RESUMEN
The virally encoded 3C-like protease (3CLpro) is a well-validated drug target for the inhibition of coronaviruses including Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2). Most inhibitors of 3CLpro are peptidomimetic, with a γ-lactam in place of Gln at the P1 position of the pseudopeptide chain. An effort was pursued to identify a viable alternative to the γ-lactam P1 mimetic which would improve physicochemical properties while retaining affinity for the target. Discovery of a 2-tetrahydrofuran as a suitable P1 replacement that is a potent enzymatic inhibitor of 3CLpro in SARS-CoV-2 virus is described herein.
Asunto(s)
Antivirales , Inhibidores de Proteasa de Coronavirus , Furanos , Antivirales/química , Antivirales/farmacología , Lactamas , Péptido Hidrolasas , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/química , SARS-CoV-2 , Furanos/química , Inhibidores de Proteasa de Coronavirus/químicaRESUMEN
GlaxoSmithKline and Astex Pharmaceuticals recently disclosed the discovery of the potent H-PGDS inhibitor GSK2894631A 1a (IC50 = 9.9 nM) as part of a fragment-based drug discovery collaboration with Astex Pharmaceuticals. This molecule exhibited good murine pharmacokinetics, allowing it to be utilized to explore H-PGDS pharmacology in vivo. Yet, with prolonged dosing at higher concentrations, 1a induced CNS toxicity. Looking to attenuate brain penetration in this series, aza-quinolines, were prepared with the intent of increasing polar surface area. Nitrogen substitutions at the 6- and 8-positions of the quinoline were discovered to be tolerated by the enzyme. Subsequent structure activity studies in these aza-quinoline scaffolds led to the identification of 1,8-naphthyridine 1y (IC50 = 9.4 nM) as a potent peripherally restricted H-PGDS inhibitor. Compound 1y is efficacious in four in vivo inflammatory models and exhibits no CNS toxicity.
Asunto(s)
Compuestos Aza/química , Inhibidores Enzimáticos/química , Quinolinas/química , Animales , Sitios de Unión , Encéfalo/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cristalografía por Rayos X , Estabilidad de Medicamentos , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Humanos , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Oxidorreductasas Intramoleculares/metabolismo , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Simulación de Dinámica Molecular , Músculo Esquelético/química , Músculo Esquelético/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
The discovery of a novel series of peptide deformylase inhibitors incorporating a piperazic acid amino acid found in nature is described. These compounds demonstrated potent in vitro enzymatic potency and antimicrobial activity. Crystal structure analysis revealed the piperazic acid optimized a key contact with the PDF protein that accounted for the increased enzymatic potency of these compounds. We describe lead optimization of the P3' region of the series that resulted in a compound with good potency against three target organisms. One molecule showed in vivo efficacy in a rat respiratory infection model but ultimately did not meet candidate progression criteria.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/farmacología , Inhibidores Enzimáticos/farmacología , Piridazinas/farmacología , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Enfermedades Cutáneas Infecciosas/tratamiento farmacológico , Amidohidrolasas/metabolismo , Antibacterianos/síntesis química , Antibacterianos/química , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/enzimología , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Estructura Molecular , Piridazinas/síntesis química , Piridazinas/química , Infecciones del Sistema Respiratorio/metabolismo , Enfermedades Cutáneas Infecciosas/metabolismo , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/enzimología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/enzimología , Relación Estructura-ActividadRESUMEN
With the goal of discovering more selective anti-inflammatory drugs, than COX inhibitors, to attenuate prostaglandin signaling, a fragment-based screen of hematopoietic prostaglandin D synthase was performed. The 76 crystallographic hits were sorted into similar groups, with the 3-cyano-quinoline 1a (FP IC50â¯=â¯220,000â¯nM, LEâ¯=â¯0.43) being a potent member of the 6,6-fused heterocyclic cluster. Employing SAR insights gained from structural comparisons of other H-PGDS fragment binding mode clusters, the initial hit 1a was converted into the 70-fold more potent quinoline 1d (IC50â¯=â¯3,100â¯nM, LEâ¯=â¯0.49). A systematic substitution of the amine moiety of 1d, utilizing structural information and array chemistry, with modifications to improve inhibitor stability, resulted in the identification of the 300-fold more active H-PGDS inhibitor tool compound 1bv (IC50â¯=â¯9.9â¯nM, LEâ¯=â¯0.42). This selective inhibitor exhibited good murine pharmacokinetics, dose-dependently attenuated PGD2 production in a mast cell degranulation assay and should be suitable to further explore H-PGDS biology.
Asunto(s)
Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Lipocalinas/antagonistas & inhibidores , Quinolinas/química , Quinolinas/farmacología , Animales , Descubrimiento de Drogas , Inhibidores Enzimáticos/farmacocinética , Humanos , Oxidorreductasas Intramoleculares/química , Oxidorreductasas Intramoleculares/metabolismo , Lipocalinas/química , Lipocalinas/metabolismo , Masculino , Ratones Endogámicos C57BL , Simulación del Acoplamiento Molecular , Quinolinas/farmacocinéticaRESUMEN
c-Abl kinase is maintained in its normal inactive state in the cell through an assembled, compact conformation. We describe two chemical series that bind to the myristoyl site of the c-Abl kinase domain and stimulate c-Abl activation. We hypothesize that these molecules activate c-Abl either by blocking the C-terminal helix from adopting a bent conformation that is critical for the formation of the autoinhibited conformation or by simply providing no stabilizing interactions to the bent conformation of this helix. Structure-based molecular modeling guided the optimization of binding and activation of c-Abl of these two chemical series and led to the discovery of c-Abl activators with nanomolar potency. The small molecule c-Abl activators reported herein could be used as molecular tools to investigate the biological functions of c-Abl and therapeutic implications of its activation.
Asunto(s)
Modelos Moleculares , Proteínas Proto-Oncogénicas c-abl/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Sitios de Unión , Cristalografía por Rayos X , Interacciones Hidrofóbicas e Hidrofílicas , Conformación Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-abl/química , Pirazoles/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-ActividadRESUMEN
Human genetic evidence shows that PDE3B is associated with metabolic and dyslipidemia phenotypes. A number of PDE3 family selective inhibitors have been approved by the FDA for various indications; however, given the undesirable proarrhythmic effects in the heart, selectivity for PDE3B inhibition over closely related family members (such as PDE3A; 48% identity) is a critical consideration for development of PDE3B therapeutics. Selectivity for PDE3B over PDE3A may be achieved in a variety of ways, including properties intrinsic to the compound or tissue-selective targeting. The high (>95%) active site homology between PDE3A and B represents a massive obstacle for obtaining selectivity at the active site; however, utilization of libraries with high molecular diversity in high throughput screens may uncover selective chemical matter. Herein, we employed a DNA-encoded library screen to identify PDE3B-selective inhibitors and identified potent and selective boronic acid compounds bound at the active site.
Asunto(s)
ADN , Corazón , Humanos , Dominio Catalítico , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 3RESUMEN
A new class of PDF inhibitor with potent, broad spectrum antibacterial activity is described. Optimization of blood stability and potency provided compounds with improved pharmacokinetics that were suitable for in vivo experiments. Compound 5c, which has robust antibacterial activity, demonstrated efficacy in two respiratory tract infection models.
Asunto(s)
Amidas/síntesis química , Amidohidrolasas/antagonistas & inhibidores , Antibacterianos/síntesis química , Proteínas Bacterianas/antagonistas & inhibidores , Prolina/análogos & derivados , Prolina/síntesis química , Infecciones del Sistema Respiratorio/tratamiento farmacológico , Administración Oral , Amidas/farmacología , Amidohidrolasas/metabolismo , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/metabolismo , Cristalografía por Rayos X , Modelos Animales de Enfermedad , Haemophilus influenzae/efectos de los fármacos , Haemophilus influenzae/crecimiento & desarrollo , Humanos , Inyecciones Intravenosas , Pruebas de Sensibilidad Microbiana , Modelos Moleculares , Prolina/farmacología , Ratas , Infecciones del Sistema Respiratorio/microbiología , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/crecimiento & desarrollo , Relación Estructura-ActividadRESUMEN
GSK3732394 is a multi-specific biologic inhibitor of HIV entry currently under clinical evaluation. A key component of this molecule is an adnectin (6940_B01) that binds to CD4 and inhibits downstream actions of gp160. Studies were performed to determine the binding site of the adnectin on CD4 and to understand the mechanism of inhibition. Using hydrogen-deuterium exchange with mass spectrometry (HDX), CD4 peptides showed differential rates of deuteration (either enhanced or slowed) in the presence of the adnectin that mapped predominantly to the interface of domains 2 and 3 (D2-D3). In addition, an X-ray crystal structure of an ibalizumab Fab/CD4(D1-D4)/adnectin complex revealed an extensive interface between the adnectin and residues on CD4 domains D2-D4 that stabilize a novel T-shaped CD4 conformation. A cryo-EM map of the gp140/CD4/GSK3732394 complex clearly shows the bent conformation for CD4 while bound to gp140. Mutagenic analyses on CD4 confirmed that amino acid F202 forms a key interaction with the adnectin. In addition, amino acid L151 was shown to be a critical indirect determinant of the specificity for binding to the human CD4 protein over related primate CD4 molecules, as it appears to modulate CD4's flexibility to adopt the adnectin-bound conformation. The significant conformational change of CD4 upon adnectin binding brings the D1 domain of CD4 in proximity to the host cell membrane surface, thereby re-orienting the gp120 binding site in a direction that is inaccessible to incoming virus due to a steric clash between gp160 trimers on the virus surface and the target cell membrane.
Asunto(s)
Fármacos Anti-VIH/farmacología , Antígenos CD4/química , Antígenos CD4/metabolismo , VIH-1/metabolismo , Acoplamiento Viral/efectos de los fármacos , Animales , Anticuerpos Monoclonales , Sitios de Unión , Modelos Moleculares , Unión Proteica , Conformación Proteica , Dominios Proteicos , Internalización del Virus/efectos de los fármacosRESUMEN
The continual bacterial adaptation to antibiotics creates an ongoing medical need for the development of novel therapeutics. Polypeptide deformylase (PDF) is a highly conserved bacterial enzyme, which is essential for viability. It has previously been shown that PDF inhibitors represent a promising new area for the development of antimicrobial agents, and that many of the best PDF inhibitors demonstrate slow, time-dependent binding. To improve our understanding of the mechanistic origin of this time-dependent inhibition, we examined in detail the kinetics of PDF catalysis and inhibition by several different PDF inhibitors. Varying pH and solvent isotope led to clear changes in time-dependent inhibition parameters, as did inclusion of NaCl, which binds to the active site metal of PDF. Quantitative analysis of these results demonstrated that the observed time dependence arises from slow binding of the inhibitors to the active site metal. However, we also found several metal binding inhibitors that exhibited rapid, non-time-dependent onset of inhibition. By a combination of structural and chemical modification studies, we show that metal binding is only slow when the rest of the inhibitor makes optimal hydrogen bonds within the subsites of PDF. Both of these interactions between the inhibitor and enzyme were found to be necessary to observe time-dependent inhibition, as elimination of either leads to its loss.
Asunto(s)
Amidohidrolasas/antagonistas & inhibidores , Amidohidrolasas/química , Antibacterianos/farmacología , Streptococcus pneumoniae/enzimología , Amidohidrolasas/farmacocinética , Antibacterianos/química , Catálisis , Dominio Catalítico/efectos de los fármacos , Cloruros/química , Cloruros/farmacología , Cristalografía por Rayos X , Medición de Intercambio de Deuterio/métodos , Ácidos Hidroxámicos/química , Ácidos Hidroxámicos/farmacocinética , Ácidos Hidroxámicos/farmacología , Marcaje Isotópico , Unión Proteica/efectos de los fármacos , Estructura Secundaria de Proteína , Solventes , Streptococcus pneumoniae/efectos de los fármacos , Zinc/químicaRESUMEN
Phosphoinositide 3-kinases have been targeted for therapeutic research because they are key components of a cell signaling cascade controlling proliferation, growth, and survival. Direct activation of the PI3Kalpha pathway contributes to the development and progression of solid tumors in breast, endometrial, colon, ovarian, and gastric cancers. In the context of a drug discovery effort, the availability of a robust crystallographic system is a means to understand the subtle differences between ATP competitive inhibitor interactions with the active site and their selectivity against other PI3Kinase enzymes. To generate a suitable recombinant design for this purpose, a p85alpha-p110alpha fusion system was developed which enabled the expression and purification of a stoichiometrically homogeneous, constitutively active enzyme for structure determination with potent ATP competitive inhibitors (Raha et al., in preparation) [56]. This approach has yielded preparations with activity and inhibition characteristics comparable to those of the full-length PI3Kalpha from which X-ray diffracting crystals were grown with inhibitors bound in the active site.
Asunto(s)
Fosfatidilinositol 3-Quinasas Clase II/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos/farmacología , Adenosina Trifosfato/metabolismo , Animales , Fusión Artificial Génica , Baculoviridae/metabolismo , Sitios de Unión , Células Cultivadas , Fosfatidilinositol 3-Quinasas Clase II/química , Fosfatidilinositol 3-Quinasas Clase II/genética , Fosfatidilinositol 3-Quinasa Clase Ia/genética , Diseño de Fármacos , Concentración 50 Inhibidora , Modelos Moleculares , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Spodoptera/citología , Spodoptera/metabolismo , Difracción de Rayos XRESUMEN
Abelson kinase (c-Abl) is a ubiquitously expressed, nonreceptor tyrosine kinase which plays a key role in cell differentiation and survival. It was hypothesized that transient activation of c-Abl kinase via displacement of the N-terminal autoinhibitory "myristoyl latch", may lead to an increased hematopoietic stem cell differentiation. This would increase the numbers of circulating neutrophils and so be an effective treatment for chemotherapy-induced neutropenia. This paper describes the discovery and optimization of a thiazole series of novel small molecule c-Abl activators, initially identified by a high throughput screening. Subsequently, a scaffold-hop, which exploited the improved physicochemical properties of a dihydropyrazole analogue, identified through fragment screening, delivered potent, soluble, cell-active c-Abl activators, which demonstrated the intracellular activation of c-Abl in vivo.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Pirazoles/farmacología , Tiazoles/farmacología , Animales , Sitios de Unión , Descubrimiento de Drogas , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Estructura Molecular , Unión Proteica , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-abl/química , Proteínas Proto-Oncogénicas c-abl/metabolismo , Pirazoles/química , Pirazoles/metabolismo , Relación Estructura-Actividad , Tiazoles/química , Tiazoles/metabolismoRESUMEN
RIP1 kinase regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including inflammatory and neurological diseases. Currently, RIP1 kinase inhibitors have advanced into early clinical trials for evaluation in inflammatory diseases such as psoriasis, rheumatoid arthritis, and ulcerative colitis and neurological diseases such as amyotrophic lateral sclerosis and Alzheimer's disease. In this paper, we report on the design of potent and highly selective dihydropyrazole (DHP) RIP1 kinase inhibitors starting from a high-throughput screen and the lead-optimization of this series from a lead with minimal rat oral exposure to the identification of dihydropyrazole 77 with good pharmacokinetic profiles in multiple species. Additionally, we identified a potent murine RIP1 kinase inhibitor 76 as a valuable in vivo tool molecule suitable for evaluating the role of RIP1 kinase in chronic models of disease. DHP 76 showed efficacy in mouse models of both multiple sclerosis and human retinitis pigmentosa.
Asunto(s)
Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Proteínas de Complejo Poro Nuclear/antagonistas & inhibidores , Pirazoles/síntesis química , Pirazoles/farmacología , Proteínas de Unión al ARN/antagonistas & inhibidores , Animales , Disponibilidad Biológica , Línea Celular , Enfermedad Crónica , Diseño de Fármacos , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Inhibidores Enzimáticos/farmacocinética , Haplorrinos , Ensayos Analíticos de Alto Rendimiento , Humanos , Ratones , Ratones Endogámicos C57BL , Modelos Moleculares , Esclerosis Múltiple/tratamiento farmacológico , Pirazoles/farmacocinética , Ratas , Retinitis Pigmentosa/tratamiento farmacológico , Relación Estructura-ActividadRESUMEN
RIP1 regulates necroptosis and inflammation and may play an important role in contributing to a variety of human pathologies, including immune-mediated inflammatory diseases. Small-molecule inhibitors of RIP1 kinase that are suitable for advancement into the clinic have yet to be described. Herein, we report our lead optimization of a benzoxazepinone hit from a DNA-encoded library and the discovery and profile of clinical candidate GSK2982772 (compound 5), currently in phase 2a clinical studies for psoriasis, rheumatoid arthritis, and ulcerative colitis. Compound 5 potently binds to RIP1 with exquisite kinase specificity and has excellent activity in blocking many TNF-dependent cellular responses. Highlighting its potential as a novel anti-inflammatory agent, the inhibitor was also able to reduce spontaneous production of cytokines from human ulcerative colitis explants. The highly favorable physicochemical and ADMET properties of 5, combined with high potency, led to a predicted low oral dose in humans.
Asunto(s)
Antiinflamatorios/química , Antiinflamatorios/farmacología , Colitis Ulcerosa/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Animales , Benzazepinas/química , Benzazepinas/farmacología , Colitis Ulcerosa/inmunología , Citocinas/inmunología , Perros , Haplorrinos , Humanos , Inflamación/inmunología , Ratones , Simulación del Acoplamiento Molecular , Conejos , Ratas , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Porcinos , Porcinos Enanos , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
The recent discovery of the role of receptor interacting protein 1 (RIP1) kinase in tumor necrosis factor (TNF)-mediated inflammation has led to its emergence as a highly promising target for the treatment of multiple inflammatory diseases. We screened RIP1 against GSK's DNA-encoded small-molecule libraries and identified a novel highly potent benzoxazepinone inhibitor series. We demonstrate that this template possesses complete monokinase selectivity for RIP1 plus unique species selectivity for primate versus nonprimate RIP1. We elucidate the conformation of RIP1 bound to this benzoxazepinone inhibitor driving its high kinase selectivity and design specific mutations in murine RIP1 to restore potency to levels similar to primate RIP1. This series differentiates itself from known RIP1 inhibitors in combining high potency and kinase selectivity with good pharmacokinetic profiles in rodents. The favorable developability profile of this benzoxazepinone template, as exemplified by compound 14 (GSK'481), makes it an excellent starting point for further optimization into a RIP1 clinical candidate.
Asunto(s)
ADN/química , Isoxazoles/farmacología , Oxazepinas/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Línea Celular Tumoral , Cristalografía por Rayos X , Relación Dosis-Respuesta a Droga , Células HT29 , Humanos , Isoxazoles/síntesis química , Isoxazoles/química , Ratones , Modelos Moleculares , Estructura Molecular , Oxazepinas/síntesis química , Oxazepinas/química , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Células U937RESUMEN
While mutations in Sox4, a member of the SRY-like HMG box gene family, have been associated with a variety of human disorders and embryonic defects in the mouse, the structure and developmental expression of Sox4 in the avian embryo has not been described. We have isolated and characterized the chicken Sox4 gene. The chicken Sox4 gene shows a high degree of sequence homology with the mouse and human Sox4 genes, particularly in the HMG-like DNA binding domain and at the carboxy terminus. Furthermore, our in situ hybridization studies document an expression pattern during embryonic development that is very similar to that described for the mouse, particularly with regards to expression in the developing heart. However, abundant expression was also detected in tissues of neural crest origin including pharyngeal arch and craniofacial mesoderm, supporting a potential primary role in neural crest cardiac pathology previously detected in Sox4 mutant mice. Furthermore, a reciprocal pattern of Sox4 and Sox11 expression in the developing neural tube was detected in the chicken compared to that seen in the mouse. These studies suggest that Sox4 plays an important and conserved role in the embryonic development of these structures in the chicken as well as in the mouse and lay the foundation for future studies of the role of Sox4 during critical events of organogenesis.
Asunto(s)
Embrión de Pollo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Transactivadores/genética , Secuencia de Aminoácidos , Animales , Northern Blotting , Embrión de Pollo/embriología , Secuencia Conservada/genética , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Corazón/embriología , Hibridación in Situ , Datos de Secuencia Molecular , Miocardio/metabolismo , Sistema Nervioso/embriología , Sistema Nervioso/metabolismo , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción SOXC , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de AminoácidoRESUMEN
The protein Keap1 is central to the regulation of the Nrf2-mediated cytoprotective response, and is increasingly recognized as an important target for therapeutic intervention in a range of diseases involving excessive oxidative stress and inflammation. The BTB domain of Keap1 plays key roles in sensing environmental electrophiles and in mediating interactions with the Cul3/Rbx1 E3 ubiquitin ligase system, and is believed to be the target for several small molecule covalent activators of the Nrf2 pathway. However, despite structural information being available for several BTB domains from related proteins, there have been no reported crystal structures of Keap1 BTB, and this has precluded a detailed understanding of its mechanism of action and interaction with antagonists. We report here the first structure of the BTB domain of Keap1, which is thought to contain the key cysteine residue responsible for interaction with electrophiles, as well as structures of the covalent complex with the antagonist CDDO/bardoxolone, and of the constitutively inactive C151W BTB mutant. In addition to providing the first structural confirmation of antagonist binding to Keap1 BTB, we also present biochemical evidence that adduction of Cys 151 by CDDO is capable of inhibiting the binding of Cul3 to Keap1, and discuss how this class of compound might exert Nrf2 activation through disruption of the BTB-Cul3 interface.
Asunto(s)
Imidazoles/química , Péptidos y Proteínas de Señalización Intracelular/química , Ácido Oleanólico/análogos & derivados , Dominios y Motivos de Interacción de Proteínas , Sitios de Unión , Humanos , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Proteína 1 Asociada A ECH Tipo Kelch , Modelos Moleculares , Conformación Molecular , Mutación , Ácido Oleanólico/química , Ácido Oleanólico/farmacología , Unión Proteica , Relación Estructura-ActividadRESUMEN
Potent inhibitors of RIP1 kinase from three distinct series, 1-aminoisoquinolines, pyrrolo[2,3-b]pyridines, and furo[2,3-d]pyrimidines, all of the type II class recognizing a DLG-out inactive conformation, were identified from screening of our in-house kinase focused sets. An exemplar from the furo[2,3-d]pyrimidine series showed a dose proportional response in protection from hypothermia in a mouse model of TNFα induced lethal shock.
RESUMEN
c-Abl kinase activity is regulated by a unique mechanism involving the formation of an autoinhibited conformation in which the N-terminal myristoyl group binds intramolecularly to the myristoyl binding site on the kinase domain and induces the bending of the αI helix that creates a docking surface for the SH2 domain. Here, we report a small-molecule c-Abl activator, DPH, that displays potent enzymatic and cellular activity in stimulating c-Abl activation. Structural analyses indicate that DPH binds to the myristoyl binding site and prevents the formation of the bent conformation of the αI helix through steric hindrance, a mode of action distinct from the previously identified allosteric c-Abl inhibitor, GNF-2, that also binds to the myristoyl binding site. DPH represents the first cell-permeable, small-molecule tool compound for c-Abl activation.
Asunto(s)
Descubrimiento de Drogas , Hidantoínas/metabolismo , Hidantoínas/farmacología , Proteínas Proto-Oncogénicas c-abl/metabolismo , Pirazoles/metabolismo , Pirazoles/farmacología , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Células Hep G2 , Humanos , Hidantoínas/química , Modelos Moleculares , Datos de Secuencia Molecular , Permeabilidad , Fosforilación/efectos de los fármacos , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-abl/química , Proteínas Proto-Oncogénicas c-crk/metabolismo , Pirazoles/químicaRESUMEN
Phosphoinositide-dependent protein kinase-1(PDK1) is a master regulator of the AGC family of kinases and an integral component of the PI3K/AKT/mTOR pathway. As this pathway is among the most commonly deregulated across all cancers, a selective inhibitor of PDK1 might have utility as an anticancer agent. Herein we describe our lead optimization of compound 1 toward highly potent and selective PDK1 inhibitors via a structure-based design strategy. The most potent and selective inhibitors demonstrated submicromolar activity as measured by inhibition of phosphorylation of PDK1 substrates as well as antiproliferative activity against a subset of AML cell lines. In addition, reduction of phosphorylation of PDK1 substrates was demonstrated in vivo in mice bearing OCl-AML2 xenografts. These observations demonstrate the utility of these molecules as tools to further delineate the biology of PDK1 and the potential pharmacological uses of a PDK1 inhibitor.
Asunto(s)
Antineoplásicos/síntesis química , Indazoles/síntesis química , Morfolinas/síntesis química , Piperidinas/síntesis química , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Pirimidinas/síntesis química , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Línea Celular Tumoral , Cristalografía por Rayos X , Ensayos de Selección de Medicamentos Antitumorales , Indazoles/química , Indazoles/farmacología , Ratones , Ratones SCID , Modelos Moleculares , Estructura Molecular , Morfolinas/química , Morfolinas/farmacología , Trasplante de Neoplasias , Fosforilación , Piperidinas/química , Piperidinas/farmacología , Unión Proteica , Pirimidinas/química , Pirimidinas/farmacología , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Estereoisomerismo , Relación Estructura-Actividad , Trasplante HeterólogoRESUMEN
Fragment screening of phosphoinositide-dependent kinase-1 (PDK1) in a biochemical kinase assay afforded hits that were characterized and prioritized based on ligand efficiency and binding interactions with PDK1 as determined by NMR. Subsequent crystallography and follow-up screening led to the discovery of aminoindazole 19, a potent leadlike PDK1 inhibitor with high ligand efficiency. Well-defined structure-activity relationships and protein crystallography provide a basis for further elaboration and optimization of 19 as a PDK1 inhibitor.