RESUMEN
BACKGROUND: Cassava leaves are an abundant global agricultural residue because the roots are a major source of dietary carbohydrates. Although cassava leaves are high in protein, the protein is not bioavailable. This work aimed to convert cassava leaves to a bioavailable protein-rich animal feed ingredient using high-protein yeasts. RESULTS: The structural proteins (ca 200 g kg-1 d.b.) from sundried cassava leaves were solubilized by mild alkali pretreatment, and the resulting cassava leaf hydrolysate (CLH) was used to screen for growth of 46 high-protein yeasts from 30 species. Promising candidates from the initial screen cultivated at a 10 mL scale demonstrated increases in relative abundance of essential amino acids over that of CLH. In particular, lysine, growth-limiting for some livestock, was increased up to 226% over the CLH content. One yeast, Pichia kudriavzevii UCDFST 11-602, was grown in 3 L of CLH in a bioreactor to examine the scale-up potential of the yeast protein production. While glucose was completely consumed, yeast growth exited log phase before depleting either carbon or nitrogen, suggesting other growth-limiting factors at the larger scale. CONCLUSIONS: High-value animal feed with enriched essential amino acid profiles can be produced by yeasts grown on agricultural residues. Yeasts convert structural protein solubilized from cassava leaves to essential amino acid-enriched, digestible protein. The low carbohydrate content of the leaves (ca 200 g kg-1 d.b.), however, necessitated glucose supplementation for yeast growth. © 2018 Society of Chemical Industry.
Asunto(s)
Manihot/microbiología , Pichia/metabolismo , Hojas de la Planta/metabolismo , Alimentación Animal/análisis , Biomasa , Biotransformación , Manihot/química , Manihot/metabolismo , Pichia/crecimiento & desarrollo , Hojas de la Planta/química , Hojas de la Planta/microbiologíaRESUMEN
We report the discovery of a novel series of ATP-competitive Janus kinase 3 (JAK3) inhibitors based on the 5H-pyrrolo[2,3-b]pyrazine scaffold. The initial leads in this series, compounds 1a and 1h, showed promising potencies, but a lack of selectivity against other isoforms in the JAK family. Computational and crystallographic analysis suggested that the phenyl ether moiety possessed a favorable vector to achieve selectivity. Exploration of this vector resulted in the identification of 12b and 12d, as potent JAK3 inhibitors, demonstrating improved JAK family and kinase selectivity.
Asunto(s)
Janus Quinasa 3/antagonistas & inhibidores , Éteres Fenílicos/química , Inhibidores de Proteínas Quinasas/química , Piridazinas/química , Pirroles/química , Sitios de Unión , Dominio Catalítico , Evaluación Preclínica de Medicamentos , Janus Quinasa 3/metabolismo , Simulación del Acoplamiento Molecular , Éteres Fenílicos/síntesis química , Éteres Fenílicos/metabolismo , Unión Proteica , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Relación Estructura-ActividadRESUMEN
Using a structure based design approach we have identified a series of indazole substituted pyrrolopyrazines, which are potent inhibitors of JAK3. Intramolecular electronic repulsion was used as a strategy to induce a strong conformational bias within the ligand. Compounds bearing this conformation participated in a favorable hydrophobic interaction with a cysteine residue in the JAK3 binding pocket, which imparted high selectivity versus the kinome and improved selectivity within the JAK family.
Asunto(s)
Diseño de Fármacos , Janus Quinasa 3/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/química , Sitios de Unión , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Indazoles/química , Janus Quinasa 1/antagonistas & inhibidores , Janus Quinasa 1/metabolismo , Janus Quinasa 2/antagonistas & inhibidores , Janus Quinasa 2/metabolismo , Janus Quinasa 3/metabolismo , Simulación del Acoplamiento Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/metabolismo , Estructura Terciaria de Proteína , Pirazinas/síntesis química , Pirazinas/química , Pirazinas/metabolismo , Relación Estructura-ActividadRESUMEN
Conformational modeling has been successfully applied to the design of cyclic bioisosteres used to replace a conformationally rigid amide bond in a series of thiophene carboxylate inhibitors of HCV NS5B polymerase. Select compounds were equipotent with the original amide series. Single-point mutant binding studies, in combination with inhibition structure-activity relationships, suggest this new series interacts at the Thumb-II domain of NS5B. Inhibitor binding at the Thumb-II site was ultimately confirmed by solving a crystal structure of 8b complexed with NS5B.
Asunto(s)
Amidas/química , Antivirales/síntesis química , Inhibidores Enzimáticos/síntesis química , Hepacivirus/efectos de los fármacos , Tiofenos/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Amidas/síntesis química , Amidas/farmacología , Antivirales/química , Antivirales/farmacología , Sitios de Unión , Cristalografía por Rayos X , Diseño de Fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Tiofenos/química , Tiofenos/farmacología , Proteínas no Estructurales Virales/metabolismoRESUMEN
A series of benzo[d]isothiazole-1,1-dioxides were designed and evaluated as inhibitors of HCV polymerase NS5B. Structure-based design led to the incorporation of a high affinity methyl sulfonamide group. Structure-activity relationship (SAR) studies of this series revealed analogues with submicromolar potencies in the HCV replicon assay and moderate pharmacokinetic properties. SAR studies combined with structure based drug design focused on the sulfonamide region led to a novel and potent cyclic analogue.
Asunto(s)
Antivirales/síntesis química , Hepacivirus/efectos de los fármacos , Tiazoles/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/química , Antivirales/farmacocinética , Sitios de Unión , Cristalografía por Rayos X , Haplorrinos , Ratas , Relación Estructura-Actividad , Tiazoles/síntesis química , Tiazoles/farmacocinética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Benzothiazine-substituted tetramic acids were discovered as highly potent non-nucleoside inhibitors of HCV NS5B polymerase. X-ray crystallography studies confirmed the binding mode of these inhibitors with HCV NS5B polymerase. Rational optimization of time dependent inactivation of CYP 3A4 and clearance was accomplished by incorporation of electron-withdrawing groups to the benzothiazine core.
Asunto(s)
Antivirales/síntesis química , Hepacivirus/efectos de los fármacos , Pirrolidinonas/química , Tiazinas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/química , Antivirales/farmacocinética , Sitios de Unión , Cristalografía por Rayos X , Pirrolidinonas/síntesis química , Pirrolidinonas/farmacocinética , Ratas , Relación Estructura-Actividad , Proteínas no Estructurales Virales/metabolismoRESUMEN
A new series of benzothiazine-substituted quinolinediones were evaluated as inhibitors of HCV polymerase NS5B. SAR studies on this series revealed a methyl sulfonamide group as a high affinity feature. Analogues with this group showed submicromolar potencies in the HCV cell based replicon assay. Pharmacokinetic and toxicology studies were also performed on a selected compound (34) to evaluate in vivo properties of this new class of inhibitors of HCV NS5B polymerase.
Asunto(s)
Antivirales/química , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Hepacivirus/efectos de los fármacos , Quinolinas/química , Quinolonas/química , Tiazinas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/síntesis química , Antivirales/farmacocinética , Simulación por Computador , Cristalografía por Rayos X , ARN Polimerasas Dirigidas por ADN/metabolismo , Perros , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacocinética , Humanos , Quinolinas/síntesis química , Quinolinas/farmacocinética , Quinolonas/síntesis química , Quinolonas/farmacología , Ratas , Relación Estructura-Actividad , Tiazinas/síntesis química , Tiazinas/farmacología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Activated factor XIIa (FXIIa) is a serine protease that has received a great deal of interest in recent years as a potential target for the development of new antithrombotics. Despite the strong interest in obtaining structural information, only the structure of the FXIIa catalytic domain in its zymogen conformation is available. In this work, reproducible experimental conditions found for the crystallization of human plasma ß-FXIIa and crystal growth optimization have led to determination of the first structure of the active form of the enzyme. Two crystal structures of human plasma ß-FXIIa complexed with small molecule inhibitors are presented herein. The first is the noncovalent inhibitor benzamidine. The second is an aminoisoquinoline containing a boronic acid-reactive group that targets the catalytic serine. Both benzamidine and the aminoisoquinoline bind in a canonical fashion typical of synthetic serine protease inhibitors, and the protease domain adopts a typical chymotrypsin-like serine protease active conformation. This novel structural data explains the basis of the FXII activation, provides insights into the enzymatic properties of ß-FXIIa, and is a great aid toward the further design of protease inhibitors for human FXIIa.
Asunto(s)
Factor XII/química , Benzamidinas/química , Ácidos Borónicos/química , Cristalización/métodos , Cristalografía por Rayos X , Bases de Datos de Proteínas , Factor XII/antagonistas & inhibidores , Humanos , Estructura Molecular , Unión Proteica , Programas InformáticosRESUMEN
We report the discovery of a new potent allosteric effector of sickle cell hemoglobin, GBT440 (36), that increases the affinity of hemoglobin for oxygen and consequently inhibits its polymerization when subjected to hypoxic conditions. Unlike earlier allosteric activators that bind covalently to hemoglobin in a 2:1 stoichiometry, 36 binds with a 1:1 stoichiometry. Compound 36 is orally bioavailable and partitions highly and favorably into the red blood cell with a RBC/plasma ratio of â¼150. This partitioning onto the target protein is anticipated to allow therapeutic concentrations to be achieved in the red blood cell at low plasma concentrations. GBT440 (36) is in Phase 3 clinical trials for the treatment of sickle cell disease (NCT03036813).
RESUMEN
The transport of 10 amino acid ester prodrugs of levovirin (LVV) was investigated in the human intestinal Caco-2 cell line in order to overcome the poor oral bioavailability of LVV, an investigational drug for the treatment of hepatitis C infection. The prodrugs were designed to improve the permeability of LVV across the intestinal epithelium by targeting the di/tri-peptide carrier, PepT1. Caco-2 cell monolayers were employed to study the transport and hydrolysis properties of the prodrugs. Among all mono amino acid ester prodrugs studied, the LVV-5'-(L)-valine prodrug (R1518) exhibited the maximum increase (48-fold) in permeability with nearly complete conversion to LVV within 1 h. Di-amino acid esters did not offer significant enhancement in permeability comparing with mono amino acid esters and exhibited slower conversion to LVV in Caco2 cell monolayers. Pharmacokinetic screening studies of the prodrugs in rats yielded the highest fold increase (6.9-fold) of AUC with R1518 and in general displayed a similar trend to that observed in increases of permeability in Caco-2 cells. Mechanisms involved in the Caco-2 cell transport of R1518 were also investigated. Results of bi-directional transport studies support the involvement of carrier-mediated transport mechanisms for R1518, but not for the LVV-5'-(D)-valine prodrug or LVV. Moreover, the permeability of R1518 was found to be proton dependent. PepT1-mediated transport of R1518 was supported by results of competitive transport studies of R1518 with the PepT1 substrates enalapril, Gly-Sar, valganciclovir, and cephalexin. R1518 was also found to inhibit the permeability of valganciclovir and cephalexin. These results suggest that R1518 is a PepT1 substrate as well as an inhibitor.
Asunto(s)
Aminoácidos/farmacocinética , Sistemas de Liberación de Medicamentos , Mucosa Intestinal/metabolismo , Profármacos/metabolismo , Protones , Simportadores/administración & dosificación , Animales , Área Bajo la Curva , Transporte Biológico , Células CACO-2 , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ésteres , Humanos , Concentración de Iones de Hidrógeno , Hidrólisis , Absorción Intestinal/efectos de los fármacos , Estructura Molecular , Monosacáridos/química , Monosacáridos/farmacología , Transportador de Péptidos 1 , Profármacos/farmacocinética , Ratas , Triazoles/química , Triazoles/farmacologíaRESUMEN
Two distinct synthetic schemes were applied to access heteroatom-containing alpha-chain lactams or lactams terminated as aryl acids. The latter lactams were devised using a pharmacophore for EP(4) receptor activity. gamma-Lactams were characterized for their prostanoid EP receptor affinities and EP(4) activity and found to be selective for the EP(2) and EP(4) receptors or selective for the EP(4) subtype. Benzoic acid 17 displayed enhanced in vivo exposure relative to 3.
Asunto(s)
Benzoatos/síntesis química , Lactamas/síntesis química , Pirrolidinas/síntesis química , Receptores de Prostaglandina E/agonistas , Animales , Benzoatos/farmacocinética , Benzoatos/farmacología , Proteínas Sanguíneas/metabolismo , Semivida , Humanos , Lactamas/farmacocinética , Lactamas/farmacología , Modelos Moleculares , Conformación Molecular , Método de Montecarlo , Oxidación-Reducción , Unión Proteica , Pirrolidinas/farmacocinética , Pirrolidinas/farmacología , Ratas , Receptores de Prostaglandina E/metabolismo , Subtipo EP2 de Receptores de Prostaglandina E , Subtipo EP4 de Receptores de Prostaglandina E , Relación Estructura-ActividadRESUMEN
Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.
Asunto(s)
Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Piridazinas/síntesis química , Piridazinas/farmacología , Bazo/enzimología , Amidas/síntesis química , Amidas/farmacología , Animales , Biología Computacional , Simulación por Computador , Diseño de Fármacos , Canales de Potasio Éter-A-Go-Go/efectos de los fármacos , Humanos , Técnicas In Vitro , Ratones , Microsomas Hepáticos/metabolismo , Modelos Moleculares , Conformación Molecular , Mutagénesis/efectos de los fármacos , Pruebas de Mutagenicidad , Inhibidores de Proteínas Quinasas/farmacocinética , Piridazinas/farmacocinética , Ratas , Bazo/efectos de los fármacos , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
The Janus kinases (JAKs) are involved in multiple signaling networks relevant to inflammatory diseases, and inhibition of one or more members of this class may modulate disease activity or progression. We optimized a new inhibitor scaffold, 3-amido-5-cyclopropylpyrrolopyrazines, to a potent example with reasonable kinome selectivity, including selectivity for JAK3 versus JAK1, and good biopharmaceutical properties. Evaluation of this analogue in cellular and in vivo models confirmed functional selectivity for modulation of a JAK3/JAK1-dependent IL-2 stimulated pathway over a JAK1/JAK2/Tyk2-dependent IL-6 stimulated pathway.