RESUMEN
The introduction of thermostable polymerases revolutionized the polymerase chain reaction (PCR) and biotechnology. However, many GC-rich genes cannot be PCR-amplified with high efficiency in water, irrespective of temperature. Although polar organic cosolvents can enhance nucleic acid polymerization and amplification by destabilizing duplex DNA and secondary structures, nature has not selected for the evolution of solvent-tolerant polymerase enzymes. Here, we used ultrahigh-throughput droplet-based selection and deep sequencing along with computational free-energy and binding affinity calculations to evolve Taq polymerase to generate enzymes that are both stable and highly active in the presence of organic cosolvents, resulting in up to 10% solvent resistance and over 100-fold increase in stability at 97.5 °C in the presence of 1,4-butanediol, as well as tolerance to up to 10 times higher concentrations of the potent cosolvents sulfolane and 2-pyrrolidone. Using these polymerases, we successfully amplified a broad spectrum of GC-rich templates containing regions with over 90% GC content, including templates recalcitrant to amplification with existing polymerases, even in the presence of cosolvents. We also demonstrated dramatically reduced GC bias in the amplification of genes with widely varying GC content in quantitative polymerase chain reaction (qPCR). By expanding the scope of solvent systems compatible with nucleic acid polymerization, these organic solvent-resistant polymerases enable a dramatic reduction of sequence bias not achievable through thermal resistance alone, with significant implications for a wide range of applications including sequencing and synthetic biology in mixed aqueous-organic media.
Asunto(s)
ADN Polimerasa Dirigida por ADN , ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Composición de Base , SolventesRESUMEN
G-protein coupled receptor kinase 2 (GRK2), which is upregulated in the failing heart, appears to play a critical role in heart failure (HF) progression in part because enhanced GRK2 activity promotes dysfunction of ß-adrenergic signaling and myocyte death. An orally bioavailable GRK2 inhibitor could offer unique therapeutic outcomes that cannot be attained by current heart failure treatments that directly target GPCRs or angiotensin-converting enzyme. Herein, we describe the discovery of a potent, selective, and orally bioavailable GRK2 inhibitor, 8h, through high-throughput screening, hit-to-lead optimization, structure-based design, molecular modelling, synthesis, and biological evaluation. In the cellular target engagement assays, 8h enhances isoproterenol-mediated cyclic adenosine 3',5'-monophosphate (cAMP) production in HEK293 cells overexpressing GRK2. Compound 8h was further evaluated in a human stem cell-derived cardiomyocyte (HSC-CM) contractility assay and potentiated isoproterenol-induced beating rate in HSC-CMs.
Asunto(s)
Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Ftalazinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Quinazolinas/farmacología , Animales , Pruebas de Enzimas , Quinasa 2 del Receptor Acoplado a Proteína-G/metabolismo , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Estructura Molecular , Miocitos Cardíacos/efectos de los fármacos , Ftalazinas/síntesis química , Ftalazinas/farmacocinética , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Inhibidores de Proteínas Quinasas/farmacocinética , Quinazolinas/síntesis química , Quinazolinas/metabolismo , Quinazolinas/farmacocinética , Relación Estructura-ActividadRESUMEN
Monoacylglycerol acyltransferase enzymes (MGAT1, MGAT2, and MGAT3) convert monoacylglycerol to diacylglycerol (DAG). MGAT1 and MGAT2 are both implicated in obesity-related metabolic diseases. Conventional MGAT enzyme assays use radioactive substrates, wherein the product of the MGAT-catalyzed reaction is usually resolved by time-consuming thin layer chromatography (TLC) analysis. Furthermore, microsomal membrane preparations typically contain endogenous diacylglycerol acyltransferase (DGAT) from the host cells, and these DGAT activities can further acylate DAG to form triglyceride (TG). Our mass spectrometry (liquid chromatography-tandem mass spectrometry, or LC/MS/MS) MGAT2 assay measures human recombinant MGAT2-catalyzed formation of didecanoyl-glycerol from 1-decanoyl-rac-glycerol and decanoyl-CoA, to produce predominantly 1,3-didecanoyl-glycerol. Unlike 1,2-DAG, 1,3-didecanoyl-glycerol is proved to be not susceptible to further acylation to TG. 1,3-Didecanoyl-glycerol product can be readily solubilized and directly subjected to high-throughput mass spectrometry (HTMS) without further extraction in a 384-well format. We also have established the LC/MS/MS MGAT activity assay in the intestinal microsomes from various species. Our assay is proved to be highly sensitive, and thus it allows measurement of endogenous MGAT activity in cell lysates and tissue preparations. The implementation of the HTMS MGAT activity assay has facilitated the robust screening and evaluation of MGAT inhibitors for the treatment of metabolic diseases.
Asunto(s)
Diglicéridos/análisis , Inhibidores Enzimáticos/análisis , Ensayos Analíticos de Alto Rendimiento , N-Acetilglucosaminiltransferasas/antagonistas & inhibidores , Acilcoenzima A/metabolismo , Animales , Chlorocebus aethiops , Cromatografía Liquida/métodos , Diglicéridos/antagonistas & inhibidores , Diglicéridos/biosíntesis , Perros , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Haplorrinos , Humanos , Intestinos/efectos de los fármacos , Intestinos/enzimología , Cinética , Ratones , Microsomas/efectos de los fármacos , Microsomas/enzimología , N-Acetilglucosaminiltransferasas/metabolismo , Ratas , Ratas Sprague-Dawley , Proteínas Recombinantes/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Diacylglycerol acyltransferase (DGAT) catalyzes the final step in triglyceride (TG) synthesis. There are two isoforms, DGAT1 and DGAT2, with distinct protein sequences and potentially different physiological functions. To date, the ability to determine clear functional differences between DGAT1 and DGAT2, especially with respect to hepatic TG synthesis, has been elusive. To dissect the roles of these two key enzymes, we pretreated HepG2 hepatoma cells with (13)C(3)-D(5)-glycerol or (13)C(18)-oleic acid, and profiled the major isotope-labeled TG species by liquid chromatography tandem mass spectrometry. Selective DGAT1 and DGAT2 inhibitors demonstrated that (13)C(3)-D(5)-glycerol-incorporated TG synthesis was mediated by DGAT2, not DGAT1. Conversely, (13)C(18)-oleoyl-incorporated TG synthesis was predominantly mediated by DGAT1. To trace hepatic TG synthesis and VLDL triglyceride (VLDL-TG) secretion in vivo, we administered D(5)-glycerol to mice and measured plasma levels of D(5)-glycerol-incorporated TG. Treatment with an antisense oligonucleotide (ASO) to DGAT2 led to a significant reduction in D(5)-glycerol incorporation into VLDL-TG. In contrast, the DGAT2 ASO had no effect on the incorporation of exogenously administered (13)C(18)-oleic acid into VLDL-TG. Thus, our results indicate that DGAT1 and DGAT2 mediate distinct hepatic functions: DGAT2 is primarily responsible for incorporating endogenously synthesized FAs into TG, whereas DGAT1 plays a greater role in esterifying exogenous FAs to glycerol.
Asunto(s)
Diacilglicerol O-Acetiltransferasa/metabolismo , Pruebas de Enzimas/métodos , Glicerol/metabolismo , Hígado/enzimología , Ácido Oléico/metabolismo , Animales , Diacilglicerol O-Acetiltransferasa/antagonistas & inhibidores , Diacilglicerol O-Acetiltransferasa/genética , Inhibidores Enzimáticos/farmacología , Esterificación/efectos de los fármacos , Ácidos Grasos/biosíntesis , Ácidos Grasos/metabolismo , Células Hep G2 , Humanos , Marcaje Isotópico , Lipoproteínas VLDL/metabolismo , Masculino , Ratones , Oligonucleótidos Antisentido/genética , Triglicéridos/biosíntesisRESUMEN
Attenuation of fructose metabolism by the inhibition of ketohexokinase (KHK; fructokinase) should reduce body weight, free fatty acids, and triglycerides, thereby offering a novel approach to treat diabetes and obesity in response to modern diets. We have identified potent, selective inhibitors of human hepatic KHK within a series of pyrimidinopyrimidines (1). For example, 8, 38, and 47 exhibited KHK IC50 values of 12, 7, and 8 nM, respectively, and also showed potent cellular KHK inhibition (IC50 < 500 nM), which relates to their intrinsic potency vs KHK and their ability to penetrate cells. X-ray cocrystal structures of KHK complexes of 3, 8, and 47 revealed the important interactions within the enzyme's adenosine 5'-triphosphate (ATP)-binding pocket.
RESUMEN
During efforts to improve the bioavailability of FMS kinase inhibitors 1 and 2, a series of saturated and aromatic 4-heterocycles of reduced basicity were prepared and evaluated in an attempt to also improve the cardiovascular safety profile over lead arylamide 1, which possessed ion channel activity. The resultant compounds retained excellent potency and exhibited diminished ion channel activity.
Asunto(s)
Amidas/farmacología , Compuestos Heterocíclicos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Amidas/síntesis química , Amidas/química , Relación Dosis-Respuesta a Droga , Compuestos Heterocíclicos/síntesis química , Compuestos Heterocíclicos/química , Modelos Moleculares , Estructura Molecular , Oxidación-Reducción , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Estereoisomerismo , Relación Estructura-ActividadRESUMEN
A series of pyrimidinopyridones has been designed, synthesized and shown to be potent and selective inhibitors of the FMS tyrosine kinase. Introduction of an amide substituent at the 6-position of the pyridone core resulted in a significant potency increase. Compound 24 effectively inhibited in vivo LPS-induced TNF in mice greater than 80%.
Asunto(s)
Antiinflamatorios/farmacología , Antineoplásicos/farmacología , Diseño de Fármacos , Inhibidores de Proteínas Quinasas/farmacología , Piridonas/farmacología , Pirimidinas/farmacología , Receptor 1 de Factores de Crecimiento Endotelial Vascular/antagonistas & inhibidores , Amidas/química , Animales , Antiinflamatorios/síntesis química , Antineoplásicos/síntesis química , Lipopolisacáridos/toxicidad , Ratones , Modelos Químicos , Inhibidores de Proteínas Quinasas/síntesis química , Piridinas/química , Piridonas/síntesis química , Pirimidinas/síntesis química , Relación Estructura-Actividad , Células Tumorales Cultivadas , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
A series of 3,4,6-substituted 2-quinolones has been synthesized and evaluated as inhibitors of the kinase domain of macrophage colony-stimulating factor-1 receptor (FMS). The fully optimized compound, 4-(4-ethyl-phenyl)-3-(2-methyl-3H-imidazol-4-yl)-2-quinolone-6-carbonitrile 21b, has an IC(50) of 2.5 nM in an in vitro assay and 5.0 nM in a bone marrow-derived macrophage cellular assay. Inhibition of FMS signaling in vivo was also demonstrated in a mouse pharmacodynamic model.
Asunto(s)
Macrófagos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Quinolonas/síntesis química , Quinolonas/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Administración Oral , Animales , Disponibilidad Biológica , Proliferación Celular/efectos de los fármacos , Polarización de Fluorescencia , Genes fos/genética , Factor Estimulante de Colonias de Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Estructura Molecular , Quinolonas/farmacocinética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Bazo/metabolismo , Relación Estructura-ActividadRESUMEN
The optimization of the arylamide lead 2 resulted in identification of a highly potent series of 2,4-disubstituted arylamides. Compound 8 (FMS kinase IC(50)=0.0008 microM) served as a proof-of-concept candidate in a collagen-induced model of arthritis in mice.
Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Animales , Artritis/inducido químicamente , Artritis/tratamiento farmacológico , Colágeno , Relación Dosis-Respuesta a Droga , Ratones , Modelos Moleculares , Estructura Molecular , Relación Estructura-Actividad , Factores de TiempoRESUMEN
A series of 2'-aminoanilides have been identified which exhibit potent and selective inhibitory activity against the cFMS tyrosine kinase. Initial SAR studies within this series are described which examine aroyl and amino group substitutions, as well as the introduction of hydrophilic substituents on the benzene core. Compound 47 inhibits the isolated enzyme (IC(50)=0.027 microM) and blocks CSF-1-induced proliferation of bone marrow-derived macrophages (IC(50)=0.11 microM) and as such, serves as a lead candidate for further optimization studies.
Asunto(s)
Anilidas/síntesis química , Anilidas/farmacología , Antiinflamatorios/farmacología , Piperidinas/química , Inhibidores de Proteínas Quinasas/farmacología , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Anilidas/química , Antiinflamatorios/síntesis química , Antiinflamatorios/química , Línea Celular , Proliferación Celular/efectos de los fármacos , Evaluación Preclínica de Medicamentos , Humanos , Concentración 50 Inhibidora , Macrófagos/efectos de los fármacos , Estructura Molecular , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Relación Estructura-ActividadRESUMEN
The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.
Asunto(s)
Inhibidores de Proteínas Quinasas/química , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/química , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Receptor de Factor Estimulante de Colonias de Macrófagos/química , Amidas/química , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Humanos , Datos de Secuencia Molecular , Proteínas Mutantes Quiméricas/antagonistas & inhibidores , Proteínas Mutantes Quiméricas/química , Estructura Terciaria de Proteína/genética , Proto-Oncogenes Mas , Quinolonas/química , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/metabolismo , Receptor TIE-2/química , Receptor TIE-2/genética , Receptores de Factores de Crecimiento de Fibroblastos/química , Receptores de Factores de Crecimiento de Fibroblastos/genéticaRESUMEN
A parallel approach to designing crystallization constructs for the c-FMS kinase domain was implemented, resulting in proteins suitable for structural studies. Sequence alignment and limited proteolysis were used to identify and eliminate unstructured and surface-exposed domains. A small library of chimeras was prepared in which the kinase insert domain of FMS was replaced with the kinase insert domain of previously crystallized receptor-tyrosine kinases. Characterization of the newly generated FMS constructs by enzymology and thermoshift assays demonstrated similar activities and compound binding to the FMS full-length cytoplasmic domain. Two chimeras were evaluated for crystallization in the presence and absence of a variety of ligands resulting in crystal structures, and leading to a successful structure-based drug design project for this important inflammation target.
Asunto(s)
Ingeniería de Proteínas , Proteínas Tirosina Quinasas Receptoras/síntesis química , Proteínas Tirosina Quinasas Receptoras/genética , Receptor de Factor Estimulante de Colonias de Macrófagos/química , Receptor de Factor Estimulante de Colonias de Macrófagos/genética , Secuencia de Aminoácidos , Animales , Células Cultivadas , Cristalización , Citoplasma/química , Citoplasma/genética , Humanos , Datos de Secuencia Molecular , Proteínas Mutantes Quiméricas/síntesis química , Proteínas Mutantes Quiméricas/genética , Inhibidores de Proteínas Quinasas/química , Estructura Terciaria de Proteína/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptor de Factor Estimulante de Colonias de Macrófagos/antagonistas & inhibidores , Alineación de Secuencia , SpodopteraRESUMEN
A new class of Aurora-A inhibitors have been identified based on the 2-amino-pyrrolo[2,3-d]pyrimidine scaffold. Here, we describe the synthesis and SAR of this novel series. We report compounds which exhibit nanomolar activity in the Aurora-A biochemical assay and are able to inhibit tumor cell proliferation. This study culminates in compound 30, an inhibitor with potent activity against Aurora A (IC50=0.008 microM), anti-proliferative activity against several tumor cell lines and induces polyploidy in H460 cells.
Asunto(s)
Antineoplásicos/síntesis química , Antineoplásicos/farmacología , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/síntesis química , Pirimidinas/farmacología , Pirroles/síntesis química , Pirroles/farmacología , Aurora Quinasas , Línea Celular Tumoral , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Concentración 50 Inhibidora , Poliploidía , Relación Estructura-ActividadRESUMEN
2-Hydroxy-4,6-diamino-[1,3,5]triazines are described which are a novel class of potent inhibitors of the VEGF-R2 (flk-1/KDR) tyrosine kinase. 4-(Benzothiazol-6-ylamino)-6-(benzyl-isopropyl-amino)-[1,3,5]triazin-2-ol (14d) exhibited low nanomolar potency in the in vitro enzyme inhibition assay (IC(50) = 18 nM) and submicromolar inhibitory activity in a KDR-induced MAP kinase autophosphorylation assay in HUVEC cells (IC(50) = 280 nM), and also demonstrated good in vitro selectivity against a panel of growth factor receptor tyrosine kinases. Further, 14d showed antiangiogenic activity in an aortic ring explant assay by blocking endothelial outgrowths in rat aortas with an IC(50) of 1 microM.