RESUMO
Naringenin (2S)-5,7-dihydroxy-2-(4-hydroxyphenyl)-3,4-dihydro-2H-1-benzopyran-4-one is a natural flavonoid found in fruits from the citrus family. Because (2S)-naringenin is known to racemize, its bioactivity might be related to one or both enantiomers. Computational studies predicted that (2R)-naringenin may act on voltage-gated ion channels, particularly the N-type calcium channel (CaV2.2) and the NaV1.7 sodium channel-both of which are key for pain signaling. Here we set out to identify the possible mechanism of action of naringenin. Naringenin inhibited depolarization-evoked Ca2+ influx in acetylcholine-, ATP-, and capsaicin-responding rat dorsal root ganglion (DRG) neurons. This was corroborated in electrophysiological recordings from DRG neurons. Pharmacological dissection of each of the voltage-gated Ca2+ channels subtypes could not pinpoint any selectivity of naringenin. Instead, naringenin inhibited NaV1.8-dependent and tetrodotoxin (TTX)-resistant while sparing tetrodotoxin sensitive (TTX-S) voltage-gated Na+ channels as evidenced by the lack of further inhibition by the NaV1.8 blocker A-803467. The effects of the natural flavonoid were validated ex vivo in spinal cord slices where naringenin decreased both the frequency and amplitude of sEPSC recorded in neurons within the substantia gelatinosa. The antinociceptive potential of naringenin was evaluated in male and female mice. Naringenin had no effect on the nociceptive thresholds evoked by heat. Naringenin's reversed allodynia was in mouse models of postsurgical and neuropathic pain. Here, driven by a call by the National Center for Complementary and Integrative Health's strategic plan to advance fundamental research into basic biological mechanisms of the action of natural products, we advance the antinociceptive potential of the flavonoid naringenin.
Assuntos
Analgésicos/farmacologia , Flavanonas/farmacologia , Gânglios Espinais/citologia , Canal de Sódio Disparado por Voltagem NAV1.8/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Células Receptoras Sensoriais/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Sódio/metabolismo , Analgésicos/química , Analgésicos/uso terapêutico , Animais , Canais de Cálcio/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Feminino , Flavanonas/química , Flavanonas/metabolismo , Flavanonas/uso terapêutico , Hiperalgesia/tratamento farmacológico , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Masculino , Camundongos , Modelos Moleculares , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/tratamento farmacológico , Dor Pós-Operatória/tratamento farmacológico , Conformação Proteica , Mapeamento de Interação de Proteínas , Ratos , Ratos Sprague-Dawley , Células Receptoras Sensoriais/classificação , Células Receptoras Sensoriais/metabolismo , Bloqueadores dos Canais de Sódio/química , Bloqueadores dos Canais de Sódio/uso terapêutico , Organismos Livres de Patógenos Específicos , Relação Estrutura-AtividadeRESUMO
Inhibition of voltage-gated calcium (CaV) channels is a potential therapy for many neurological diseases including chronic pain. Neuronal CaV1/CaV2 channels are composed of α, ß, γ and α2δ subunits. The ß subunits of CaV channels are cytoplasmic proteins that increase the surface expression of the pore-forming α subunit of CaV. We targeted the high-affinity protein-protein interface of CaVß's pocket within the CaVα subunit. Structure-based virtual screening of 50,000 small molecule library docked to the ß subunit led to the identification of 2-(3,5-dimethylisoxazol-4-yl)-N-((4-((3-phenylpropyl)amino)quinazolin-2-yl)methyl)acetamide (IPPQ). This small molecule bound to CaVß and inhibited its coupling with N-type voltage-gated calcium (CaV2.2) channels, leading to a reduction in CaV2.2 currents in rat dorsal root ganglion sensory neurons, decreased presynaptic localization of CaV2.2 in vivo, decreased frequency of spontaneous excitatory postsynaptic potentials and miniature excitatory postsynaptic potentials, and inhibited release of the nociceptive neurotransmitter calcitonin gene-related peptide from spinal cord. IPPQ did not target opioid receptors nor did it engage inhibitory G protein-coupled receptor signaling. IPPQ was antinociceptive in naive animals and reversed allodynia and hyperalgesia in models of acute (postsurgical) and neuropathic (spinal nerve ligation, chemotherapy- and gp120-induced peripheral neuropathy, and genome-edited neuropathy) pain. IPPQ did not cause akinesia or motor impairment, a common adverse effect of CaV2.2 targeting drugs, when injected into the brain. IPPQ, a quinazoline analog, represents a novel class of CaV2.2-targeting compounds that may serve as probes to interrogate CaVα-CaVß function and ultimately be developed as a nonopioid therapeutic for chronic pain.