RESUMEN
BACKGROUND: Inflammation is an essential component of arthritis pain. Nerve growth factor (NGF) plays a key role in acute and chronic pain states especially those associated with inflammation. NGF acts through tropomyosin-receptor-kinase A (TrkA). NGF blockade has reduced arthritis pain in clinical trials. We explored the mechanisms within the joint which may contribute to the analgesic effects of NGF by selectively inhibiting TrkA in carrageenan-induced or collagen-induced joint pain behaviour. The goal of the current study was to elucidate whether inflammation is central to the efficacy for NGF blockade. METHODS: Rats were injected in their left knees with 2 % carrageenan or saline. Collagen-induced arthritis (CIA) was induced by intradermal injections of a mixture of bovine type II collagen (0.2 mg) and incomplete Freund's adjuvant (0.2 mg). Oral doses (30 mg/kg) of AR786 or vehicle control were given twice daily after arthritis induction. Ibuprofen-treated (35 mg/kg, orally, once daily) rats with CIA were used as positive analgesic controls. Pain behaviour was measured as hind-limb weight-bearing asymmetry and hind-paw withdrawal thresholds to von Frey hair stimulation (carrageenan synovitis), or withdrawal to joint compression using a Randall Selitto device (CIA). Inflammation was measured as increased knee joint diameter and by histopathological analysis. RESULTS: Intra-articular injections of carrageenan or induction of CIA was each associated with pain behaviour and synovial inflammation. Systemic administration of the TrkA inhibitor AR786 reduced carrageenan-induced or CIA-induced pain behaviour to control values, and inhibited joint swelling and histological evidence of synovial inflammation and joint damage. CONCLUSIONS: By using two models of varying inflammation we demonstrate for the first time that selective inhibition of TrkA may reduce carrageenan-induced or CIA-induced pain behaviour in rats, in part through potentially inhibiting synovial inflammation, although direct effects on sensory nerves are also likely. Our observations suggest that inflammatory arthritis causes pain and the presence of inflammation is fundamental to the beneficial effects (reduction in pain and pathology) of NGF blockade. Further research should determine whether TrkA inhibition may ameliorate human inflammatory arthritis.
Asunto(s)
Artritis Experimental/patología , Artritis Reumatoide/patología , Inhibidores Enzimáticos/farmacología , Receptor trkA/antagonistas & inhibidores , Animales , Masculino , Dolor/etiología , Ratas , Ratas Sprague-DawleyRESUMEN
HCV serine protease NS3 represents an attractive drug target because it is not only essential for viral replication but also implicated in the viral evasion of the host immune response pathway through direct cleavage of key proteins in the human innate immune system. Through structure-based drug design and optimization, macrocyclic peptidomimetic molecules bearing both a lipophilic P2 isoindoline carbamate and a P1/P1' acylsulfonamide/acylsulfamide carboxylic acid bioisostere were prepared that possessed subnanomolar potency against the NS3 protease in a subgenomic replicon-based cellular assay (Huh-7). Danoprevir (compound 49) was selected as the clinical development candidate for its favorable potency profile across multiple HCV genotypes and key mutant strains and for its good in vitro ADME profiles and in vivo target tissue (liver) exposures across multiple animal species. X-ray crystallographic studies elucidated several key features in the binding of danoprevir to HCV NS3 protease and proved invaluable to our iterative structure-based design strategy.
Asunto(s)
Antivirales/uso terapéutico , Descubrimiento de Drogas , Lactamas/uso terapéutico , Inhibidores de Proteasas/uso terapéutico , Sulfonamidas/uso terapéutico , Proteínas no Estructurales Virales/antagonistas & inhibidores , Animales , Antivirales/química , Antivirales/farmacología , Cristalografía por Rayos X , Ciclopropanos , Perros , Isoindoles , Lactamas/química , Lactamas/farmacología , Lactamas Macrocíclicas , Macaca fascicularis , Estructura Molecular , Prolina/análogos & derivados , Inhibidores de Proteasas/farmacología , Ratas , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
Aggrecanases, particularly aggrecanase-1 (ADAMTS-4) and aggrecanase-2 (ADAMTS-5), are believed to be key enzymes involved in the articular cartilage breakdown that leads to osteoarthritis. Thus, aggrecanases are considered to be viable drug targets for the treatment of this debilitating disease. A series of (1S,2R,3R)-2,3-dimethyl-2-phenyl-1-sulfamidocyclopropanecarboxylates was discovered to be potent, highly selective, and orally bioavailable aggrecanase inhibitors. These compounds have unique P1' groups comprising novel piperidine- or piperazine-based heterocycles that are connected to a cyclopropane amino acid scaffold via a sulfamido linkage. These P1' groups are quite effective in imparting selectivity over other MMPs, and this selectivity was further increased by incorporation of a methyl substituent in the 2-position of the cyclopropane ring. In contrast to classical hydroxamate-based inhibitors that tend to lack metabolic stability, our aggrecanase inhibitors bear a carboxylate zinc-binding group and have good oral bioavailability. Lead compound 13b, characterized by the novel P1' portion of 1,2,3,4-tetrahydropyrido[3',4':4,5]imidazo[1,2-a]pyridine ring, is a potent and selective aggrecanse inhibitor with excellent pharmacokinetic profiles.
Asunto(s)
Ciclopropanos/química , Ciclopropanos/farmacología , Descubrimiento de Drogas , Endopeptidasas/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Animales , Cristalografía por Rayos X , Ciclopropanos/farmacocinética , Inhibidores Enzimáticos/farmacocinética , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética , Ratones , Ratones Noqueados , Modelos Moleculares , Relación Estructura-ActividadRESUMEN
Current therapies for treating skeletal pain have significant limitations as available drugs (non-steroidal anti-inflammatory drugs and opiates) have significant unwanted side effects. Targeting nerve growth factor (NGF) or its cognate receptor tropomysin receptor kinase A (TrkA) has recently become an attractive target for inhibition of adult skeletal pain. Here we explore whether sustained administration of a selective small molecule Trk inhibitor that blocks TrkA, TrkB and TrkC kinase activity with nanomolar affinity reduces skeletal pain while allowing the maintenance of sensory and sympathetic neurons in the adult mouse. Twice-daily administration of a Trk inhibitor was begun 1 day post fracture and within 8 h of acute administration fracture pain-related behaviors were reduced by 50% without significant sedation, weight gain or inhibition of fracture healing. Following administration of the Trk inhibitor for 7 weeks, there was no significant decline in the density of unmyelinated or myelinated sensory nerve fibers, sympathetic nerve fibers, measures of acute thermal pain, acute mechanical pain, or general neuromuscular function. The present results suggest that sustained administration of a peripherally selective TrkA, B and C inhibitor significantly reduces skeletal pain without having any obvious detrimental effects on adult sensory and sympathetic nerve fibers or early fracture healing. As with any potential therapeutic advance, understanding whether the benefits of Trk blockade are associated with any risks or unexpected effects will be required to fully appreciate the patient populations that may benefit from this therapeutic approach.
Asunto(s)
Inhibidores Enzimáticos/uso terapéutico , Dolor/tratamiento farmacológico , Receptor trkA/antagonistas & inhibidores , Receptor trkB/antagonistas & inhibidores , Receptor trkC/antagonistas & inhibidores , Células Receptoras Sensoriales/efectos de los fármacos , Sistema Nervioso Simpático/efectos de los fármacos , Animales , Fracturas Óseas/fisiopatología , Masculino , RatonesRESUMEN
A series of 1-sulfonylaminocyclopropanecarboxylates was synthesized as ADAMTS-5 (Aggrecanase-2) inhibitors. After an intensive investigation of the central cyclopropane core including its absolute stereochemistry and substituents, we found compound 22 with an Agg-2 IC50=7.4 nM, the most potent ADAMTS-5 inhibitor reported so far.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Ciclopropanos/síntesis química , Inhibidores de Proteasas/síntesis química , Sulfonamidas/síntesis química , Proteínas ADAM/metabolismo , Proteína ADAMTS5 , Animales , Sitios de Unión , Simulación por Computador , Ciclopropanos/química , Ciclopropanos/farmacocinética , Ciclopropanos/farmacología , Diseño de Fármacos , Humanos , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacocinética , Unión Proteica , Ratas , Relación Estructura-Actividad , Sulfonamidas/química , Sulfonamidas/farmacologíaRESUMEN
The protease activity of hepatitis C virus nonstructural protein 3 (NS3) is essential for viral replication. ITMN-191, a macrocyclic inhibitor of the NS3 protease active site, promotes rapid, multilog viral load reductions in chronic HCV patients. Here, ITMN-191 is shown to be a potent inhibitor of NS3 with a two-step binding mechanism. Progress curves are consistent with the formation of an initial collision complex (EI) that isomerizes to a highly stable complex (EI*) from which ITMN-191 dissociates very slowly. K(i), the dissociation constant of EI, is 100 nM, and the rate constant for conversion of EI to EI* is 6.2 x 10(-2) s(-1). Binding experiments using protein fluorescence confirm this isomerization rate. From progress curve analysis, the rate constant for dissociation of ITMN-191 from the EI* complex is 3.8 x 10(-5) s(-1) with a calculated complex half-life of approximately 5 h and a true biochemical potency (K(i)*) of approximately 62 pM. Surface plasmon resonance studies and assessment of enzyme reactivation following dilution of the EI* complex confirm slow dissociation and suggest that the half-life may be considerably longer. Abrogation of the tight binding and slow dissociative properties of ITMN-191 is observed with proteases that carry the R155K or D168A substitution, each of which is likely in drug resistant mutants. Slow dissociation is not observed with closely related macrocyclic inhibitors of NS3, suggesting that members of this class may display distinct binding kinetics.
Asunto(s)
Hepacivirus/enzimología , Inhibidores de Proteasas/química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Sustitución de Aminoácidos , Hepacivirus/química , Hepacivirus/genética , Cinética , Inhibidores de Proteasas/síntesis química , Unión Proteica , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
A series of N-substituted sulfonylamino-alkanecarboxylate ADAMTS-5 (Aggrecanase-2) inhibitors has been synthesized and the in vitro enzyme SAR is discussed. This report is the first example of carboxylate-based ADAMTS-5 inhibitors which show strong potency of IC(50)<0.1muM with excellent selectivity over MMP-1 and TACE.
Asunto(s)
Proteínas ADAM/antagonistas & inhibidores , Inhibidores de Proteasas/síntesis química , Proteína ADAMTS5 , Ácidos Carboxílicos/química , Diseño de Fármacos , Humanos , Concentración 50 Inhibidora , Modelos Químicos , Conformación Molecular , Estructura Molecular , Osteoartritis/tratamiento farmacológico , Inhibidores de Proteasas/farmacología , Estructura Terciaria de Proteína , Relación Estructura-ActividadRESUMEN
Future treatments for chronic hepatitis C virus (HCV) infection are likely to include agents that target viral components directly. Here, the preclinical characteristics of ITMN-191, a peptidomimetic inhibitor of the NS3/4A protease of HCV, are described. ITMN-191 inhibited a reference genotype 1 NS3/4A protein in a time-dependent fashion, a hallmark of an inhibitor with a two-step binding mechanism and a low dissociation rate. Under preequilibrium conditions, 290 pM ITMN-191 half-maximally inhibited the reference NS3/4A protease, but a 35,000-fold-higher concentration did not appreciably inhibit a panel of 79 proteases, ion channels, transporters, and cell surface receptors. Subnanomolar biochemical potency was maintained against NS3/4A derived from HCV genotypes 4, 5, and 6, while single-digit nanomolar potency was observed against NS3/4A from genotypes 2b and 3a. Dilution of a preformed enzyme inhibitor complex indicated ITMN-191 remained bound to and inhibited NS3/4A for more than 5 h after its initial association. In cell-based potency assays, half-maximal reduction of genotype 1b HCV replicon RNA was afforded by 1.8 nM; 45 nM eliminated the HCV replicon from cells. Peginterferon alfa-2a displayed a significant degree of antiviral synergy with ITMN-191 and reduced the concentration of ITMN-191 required for HCV replicon elimination. A 30-mg/kg of body weight oral dose administered to rats or monkeys yielded liver concentrations 12 h after dosing that exceeded the ITMN-191 concentration required to eliminate replicon RNA from cells. These preclinical characteristics compare favorably to those of other inhibitors of NS3/4A in clinical development and therefore support the clinical investigation of ITMN-191 for the treatment of chronic hepatitis C.