RESUMEN
Gram-negative bacteria possess a characteristic outer membrane, of which the lipid A constituent elicits a strong host immune response through the Toll-like receptor 4 complex, and acts as a component of the permeability barrier to prevent uptake of bactericidal compounds. Lipid A species comprise the bulk of the outer leaflet of the outer membrane and are produced through a multistep biosynthetic pathway conserved in most Gram-negative bacteria. The final steps in this pathway involve the secondary acylation of lipid A precursors. These are catalyzed by members of a superfamily of enzymes known as lysophospholipid acyltransferases (LPLATs), which are present in all domains of life and play important roles in diverse biological processes. To date, characterization of this clinically important class of enzymes has been limited by a lack of structural information and the availability of only low-throughput biochemical assays. In this work, we present the structure of the bacterial LPLAT protein LpxM, and we describe a high-throughput, label-free mass spectrometric assay to characterize acyltransferase enzymatic activity. Using our structure and assay, we identify an LPLAT thioesterase activity, and we provide experimental evidence to support an ordered-binding and "reset" mechanistic model for LpxM function. This work enables the interrogation of other bacterial acyltransferases' structure-mechanism relationships, and the assay described herein provides a foundation for quantitatively characterizing the enzymology of any number of clinically relevant LPLAT proteins.
Asunto(s)
Aciltransferasas/química , Aciltransferasas/metabolismo , Lípido A/química , Lípido A/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Secuencia de Consenso , Activación Enzimática , Bacterias Gramnegativas , Interacciones Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Posición Específica de Matrices de Puntuación , Unión Proteica , Conformación Proteica , Relación Estructura-Actividad , Tioléster Hidrolasas/química , Tioléster Hidrolasas/metabolismoRESUMEN
Autophagy is a dynamic process that regulates lysosomal-dependent degradation of cellular components. Until recently the study of autophagy has been hampered by the lack of reliable pharmacological tools, but selective inhibitors are now available to modulate the PI 3-kinase VPS34, which is required for autophagy. Here we describe the discovery of potent and selective VPS34 inhibitors, their pharmacokinetic (PK) properties, and ability to inhibit autophagy in cellular and mouse models.
RESUMEN
Alterations in PI3K/AKT signaling are known to be implicated with tumorigenesis. The PI3 kinases family of lipid kinases has been an attractive therapeutic target for cancer treatment. Imidazopyridine compound 1, a potent, selective, and orally available pan-PI3K inhibitor, identified by scaffold morphing of a benzothiazole hit, was further optimized in order to achieve efficacy in a PTEN-deleted A2780 ovarian cancer mouse xenograft model. With a hypothesis that a planar conformation between the core and the 6-heteroaryl ring will allow for the accommodation of larger 5'-substituents in a hydrophobic area under P-loop, SAR efforts focused on 5'-alkoxy heteroaryl rings at the 6-position of imidazopyridine and imidazopyridazine cores that have the same dihedral angle of zero degrees. 6'-Alkoxy 5'-aminopyrazines in the imidazopyridine series were identified as the most potent compounds in the A2780 cell line. Compound 14 with 1,1,1-trifluoroisopropoxy group at 6'-position demonstrated excellent potency and selectivity, good oral exposure in rats and in vivo efficacy in A2780 tumor-bearing mouse. Also, we disclose the X-ray co-crystal structure of one enantiomer of compound 14 in PI3Kα, confirming that the trifluoromethyl group fits nicely in the hydrophobic hot spot under P-loop.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Piridinas/química , Piridinas/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Sitios de Unión , Línea Celular Tumoral , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Activación Enzimática/efectos de los fármacos , Femenino , Semivida , Xenoinjertos , Humanos , Ratones , Simulación del Acoplamiento Molecular , Neoplasias Ováricas/tratamiento farmacológico , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Inhibidores de Proteínas Quinasas/uso terapéutico , Estructura Terciaria de Proteína , Piridinas/farmacocinética , Piridinas/uso terapéutico , Ratas , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
Cells rely on autophagy to clear misfolded proteins and damaged organelles to maintain cellular homeostasis. In this study we use the new autophagy inhibitor PIK-III to screen for autophagy substrates. PIK-III is a selective inhibitor of VPS34 that binds a unique hydrophobic pocket not present in related kinases such as PI(3)Kα. PIK-III acutely inhibits autophagy and de novo lipidation of LC3, and leads to the stabilization of autophagy substrates. By performing ubiquitin-affinity proteomics on PIK-III-treated cells we identified substrates including NCOA4, which accumulates in ATG7-deficient cells and co-localizes with autolysosomes. NCOA4 directly binds ferritin heavy chain-1 (FTH1) to target the iron-binding ferritin complex with a relative molecular mass of 450,000 to autolysosomes following starvation or iron depletion. Interestingly, Ncoa4(-/-) mice exhibit a profound accumulation of iron in splenic macrophages, which are critical for the reutilization of iron from engulfed red blood cells. Taken together, the results of this study provide a new mechanism for selective autophagy of ferritin and reveal a previously unappreciated role for autophagy and NCOA4 in the control of iron homeostasis in vivo.
Asunto(s)
Autofagia/fisiología , Fosfatidilinositol 3-Quinasas Clase III/antagonistas & inhibidores , Ferritinas/metabolismo , Homeostasis/fisiología , Hierro/metabolismo , Coactivadores de Receptor Nuclear/metabolismo , Animales , Autofagia/efectos de los fármacos , Células Cultivadas , Humanos , Lisosomas/metabolismo , Ratones , Fagosomas/metabolismo , Unión ProteicaRESUMEN
PI3 kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration and differentiation. The PI3 Kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe how the potency of a benzothiazole fragment hit was quickly improved based on structural information and how this early chemotype was further optimized through scaffold hopping. This effort led to the identification of a series of 2-acetamido-5-heteroaryl imidazopyridines showing potent in vitro activity against all class I PI3Ks and attractive pharmacokinetic properties.
Asunto(s)
Compuestos Azo/síntesis química , Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/síntesis química , Piridinas/farmacología , Compuestos Azo/química , Compuestos Azo/farmacología , Benzotiazoles/química , Benzotiazoles/farmacología , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Femenino , Humanos , Imidas/síntesis química , Imidas/química , Imidas/farmacología , Concentración 50 Inhibidora , Modelos Moleculares , Piridinas/química , Solubilidad , Relación Estructura-ActividadRESUMEN
PI3 Kinases are a family of lipid kinases mediating numerous cell processes such as proliferation, migration, and differentiation. The PI3 kinase pathway is often de-regulated in cancer through PI3Kα overexpression, gene amplification, mutations, and PTEN phosphatase deletion. PI3K inhibitors represent therefore an attractive therapeutic modality for cancer treatment. Herein we describe a novel series of PI3K inhibitors sharing a pyrimidine core and showing significant potency against class I PI3 kinases in the biochemical assay and in cells. The discovery, synthesis and SAR of this chemotype are described.