RESUMEN
BACKGROUND AND PURPOSE: Asthma is a heterogenous disease strongly associated with inflammation that has many different causes and triggers. Current asthma treatments target symptoms such as bronchoconstriction and airway inflammation. Despite recent advances in biological therapies, there remains a need for new classes of therapeutic agents with novel, upstream targets. The proteinase-activated receptor-2 (PAR2) has long been implicated in allergic airway inflammation and asthma and it remains an intriguing target for novel therapies. Here, we describe the actions of C781, a newly developed low MW PAR2 biased antagonist, in vitro and in vivo in the context of acute allergen exposure. EXPERIMENTAL APPROACH: A human bronchial epithelial cell line expressing PAR2 (16HBE14o- cells) was used to evaluate the modulation in vitro, by C781, of physiological responses to PAR2 activation and downstream ß-arrestin/MAPK and Gq/Ca2+ signalling. Acute Alternaria alternata sensitized and challenged mice were used to evaluate C781 as a prophylactically administered modulator of airway hyperresponsiveness, inflammation and mucus overproduction in vivo. KEY RESULTS: C781 reduced in vitro physiological signalling in response to ligand and proteinase activation. C781 effectively antagonized ß-arrestin/MAPK signalling without significant effect on Gq/Ca2+ signalling in vitro. Given prophylactically, C781 modulated airway hyperresponsiveness, airway inflammation and mucus overproduction of the small airways in an acute allergen-challenged mouse model. CONCLUSION AND IMPLICATIONS: Our work demonstrates the first biased PAR2 antagonist for ß-arrestin/MAPK signalling. C781 is efficacious as a prophylactic treatment for allergen-induced airway hyperresponsiveness and inflammation in mice. It exemplifies a key pharmacophore for PAR2 that can be optimized for clinical development.
Asunto(s)
Asma , Hiperreactividad Bronquial , Hipersensibilidad Respiratoria , Ratones , Humanos , Animales , Alérgenos , Receptor PAR-2 , beta-Arrestinas , Asma/tratamiento farmacológico , Hipersensibilidad Respiratoria/tratamiento farmacológico , beta-Arrestina 1 , Inflamación/tratamiento farmacológico , Ratones Endogámicos BALB C , Pulmón , Hiperreactividad Bronquial/tratamiento farmacológicoRESUMEN
Biochemical and X-ray crystallographic studies confirmed that hydroxyquinoline derivatives identified by virtual screening were actually covalent inhibitors of the MIF tautomerase. Adducts were formed by N-alkylation of the Pro-1 at the catalytic site with a loss of an amino group of the inhibitor.
Asunto(s)
Descubrimiento de Drogas , Evaluación Preclínica de Medicamentos/métodos , Inhibidores Enzimáticos/farmacología , Hidroxiquinolinas/farmacología , Oxidorreductasas Intramoleculares/antagonistas & inhibidores , Factores Inhibidores de la Migración de Macrófagos/antagonistas & inhibidores , Cristalografía por Rayos X , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Hidroxiquinolinas/síntesis química , Hidroxiquinolinas/química , Modelos Moleculares , Estructura Molecular , Relación Estructura-ActividadRESUMEN
BACKGROUND: Large-scale expansion of myogenic progenitors is necessary to support the development of high-throughput cellular assays in vitro and to advance genetic engineering approaches necessary to develop cellular therapies for rare muscle diseases. However, optimization has not been performed in order to maintain the differentiation capacity of myogenic cells undergoing long-term cell culture. Multiple extracellular matrices have been utilized for myogenic cell studies, but it remains unclear how different matrices influence long-term myogenic activity in culture. To address this challenge, we have evaluated multiple extracellular matrices in myogenic studies over long-term expansion. METHODS: We evaluated the consequence of propagating mouse and human myogenic stem cell progenitors on various extracellular matrices to determine if they could enhance long-term myogenic potential. For the first time reported, we comprehensively examine the effect of physiologically relevant laminins, laminin 211 and laminin 521, compared to traditionally utilized ECMs (e.g., laminin 111, gelatin, and Matrigel) to assess their capacity to preserve myogenic differentiation potential. RESULTS: Laminin 521 supported increased proliferation in early phases of expansion and was the only substrate facilitating high-level fusion following eight passages in mouse myoblast cell cultures. In human myoblast cell cultures, laminin 521 supported increased proliferation during expansion and superior differentiation with myotube hypertrophy. Counterintuitively however, laminin 211, the native laminin isoform in resting skeletal muscle, resulted in low proliferation and poor differentiation in mouse and human cultures. Matrigel performed excellent in short-term mouse studies but showed high amounts of variability following long-term expansion. CONCLUSIONS: These results demonstrate laminin 521 is a superior substrate for both short-term and long-term myogenic cell culture applications compared to other commonly utilized substrates. Since Matrigel cannot be used for clinical applications, we propose that laminin 521 could possibly be employed in the future to provide myoblasts for cellular therapy directed clinical studies.
Asunto(s)
Diferenciación Celular , Laminina/farmacología , Mioblastos/citología , Células Satélite del Músculo Esquelético/citología , Animales , Proliferación Celular , Células Cultivadas , Humanos , Masculino , Ratones , Ratones Endogámicos DBA , Mioblastos/efectos de los fármacos , Células Satélite del Músculo Esquelético/efectos de los fármacosRESUMEN
Multiple Sclerosis is a demyelinating disease of the CNS and the primary cause of neurological disability in young adults. Loss of myelinating oligodendrocytes leads to neuronal dysfunction and death and is an important contributing factor to this disease. Endogenous oligodendrocyte precursor cells (OPCs), which on differentiation are responsible for replacing myelin, are present in the adult CNS. As such, therapeutic agents that can stimulate OPCs to differentiate and remyelinate demyelinated axons under pathologic conditions may improve neuronal function and clinical outcome. We describe the details of an automated, cell-based, morphometric-based, high-content screen that is used to identify small molecules eliciting the differentiation of OPCs after 3 days. Primary screening was performed using rat CG-4 cells maintained in culture conditions that normally support a progenitor cell-like state. From a library of 73,000 diverse small molecules within the Sanofi collection, 342 compounds were identified that increased OPC morphological complexity as an indicator of oligodendrocyte maturation. Subsequent to the primary high-content screen, a suite of cellular assays was established that identified 22 nontoxic compounds that selectively stimulated primary rat OPCs but not C2C12 muscle cell differentiation. This rigorous triaging yielded several chemical series for further expansion and bio- or cheminformatics studies, and their compelling biological activity merits further investigation.