RESUMEN
Botulism is caused by potent and specific bacterial neurotoxins that infect host neurons and block neurotransmitter release. Treatment for botulism is limited to administration of an antitoxin within a short time window, before the toxin enters neurons. Alternatively, current botulism drug development targets the toxin light chain, which is a zinc-dependent metalloprotease that is delivered into neurons and mediates long-term pathology. Several groups have identified inhibitory small molecules, peptides, or aptamers, although no molecule has advanced to the clinic due to a lack of efficacy in advanced models. Here we used a homogeneous high-throughput enzyme assay to screen three libraries of drug-like small molecules for new chemotypes that modulate recombinant botulinum neurotoxin light chain activity. High-throughput screening of 97088 compounds identified numerous small molecules that activate or inhibit metalloprotease activity. We describe four major classes of inhibitory compounds identified, detail their structure-activity relationships, and assess their relative inhibitory potency. A previously unreported chemotype in any context of enzyme inhibition is described with potent submicromolar inhibition (Ki = 200-300 nM). Additional detailed kinetic analyses and cellular cytotoxicity assays indicate the best compound from this series is a competitive inhibitor with cytotoxicity values around 4-5 µM. Given the potency and drug-like character of these lead compounds, further studies, including cellular activity assays and DMPK analysis, are justified.
Asunto(s)
Toxinas Botulínicas/antagonistas & inhibidores , Inhibidores de Proteasas/química , Bibliotecas de Moléculas Pequeñas/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos Analíticos de Alto Rendimiento , Humanos , Cinética , Ratones , Inhibidores de Proteasas/farmacología , Inhibidores de Proteasas/toxicidad , Pirazoles/química , Piridinas/química , Quinolinas/química , Relación Estructura-Actividad , Tiadiazoles/químicaRESUMEN
Peripheral processes that mediate beneficial effects of exercise on the brain remain sparsely explored. Here, we show that a muscle secretory factor, cathepsin B (CTSB) protein, is important for the cognitive and neurogenic benefits of running. Proteomic analysis revealed elevated levels of CTSB in conditioned medium derived from skeletal muscle cell cultures treated with AMP-kinase agonist AICAR. Consistently, running increased CTSB levels in mouse gastrocnemius muscle and plasma. Furthermore, recombinant CTSB application enhanced expression of brain-derived neurotrophic factor (BDNF) and doublecortin (DCX) in adult hippocampal progenitor cells through a mechanism dependent on the multifunctional protein P11. In vivo, in CTSB knockout (KO) mice, running did not enhance adult hippocampal neurogenesis and spatial memory function. Interestingly, in Rhesus monkeys and humans, treadmill exercise elevated CTSB in plasma. In humans, changes in CTSB levels correlated with fitness and hippocampus-dependent memory function. Our findings suggest CTSB as a mediator of effects of exercise on cognition.
Asunto(s)
Catepsina B/metabolismo , Memoria/fisiología , Condicionamiento Físico Animal , Carrera/fisiología , Adulto , Afecto , Envejecimiento/fisiología , Animales , Conducta Animal , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Catepsina B/sangre , Catepsina B/genética , Cognición , Proteínas de Dominio Doblecortina , Proteína Doblecortina , Prueba de Esfuerzo , Femenino , Hipocampo/fisiología , Humanos , Macaca mulatta , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Actividad Motora , Neurogénesis , Neuronas/metabolismo , Neuropéptidos/metabolismo , Reproducibilidad de los Resultados , Conducta Sedentaria , Adulto JovenRESUMEN
Botulinum neurotoxins (BoNT) are the most potent toxins known and a significant bioterrorist threat. Few small molecule compounds have been identified that are active in cell-based or animal models, potentially due to toxin enzyme plasticity. Here we screened commercially available quinolinols, as well as synthesized hydroxyquinolines. Seventy-two compounds had IC50 values below 10 µM, with the best compound exhibiting submicromolar inhibition (IC50 = 0.8 µM). Structure-activity relationship trends showed that the enzyme tolerates various substitutions at R1 but has a clear preference for bulky aryl amide groups at R2, while methylation at R3 increased inhibitor potency. Evaluation of the most potent compounds in an ADME panel showed that these compounds possess poor solubility at pH 6.8, but display excellent solubility at low pH, suggesting that oral dosing may be possible. Our data show the potential of quinolinol compounds as BoNT therapeutics due to their good in vitro potencies and favorable ADME properties.