RESUMEN
We devised a high-throughput chemoproteomics method that enabled multiplexed screening of 16,000 compounds against native protein and lipid kinases in cell extracts. Optimization of one chemical series resulted in CZC24832, which is to our knowledge the first selective inhibitor of phosphoinositide 3-kinase γ (PI3Kγ) with efficacy in in vitro and in vivo models of inflammation. Extensive target- and cell-based profiling of CZC24832 revealed regulation of interleukin-17-producing T helper cell (T(H)17) differentiation by PI3Kγ, thus reinforcing selective inhibition of PI3Kγ as a potential treatment for inflammatory and autoimmune diseases.
Asunto(s)
Antiinflamatorios no Esteroideos/farmacología , Diferenciación Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Interleucina-17/inmunología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Bibliotecas de Moléculas Pequeñas/farmacología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Animales , Antiinflamatorios no Esteroideos/química , Antiinflamatorios no Esteroideos/farmacocinética , Antiinflamatorios no Esteroideos/uso terapéutico , Artritis Experimental/tratamiento farmacológico , Artritis Experimental/inmunología , Artritis Experimental/patología , Unión Competitiva , Línea Celular , Movimiento Celular/efectos de los fármacos , Fosfatidilinositol 3-Quinasa Clase Ib , Descubrimiento de Drogas , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacocinética , Inhibidores Enzimáticos/uso terapéutico , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Estructura Molecular , Ratas , Ratas Wistar , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacocinética , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Relación Estructura-Actividad , Linfocitos T Colaboradores-Inductores/citología , Linfocitos T Colaboradores-Inductores/enzimología , Linfocitos T Colaboradores-Inductores/inmunologíaRESUMEN
Herein, we disclose the discovery of a series of 7-substituted triazolopyridines which culminated in the identification of 14 (CZC24758), a potent, orally bioavailable small-molecule inhibitor of PI3Kγ, an attractive drug target for inflammatory and autoimmune disorders. Compound 14 has excellent selectivity across the kinome, demonstrates good potency in cell based assays and furthermore exhibits in vivo efficacy in a collagen induced arthritis model in mouse after oral dosing.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Animales , Descubrimiento de Drogas , Granulocitos/efectos de los fármacos , Enlace de Hidrógeno , Inflamación/tratamiento farmacológico , Masculino , Ratones , Microsomas/efectos de los fármacos , Microsomas/enzimología , Modelos Moleculares , Estructura Molecular , Piridinas/química , Piridinas/uso terapéutico , Ratas , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/uso terapéutico , Triazoles/química , Triazoles/uso terapéuticoRESUMEN
Dual PI3Kγ/δ inhibitors have recently been shown to be suitable targets for inflammatory and respiratory diseases. In a recent study we described the discovery of selective PI3Kγ inhibitors based on a triazolopyridine scaffold. Herein, we describe the elaboration of this structural class into dual PI3Kγ/δ inhibitors with excellent selectivity over the other PI3K isoforms and the general kinome. Structural optimization led to the identification of two derivatives which showed significant efficacy in an acute model of lung inflammation.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/química , Descubrimiento de Drogas , Inflamación/tratamiento farmacológico , Modelos Moleculares , Estructura Molecular , Inhibidores de Proteínas Quinasas/farmacología , Relación Estructura-ActividadRESUMEN
Herein we describe the SAR of a novel series of 6-aryl-2-amino-triazolopyridines as potent and selective PI3Kγ inhibitors. The 6-aryl-triazolopyridine core was identified by chemoproteomic screening of a kinase focused library. Rapid chemical expansion around a bi-functional core identified the key features required for PI3Kγ activity and selectivity. The series was optimized to afford 43 (CZC19945), a potent PI3Kγ inhibitor with high oral bioavailability and selectivity over PI3Kα and PI3Kδ. Modification to the core afforded 53 (CZC24832) which showed increased selectivity over the entire kinome in particular over PI3Kß.
Asunto(s)
Inhibidores de las Quinasa Fosfoinosítidos-3 , Piridinas/química , Administración Oral , Animales , Artritis/tratamiento farmacológico , Sitios de Unión , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase Ib/metabolismo , Simulación por Computador , Modelos Animales de Enfermedad , Semivida , Humanos , Ratones , Microsomas/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/metabolismo , Estructura Terciaria de Proteína , Piridinas/farmacocinética , Piridinas/uso terapéutico , Ratas , Relación Estructura-ActividadRESUMEN
The identification and hit-to-lead exploration of a novel, potent and selective series of histamine H(4) receptor inverse agonists is described. The initial hit, 3A (IC(50) 19 nM) was identified by means of a ligand-based virtual screening approach. Subsequent medicinal chemistry exploration yielded 18I which possessed increased potency (R-enantiomer IC(50) 1 nM) as well as enhanced microsomal stability.
Asunto(s)
Antagonistas de los Receptores Histamínicos/farmacología , Receptores Acoplados a Proteínas G/antagonistas & inhibidores , Animales , Disponibilidad Biológica , Descubrimiento de Drogas , Antagonistas de los Receptores Histamínicos/química , Antagonistas de los Receptores Histamínicos/farmacocinética , Concentración 50 Inhibidora , Macaca fascicularis , Ratas , Receptores Histamínicos , Receptores Histamínicos H4 , EstereoisomerismoRESUMEN
We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.
Asunto(s)
Inhibidores de Proteínas Quinasas/farmacología , Proteómica/métodos , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Benzamidas , Extractos Celulares , Cromatografía de Afinidad , Receptor con Dominio Discoidina 1 , Enzimas Inmovilizadas/antagonistas & inhibidores , Células HeLa , Humanos , Mesilato de Imatinib , Concentración 50 Inhibidora , Células K562 , Preparaciones Farmacéuticas , Fosforilación/efectos de los fármacos , Piperazinas/farmacología , Pirimidinas/farmacología , Quinona Reductasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Transducción de Señal/efectos de los fármacosRESUMEN
The 2-oxoglutarate-dependent dioxygenase target class comprises around 60 enzymes including several subfamilies with relevance to human disease, such as the prolyl hydroxylases and the Jumonji-type lysine demethylases. Current drug discovery approaches are largely based on small molecule inhibitors targeting the iron/2-oxoglutarate cofactor binding site. We have devised a chemoproteomics approach based on a combination of unselective active-site ligands tethered to beads, enabling affinity capturing of around 40 different dioxygenase enzymes from human cells. Mass-spectrometry-based quantification of bead-bound enzymes using a free-ligand competition-binding format enabled the comprehensive determination of affinities for the cosubstrate 2-oxoglutarate and for oncometabolites such as 2-hydroxyglutarate. We also profiled a set of representative drug-like inhibitor compounds. The results indicate that intracellular competition by endogenous cofactors and high active site similarity present substantial challenges for drug discovery for this target class.
Asunto(s)
Dioxigenasas/metabolismo , Ácidos Cetoglutáricos/metabolismo , ProteómicaRESUMEN
Leucine-rich repeat kinase-2 (LRRK2) mutations are the most important cause of familial Parkinson's disease, and non-selective inhibitors are protective in rodent disease models. Because of their poor potency and selectivity, the neuroprotective mechanism of these tool compounds has remained elusive so far, and it is still unknown whether selective LRRK2 inhibition can attenuate mutant LRRK2-dependent toxicity in human neurons. Here, we employ a chemoproteomics strategy to identify potent, selective, and metabolically stable LRRK2 inhibitors. We demonstrate that CZC-25146 prevents mutant LRRK2-induced injury of cultured rodent and human neurons with mid-nanomolar potency. These precise chemical probes further validate this emerging therapeutic strategy. They will enable more detailed studies of LRRK2-dependent signaling and pathogenesis and accelerate drug discovery.
Asunto(s)
Diseño de Fármacos , Neuronas/efectos de los fármacos , Enfermedad de Parkinson/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteómica/métodos , Animales , Células Cultivadas , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Mutación , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/patología , Inhibidores de Proteínas Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , RatasRESUMEN
The development of selective histone deacetylase (HDAC) inhibitors with anti-cancer and anti-inflammatory properties remains challenging in large part owing to the difficulty of probing the interaction of small molecules with megadalton protein complexes. A combination of affinity capture and quantitative mass spectrometry revealed the selectivity with which 16 HDAC inhibitors target multiple HDAC complexes scaffolded by ELM-SANT domain subunits, including a novel mitotic deacetylase complex (MiDAC). Inhibitors clustered according to their target profiles with stronger binding of aminobenzamides to the HDAC NCoR complex than to the HDAC Sin3 complex. We identified several non-HDAC targets for hydroxamate inhibitors. HDAC inhibitors with distinct profiles have correspondingly different effects on downstream targets. We also identified the anti-inflammatory drug bufexamac as a class IIb (HDAC6, HDAC10) HDAC inhibitor. Our approach enables the discovery of novel targets and inhibitors and suggests that the selectivity of HDAC inhibitors should be evaluated in the context of HDAC complexes and not purified catalytic subunits.