RESUMEN
In cystic fibrosis (CF), excessive furin activity plays a critical role in the activation of the epithelial sodium channel (ENaC), dysregulation of which contributes to airway dehydration, ineffective mucociliary clearance (MCC), and mucus obstruction. Here, we report a highly selective, cell-permeable furin inhibitor, BOS-318, that derives selectivity by eliciting the formation of a new, unexpected binding pocket independent of the active site catalytic triad. Using human ex vivo models, BOS-318 showed significant suppression of ENaC, which led to enhanced airway hydration and an â¼30-fold increase in MCC rate. Furin inhibition also protected ENaC from subsequent activation by neutrophil elastase, a soluble protease dominant in CF airways. Additional therapeutic benefits include protection against epithelial cell death induced by Pseudomonas aeruginosa exotoxin A. Our findings demonstrate the utility of selective furin inhibition as a mutation-agnostic approach that can correct features of CF airway pathophysiology in a manner expected to deliver therapeutic value.
Asunto(s)
Fibrosis Quística , Furina , Fibrosis Quística/tratamiento farmacológico , Fibrosis Quística/metabolismo , Regulador de Conductancia de Transmembrana de Fibrosis Quística/metabolismo , Canales Epiteliales de Sodio/genética , Canales Epiteliales de Sodio/metabolismo , Furina/antagonistas & inhibidores , Humanos , Depuración MucociliarRESUMEN
DNA methylation, a key epigenetic driver of transcriptional silencing, is universally dysregulated in cancer. Reversal of DNA methylation by hypomethylating agents, such as the cytidine analogs decitabine or azacytidine, has demonstrated clinical benefit in hematologic malignancies. These nucleoside analogs are incorporated into replicating DNA where they inhibit DNA cytosine methyltransferases DNMT1, DNMT3A and DNMT3B through irreversible covalent interactions. These agents induce notable toxicity to normal blood cells thus limiting their clinical doses. Herein we report the discovery of GSK3685032, a potent first-in-class DNMT1-selective inhibitor that was shown via crystallographic studies to compete with the active-site loop of DNMT1 for penetration into hemi-methylated DNA between two CpG base pairs. GSK3685032 induces robust loss of DNA methylation, transcriptional activation and cancer cell growth inhibition in vitro. Due to improved in vivo tolerability compared with decitabine, GSK3685032 yields superior tumor regression and survival mouse models of acute myeloid leukemia.
Asunto(s)
Azacitidina , Leucemia Mieloide Aguda , Animales , Azacitidina/farmacología , ADN/metabolismo , Metilación de ADN , Metilasas de Modificación del ADN/genética , Decitabina/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , RatonesRESUMEN
Indoleamine-2,3-dioxygenase 1 (IDO1) is a heme-containing enzyme that catalyzes the rate-limiting step in the kynurenine pathway of tryptophan (TRP) metabolism. As it is an inflammation-induced immunoregulatory enzyme, pharmacological inhibition of IDO1 activity is currently being pursued as a potential therapeutic tool for the treatment of cancer and other disease states. As such, a detailed understanding of the mechanism of action of IDO1 inhibitors with various mechanisms of inhibition is of great interest. Comparison of an apo-form-binding IDO1 inhibitor (GSK5628) to the heme-coordinating compound, epacadostat (Incyte), allows us to explore the details of the apo-binding inhibition of IDO1. Herein, we demonstrate that GSK5628 inhibits IDO1 by competing with heme for binding to a heme-free conformation of the enzyme (apo-IDO1), whereas epacadostat coordinates its binding with the iron atom of the IDO1 heme cofactor. Comparison of these two compounds in cellular systems reveals a long-lasting inhibitory effect of GSK5628, previously undescribed for other known IDO1 inhibitors. Detailed characterization of this apo-binding mechanism for IDO1 inhibition might help design superior inhibitors or could confer a unique competitive advantage over other IDO1 inhibitors vis-à-vis specificity and pharmacokinetic parameters.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Indolamina-Pirrol 2,3,-Dioxigenasa/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Conformación MolecularRESUMEN
Iron overload disorders are characterized by the body's inability to regulate iron absorption and its storage which can lead to organ failures. Accumulated evidence has revealed that hepcidin, the master regulator of iron homeostasis, is negatively modulated by TMPRSS6 (matriptase-2), a liver-specific type II transmembrane serine protease (TTSP). Here, we report that treatment with a peptidomimetic inhibitor affecting TMPRSS6 activity increases hepcidin production in hepatic cells. Moreover, similar effects were observed when using non-peptidic inhibitors obtained through optimization of hits from high-throughput screening. Using HepG2 cells and human primary hepatocytes, we show that TMPRSS6 inhibitors block TMPRSS6-dependent hemojuvelin cleavage and increase HAMP expression and levels of secreted hepcidin.
Asunto(s)
Evaluación Preclínica de Medicamentos , Hepcidinas/metabolismo , Proteínas de la Membrana/antagonistas & inhibidores , Inhibidores de Serina Proteinasa/química , Benzotiazoles/química , Sitios de Unión , Dominio Catalítico , Supervivencia Celular/efectos de los fármacos , Proteínas Ligadas a GPI/metabolismo , Proteína de la Hemocromatosis/metabolismo , Células Hep G2 , Hepatocitos/citología , Hepatocitos/metabolismo , Ensayos Analíticos de Alto Rendimiento , Humanos , Hierro/metabolismo , Proteínas de la Membrana/metabolismo , Simulación de Dinámica Molecular , Peptidomiméticos , Proteolisis/efectos de los fármacos , Serina Endopeptidasas/química , Serina Endopeptidasas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
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
Furin, also called proprotein convertase subtilisin/kexin 3 (PCSK3), is a calcium-dependent serine endoprotease that processes a wide variety of proproteins involved in cell function and homeostasis. Dysregulation of furin has been implicated in numerous disease states, including cancer and fibrosis. Mammalian cell expression of the furin ectodomain typically produces a highly glycosylated, heterogeneous protein, which can make crystallographic studies difficult. Here, the expression and purification of nonglycosylated human furin using the BacMam technology and site-directed mutagenesis of the glycosylation sites is reported. Nonglycosylated furin produced using this system retains full proteolytic activity indistinguishable from that of the glycosylated protein. Importantly, the nonglycosylated furin protein reliably forms extremely durable apo crystals that diffract to high resolution. These crystals can be soaked with a wide variety of inhibitors to enable a structure-guided drug-discovery campaign.
Asunto(s)
Apoproteínas/química , Bioquímica/métodos , Furina/química , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Cristalografía por Rayos X , Glicosilación , Células HEK293 , Humanos , Dominios Proteicos , Estructura Secundaria de ProteínaRESUMEN
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
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
Therapies that suppress RIPK1 kinase activity are emerging as promising therapeutic agents for the treatment of multiple inflammatory disorders. The ability to directly measure drug binding of a RIPK1 inhibitor to its target is critical for providing insight into pharmacokinetics, pharmacodynamics, safety and clinical efficacy, especially for a first-in-class small-molecule inhibitor where the mechanism has yet to be explored. Here, we report a novel method for measuring drug binding to RIPK1 protein in cells and tissues. This TEAR1 (Target Engagement Assessment for RIPK1) assay is a pair of immunoassays developed on the principle of competition, whereby a first molecule (ie, drug) prevents the binding of a second molecule (ie, antibody) to the target protein. Using the TEAR1 assay, we have validated the direct binding of specific RIPK1 inhibitors in cells, blood and tissues following treatment with benzoxazepinone (BOAz) RIPK1 inhibitors. The TEAR1 assay is a valuable tool for facilitating the clinical development of the lead RIPK1 clinical candidate compound, GSK2982772, as a first-in-class RIPK1 inhibitor for the treatment of inflammatory disease.
Asunto(s)
Anticuerpos/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Animales , Células HT29 , Humanos , Inmunoensayo , Macaca fascicularis , Masculino , Unión Proteica/efectos de los fármacos , Proteína Serina-Treonina Quinasas de Interacción con Receptores/inmunología , Bibliotecas de Moléculas Pequeñas/farmacologíaRESUMEN
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
A novel series of potent and selective hexokinase 2 (HK2) inhibitors, 2,6-disubstituted glucosamines, has been identified based on HTS hits, exemplified by compound 1. Inhibitor-bound crystal structures revealed that the HK2 enzyme could adopt an "induced-fit" conformation. The SAR study led to the identification of potent HK2 inhibitors, such as compound 34 with greater than 100-fold selectivity over HK1. Compound 25 inhibits in situ glycolysis in a UM-UC-3 bladder tumor cell line via (13)CNMR measurement of [3-(13)C]lactate produced from [1,6-(13)C2]glucose added to the cell culture.
RESUMEN
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
Activation of the inositol-requiring enzyme-1 alpha (IRE1α) protein caused by endoplasmic reticulum stress results in the homodimerization of the N-terminal endoplasmic reticulum luminal domains, autophosphorylation of the cytoplasmic kinase domains, and conformational changes to the cytoplasmic endoribonuclease (RNase) domains, which render them functional and can lead to the splicing of X-box binding protein 1 (XBP 1) mRNA. Herein, we report the first crystal structures of the cytoplasmic portion of a human phosphorylated IRE1α dimer in complex with (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide, a novel, IRE1α-selective kinase inhibitor, and staurosporine, a broad spectrum kinase inhibitor. (R)-2-(3,4-dichlorobenzyl)-N-(4-methylbenzyl)-2,7-diazaspiro(4.5)decane-7-carboxamide inhibits both the kinase and RNase activities of IRE1α. The inhibitor interacts with the catalytic residues Lys599 and Glu612 and displaces the kinase activation loop to the DFG-out conformation. Inactivation of IRE1α RNase activity appears to be caused by a conformational change, whereby the αC helix is displaced, resulting in the rearrangement of the kinase domain-dimer interface and a rotation of the RNase domains away from each other. In contrast, staurosporine binds at the ATP-binding site of IRE1α, resulting in a dimer consistent with RNase active yeast Ire1 dimers. Activation of IRE1α RNase activity appears to be promoted by a network of hydrogen bond interactions between highly conserved residues across the RNase dimer interface that place key catalytic residues poised for reaction. These data implicate that the intermolecular interactions between conserved residues in the RNase domain are required for activity, and that the disruption of these interactions can be achieved pharmacologically by small molecule kinase domain inhibitors.
Asunto(s)
Endorribonucleasas/antagonistas & inhibidores , Endorribonucleasas/metabolismo , Inhibidores de Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Línea Celular Tumoral , Cristalización , Endorribonucleasas/química , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Humanos , Conformación Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/química , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Hydrogen deuterium exchange mass spectrometry (HDX-MS) has become an increasingly routine technology for probing the solution structure and dynamics of proteins. HDX-MS measures the exchange of deuterium with the amide proton on the protein backbone chain. HDX-MS results can be used to aid in construct design for biophysical studies, to probe protein-ligand interactions and to characterize the dynamics of proteins. This digest highlights recent progress which makes this technology accessible and explores examples of HDX-MS in drug discovery and solution state structural biology.
Asunto(s)
Medición de Intercambio de Deuterio , Descubrimiento de Drogas/métodos , Espectrometría de MasasRESUMEN
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
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
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
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
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.