RESUMEN
Increasing numbers of target protein structures available for computational studies makes the structure-based screening paradigm more attractive for initial hit indentification. We have developed a novel in silico screening methodology incorporating Molecular Mechanics (MM)/implicit solvent methods to evaluate binding free energies and applied this technology to the identification of inhibitors of the TLR4/MD-2 interaction.
Asunto(s)
Diseño de Fármacos , Ensayos Analíticos de Alto Rendimiento/métodos , Antígeno 96 de los Linfocitos/antagonistas & inhibidores , Antígeno 96 de los Linfocitos/metabolismo , Mapeo de Interacción de Proteínas/métodos , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/metabolismo , Animales , Línea Celular , Humanos , Lipopolisacáridos/inmunología , Antígeno 96 de los Linfocitos/química , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Ratones , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Receptor Toll-Like 4/química , Receptor Toll-Like 4/inmunologíaRESUMEN
Opioid-induced proinflammatory glial activation modulates wide-ranging aspects of opioid pharmacology including: opposition of acute and chronic opioid analgesia, opioid analgesic tolerance, opioid-induced hyperalgesia, development of opioid dependence, opioid reward, and opioid respiratory depression. However, the mechanism(s) contributing to opioid-induced proinflammatory actions remains unresolved. The potential involvement of toll-like receptor 4 (TLR4) was examined using in vitro, in vivo, and in silico techniques. Morphine non-stereoselectively induced TLR4 signaling in vitro, blocked by a classical TLR4 antagonist and non-stereoselectively by naloxone. Pharmacological blockade of TLR4 signaling in vivo potentiated acute intrathecal morphine analgesia, attenuated development of analgesic tolerance, hyperalgesia, and opioid withdrawal behaviors. TLR4 opposition to opioid actions was supported by morphine treatment of TLR4 knockout mice, which revealed a significant threefold leftward shift in the analgesia dose response function, versus wildtype mice. A range of structurally diverse clinically-employed opioid analgesics was found to be capable of activating TLR4 signaling in vitro. Selectivity in the response was identified since morphine-3-glucuronide, a morphine metabolite with no opioid receptor activity, displayed significant TLR4 activity, whilst the opioid receptor active metabolite, morphine-6-glucuronide, was devoid of such properties. In silico docking simulations revealed ligands bound preferentially to the LPS binding pocket of MD-2 rather than TLR4. An in silico to in vitro prediction model was built and tested with substantial accuracy. These data provide evidence that select opioids may non-stereoselectively influence TLR4 signaling and have behavioral consequences resulting, in part, via TLR4 signaling.
Asunto(s)
Analgésicos Opioides/farmacología , Antígeno 96 de los Linfocitos/efectos de los fármacos , Receptor Toll-Like 4/efectos de los fármacos , Analgesia , Animales , Línea Celular , Simulación por Computador , Calor , Hiperalgesia/psicología , Bombas de Infusión , Inyecciones Espinales , Antígeno 96 de los Linfocitos/agonistas , Antígeno 96 de los Linfocitos/antagonistas & inhibidores , Macrófagos/efectos de los fármacos , Masculino , Ratones , Naloxona/farmacología , Antagonistas de Narcóticos/farmacología , Dimensión del Dolor , Ratas , Ratas Sprague-Dawley , Tiempo de Reacción/efectos de los fármacos , Receptores Opioides mu/agonistas , Receptores Opioides mu/antagonistas & inhibidores , Receptores Opioides mu/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Síndrome de Abstinencia a Sustancias/psicología , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 4/antagonistas & inhibidores , TransfecciónRESUMEN
Toll-like receptors are an integral part of innate immunity in the central nervous system (CNS); they orchestrate a robust defense in response to both exogenous and endogenous danger signals. Recently, toll-like receptor 4 (TLR4) has emerged as a therapeutic target for the treatment of CNS-related diseases such as sepsis and chronic pain. We herein report a chemical biology approach by using a rationally designed peptide inhibitor to disrupt the TLR4-MD2 association, thereby blocking TLR4 signaling.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Péptidos/farmacología , Receptor Toll-Like 4/antagonistas & inhibidores , Receptor Toll-Like 4/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Línea Celular , Biología Computacional , Antígeno 96 de los Linfocitos , Ratones , Modelos Moleculares , Péptidos/síntesis química , Unión Proteica/efectos de los fármacos , Conformación Proteica , Receptor Toll-Like 4/químicaRESUMEN
Although activated spinal cord glia contribute importantly to neuropathic pain, how nerve injury activates glia remains controversial. It has recently been proposed, on the basis of genetic approaches, that toll-like receptor 4 (TLR4) may be a key receptor for initiating microglial activation following L5 spinal nerve injury. The present studies extend this idea pharmacologically by showing that TLR4 is key for maintaining neuropathic pain following sciatic nerve chronic constriction injury (CCI). Established neuropathic pain was reversed by intrathecally delivered TLR4 receptor antagonists derived from lipopolysaccharide. Additionally, (+)-naltrexone, (+)-naloxone, and (-)-naloxone, which we show here to be TLR4 antagonists in vitro on both stably transfected HEK293-TLR4 and microglial cell lines, suppressed neuropathic pain with complete reversal upon chronic infusion. Immunohistochemical analyses of spinal cords following chronic infusion revealed suppression of CCI-induced microglial activation by (+)-naloxone and (-)-naloxone, paralleling reversal of neuropathic pain. Together, these CCI data support the conclusion that neuron-to-glia signaling through TLR4 is important not only for initiating neuropathic pain, as suggested previously, but also for maintaining established neuropathic pain. Furthermore, these studies suggest that the novel TLR4 antagonists (+)-naloxone and (-)-naloxone can each fully reverse established neuropathic pain upon multi-day administration. This finding with (+)-naloxone is of potential clinical relevance. This is because (+)-naloxone is an antagonist that is inactive at the (-)-opioid selective receptors on neurons that produce analgesia. Thus, these data suggest that (+)-opioid antagonists such as (+)-naloxone may be useful clinically to suppress glial activation, yet (-)-opioid agonists suppress pain.