicas Combinatorias , Cristalografía por Rayos X , Humanos , Conformación Molecular , Estructura Molecular , Inhibidores de Proteínas Quinasas/química , Proteínas Quinasas/química , Proteínas Quinasas/metabolismo , Quinazolinas/químicaRESUMEN
Expression and purification of human beta-secretase (BACE1) in bacteria have been plagued with issues concerning solubility, inhomogeneous N-terminus, and lack of enzymic activity. Several forms of the mature human BACE1 have been expressed in Escherichia coli with different N-terminal extensions and without the C-terminus transmembrane domain. Although each of the proteins expresses in inclusion bodies, a generalized protocol has been developed to solubilize, refold, and purify these BACE1 variants. The resultant proteins are homogeneous and monodispersed in solution. Each possesses a unique N-terminus. Activity assays using the peptide substrate 7-methoxycoumarin-4-yl-SEVNLDAEFK-2,4-dinitrophenyl-RR, corresponding to the beta-secretase cleavage sequence in the amyloid precursor protein with the Swedish mutations of N(670)L(671) substituting for the residues K(670)M(671), reveal a kcat and KM of 9.3 min(-1) and 55 microM, respectively.