RESUMEN
Voltage-gated sodium (Nav) channels are targets of disease mutations, toxins, and therapeutic drugs. Despite recent advances, the structural basis of voltage sensing, electromechanical coupling, and toxin modulation remains ill-defined. Protoxin-II (ProTx2) from the Peruvian green velvet tarantula is an inhibitor cystine-knot peptide and selective antagonist of the human Nav1.7 channel. Here, we visualize ProTx2 in complex with voltage-sensor domain II (VSD2) from Nav1.7 using X-ray crystallography and cryoelectron microscopy. Membrane partitioning orients ProTx2 for unfettered access to VSD2, where ProTx2 interrogates distinct features of the Nav1.7 receptor site. ProTx2 positions two basic residues into the extracellular vestibule to antagonize S4 gating-charge movement through an electrostatic mechanism. ProTx2 has trapped activated and deactivated states of VSD2, revealing a remarkable â¼10 Å translation of the S4 helix, providing a structural framework for activation gating in voltage-gated ion channels. Finally, our results deliver key templates to design selective Nav channel antagonists.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Canal de Sodio Activado por Voltaje NAV1.7/ultraestructura , Péptidos/metabolismo , Venenos de Araña/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Células CHO , Cricetulus , Microscopía por Crioelectrón/métodos , Cristalografía por Rayos X/métodos , Células HEK293 , Humanos , Activación del Canal Iónico , Péptidos/toxicidad , Dominios Proteicos , Venenos de Araña/toxicidad , Arañas , Bloqueadores del Canal de Sodio Activado por Voltaje , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
Sterile alpha and TIR motif containing 1 (SARM1) is an inducible NADase that localizes to mitochondria throughout neurons and senses metabolic changes that occur after injury. Minimal proteomic changes are observed upon either SARM1 depletion or activation, suggesting that SARM1 does not exert broad effects on neuronal protein homeostasis. However, whether SARM1 activation occurs throughout the neuron in response to injury and cell stress remains largely unknown. Using a semiautomated imaging pipeline and a custom-built deep learning scoring algorithm, we studied degeneration in both mixed-sex mouse primary cortical neurons and male human-induced pluripotent stem cell-derived cortical neurons in response to a number of different stressors. We show that SARM1 activation is differentially restricted to specific neuronal compartments depending on the stressor. Cortical neurons undergo SARM1-dependent axon degeneration after mechanical transection, and SARM1 activation is limited to the axonal compartment distal to the injury site. However, global SARM1 activation following vacor treatment causes both cell body and axon degeneration. Context-specific stressors, such as microtubule dysfunction and mitochondrial stress, induce axonal SARM1 activation leading to SARM1-dependent axon degeneration and SARM1-independent cell body death. Our data reveal that compartment-specific SARM1-mediated death signaling is dependent on the type of injury and cellular stressor.
Asunto(s)
Proteínas del Dominio Armadillo , Corteza Cerebral , Proteínas del Citoesqueleto , Células Madre Pluripotentes Inducidas , Neuronas , Proteínas del Dominio Armadillo/metabolismo , Proteínas del Dominio Armadillo/genética , Animales , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/genética , Ratones , Neuronas/metabolismo , Neuronas/patología , Masculino , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Humanos , Femenino , Células Madre Pluripotentes Inducidas/metabolismo , Degeneración Nerviosa/patología , Degeneración Nerviosa/metabolismo , Degeneración Nerviosa/genética , Células Cultivadas , Ratones Endogámicos C57BL , Estrés Fisiológico/fisiología , Axones/metabolismo , Axones/patología , Mitocondrias/metabolismoRESUMEN
The transient receptor potential ankyrin 1 (TRPA1) channel functions as an irritant sensor and is a therapeutic target for treating pain, itch, and respiratory diseases. As a ligand-gated channel, TRPA1 can be activated by electrophilic compounds such as allyl isothiocyanate (AITC) through covalent modification or activated by noncovalent agonists through ligand binding. However, how covalent modification leads to channel opening and, importantly, how noncovalent binding activates TRPA1 are not well-understood. Here we report a class of piperidine carboxamides (PIPCs) as potent, noncovalent agonists of human TRPA1. Based on their species-specific effects on human and rat channels, we identified residues critical for channel activation; we then generated binding modes for TRPA1-PIPC interactions using structural modeling, molecular docking, and mutational analysis. We show that PIPCs bind to a hydrophobic site located at the interface of the pore helix 1 (PH1) and S5 and S6 transmembrane segments. Interestingly, this binding site overlaps with that of known allosteric modulators, such as A-967079 and propofol. Similar binding sites, involving π-helix rearrangements on S6, have been recently reported for other TRP channels, suggesting an evolutionarily conserved mechanism. Finally, we show that for PIPC analogs, predictions from computational modeling are consistent with experimental structure-activity studies, thereby suggesting strategies for rational drug design.
Asunto(s)
Simulación del Acoplamiento Molecular , Piperidinas/farmacología , Canal Catiónico TRPA1/química , Canal Catiónico TRPA1/efectos de los fármacos , Animales , Sitios de Unión , Canales de Calcio/química , Canales de Calcio/metabolismo , Diseño de Fármacos , Humanos , Isotiocianatos , Ligandos , Modelos Estructurales , Mutagénesis , Oximas/farmacología , Propofol/farmacología , Dominios Proteicos , Ratas , Especificidad de la Especie , Canal Catiónico TRPA1/metabolismoRESUMEN
Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non-pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.
Asunto(s)
Descubrimiento de Drogas/métodos , Terapia Molecular Dirigida , Bloqueadores del Canal de Sodio Activado por Voltaje/análisis , Animales , Ensayos Analíticos de Alto Rendimiento , Humanos , Proteínas de Insectos , Potenciales de la Membrana , Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.7/genética , Ratas , Veratridina , Venenos de AvispasRESUMEN
Strong human genetic evidence points to an essential contribution of the voltage-gated sodium channel Nav1.7 to pain sensation: loss of Nav1.7 function leads to congenital insensitivity to pain, whereas gain-of-function mutations in the SCN9A gene that encodes Nav1.7 cause painful neuropathies, such as inherited erythromelalgia, a syndrome characterized by episodic spontaneous pain. Selective Nav1.7 channel blockers thus hold promise as potential painkillers with improved safety and reduced unwanted side effects compared with existing therapeutics. To determine the maximum effect of a theoretically perfectly selective Nav1.7 inhibitor, we generated a tamoxifen-inducible KO mouse model enabling genetic deletion of Nav1.7 from adult mice. Electrophysiological recordings of sensory neurons from these mice following tamoxifen injection demonstrated the loss of Nav1.7 channel current and the resulting decrease in neuronal excitability of small-diameter neurons. We found that behavioral responses to most, but surprisingly not all, modalities of noxious stimulus are abolished following adult deletion of Nav1.7, pointing toward indications where Nav1.7 blockade should be efficacious. Furthermore, we demonstrate that isoform-selective acylsulfonamide Nav1.7 inhibitors show robust analgesic and antinociceptive activity acutely after a single dose in mouse pain models shown to be Nav1.7-dependent. All experiments were done with both male and female mice. Collectively, these data expand the depth of knowledge surrounding Nav1.7 biology as it relates to pain, and provide preclinical proof of efficacy that lays a clear path toward translation for the therapeutic use of Nav1.7-selective inhibitors in humans.SIGNIFICANCE STATEMENT Loss-of-function mutations in the sodium channel Nav1.7 cause congenital insensitivity to pain in humans, making Nav1.7 a top target for novel pain drugs. Targeting Nav1.7 selectively has been challenging, however, in part due to uncertainties in which rodent pain models are dependent on Nav1.7. We have developed and characterized an adult-onset Nav1.7 KO mouse model that allows us to determine the expected effects of a theoretically perfect Nav1.7 blocker. Importantly, many commonly used pain models, such as mechanical allodynia after nerve injury, appear to not be dependent on Nav1.7 in the adult. By defining which models are Nav1.7 dependent, we demonstrate that selective Nav1.7 inhibitors can approximate the effects of genetic loss of function, which previously has not been directly established.
Asunto(s)
Canal de Sodio Activado por Voltaje NAV1.7/deficiencia , Insensibilidad Congénita al Dolor/metabolismo , Percepción del Dolor/fisiología , Dolor/metabolismo , Bloqueadores de los Canales de Sodio/uso terapéutico , Animales , Células Cultivadas , Femenino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Células HEK293 , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.7/genética , Dolor/tratamiento farmacológico , Dolor/genética , Insensibilidad Congénita al Dolor/tratamiento farmacológico , Insensibilidad Congénita al Dolor/genética , Percepción del Dolor/efectos de los fármacos , Bloqueadores de los Canales de Sodio/farmacologíaRESUMEN
The voltage-gated sodium (Nav) channel Nav1.7 has been the focus of intense investigation in recent years. Human genetics studies of individuals with gain-of-function and loss-of-function mutations in the Nav1.7 channel have implicated Nav1.7 as playing a critical role in pain. Therefore, selective inhibition of Nav1.7 represents a potentially new analgesic strategy that is expected to be devoid of the significant liabilities associated with available treatment options. Although the identification and development of selective Nav channel modulators have historically been challenging, a number of recent publications has demonstrated progression of increasingly subtype-selective small molecules and peptides toward potential use in preclinical or clinical studies. In this respect, we focus on three binding sites that appear to offer the highest potential for the discovery and optimization of Nav1.7-selective inhibitors: the extracellular vestibule of the pore, the extracellular loops of voltage-sensor domain II (VSD2), and the extracellular loops of voltage-sensor domain IV (VSD4). Notably, these three receptor sites on Nav1.7 can all be defined as extracellular druggable sites, suggesting that non-small molecule formats are potential therapeutic options. In this chapter, we will review specific considerations and challenges underlying the identification and optimization of selective, potential therapeutics targeting Nav1.7 for chronic pain indications.
Asunto(s)
Descubrimiento de Drogas , Canal de Sodio Activado por Voltaje NAV1.7/efectos de los fármacos , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Animales , Sitios de Unión , Dolor Crónico/tratamiento farmacológico , Humanos , Ligandos , Canal de Sodio Activado por Voltaje NAV1.7/química , Canal de Sodio Activado por Voltaje NAV1.7/fisiologíaRESUMEN
A series of α-aryl pyrrolidine sulfonamide TRPA1 antagonists were advanced from an HTS hit to compounds that were stable in liver microsomes with retention of TRPA1 potency. Metabolite identification studies and physicochemical properties were utilized as a strategy for compound design. These compounds serve as starting points for further compound optimization.
Asunto(s)
Proteínas del Tejido Nervioso/antagonistas & inhibidores , Pirrolidinas/farmacología , Sulfonamidas/farmacología , Canales de Potencial de Receptor Transitorio/antagonistas & inhibidores , Animales , Canales de Calcio , Humanos , Microsomas Hepáticos/metabolismo , Pirrolidinas/síntesis química , Ratas , Estereoisomerismo , Relación Estructura-Actividad , Sulfonamidas/síntesis química , Canal Catiónico TRPA1RESUMEN
Histone deacetylase 2 (HDAC2) negatively regulates excitatory synapse number and memory performance. However, whether HDAC2 regulation of excitatory synapses occurs in a cell-autonomous manner and whether HDAC2 regulates inhibitory synaptic functions are not well understood. To examine these aspects of HDAC2 function, we used sparse transfection of rat hippocampal slice cultures and whole-cell recordings in pyramidal neurons. HDAC2 knockdown (KD) in single postsynaptic pyramidal neurons enhanced, whereas HDAC2 overexpression (OE) reduced, excitatory synaptic transmission. Postsynaptic KD of HDAC2 also facilitated expression of long-term potentiation induced by subthreshold induction stimuli, without altering long-term depression. In contrast, HDAC2 KD reduced, whereas HDAC2 OE enhanced, inhibitory synaptic transmission. Alterations of postsynaptic GABA(A) receptors (GABA(A)Rs) likely underlie the impact of HDAC2 on inhibitory transmission. Consistent with this, we observed reduced transcript and protein levels of the GABA(A)R γ2 subunit and reduced surface expression of the α2 subunit after HDAC2 KD. Furthermore, we observed a reduction in synaptic but not tonic GABA(A)R currents by HDAC2 KD, suggesting that HDAC2 selectively affects synaptic abundance of functional GABA(A)Rs. Immunostaining for postsynaptic GABA(A)Rs confirmed that HDAC2 KD and OE can regulate the synaptic abundance of these receptors. Together, these results highlight a role for HDAC2 in suppressing synaptic excitation and enhancing synaptic inhibition of hippocampal neurons. Therefore, a shift in the balance of synaptic excitation versus inhibition favoring excitation could contribute to the beneficial effects of reducing HDAC2 function in wild-type mice or of inhibiting HDACs in models of cognitive impairment.
Asunto(s)
Región CA1 Hipocampal/citología , Potenciales Postsinápticos Excitadores/fisiología , Histona Desacetilasa 2/metabolismo , Potenciales Postsinápticos Inhibidores/fisiología , Animales , Animales Recién Nacidos , Línea Celular Transformada , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/genética , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Proteínas Fluorescentes Verdes/genética , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/genética , Humanos , Técnicas In Vitro , Potenciales Postsinápticos Inhibidores/efectos de los fármacos , Potenciales Postsinápticos Inhibidores/genética , Masculino , Neuronas , Neurotransmisores/farmacología , Técnicas de Placa-Clamp , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Transfección , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
BACKGROUND AND AIMS: This study aimed to identify microbial drivers of inflammatory bowel disease [IBD], by investigating mucosal-associated bacteria and their detrimental products in IBD patients. METHODS: We directly cultured bacterial communities from mucosal biopsies from paediatric gastrointestinal patients and examined for pathogenicity-associated traits. Upon identifying Clostridium perfringens as toxigenic bacteria present in mucosal biopsies, we isolated strains and further characterized toxicity and prevalence. RESULTS: Mucosal biopsy microbial composition differed from corresponding stool samples. C. perfringens was present in eight of nine patients' mucosal biopsies, correlating with haemolytic activity, but was not present in all corresponding stool samples. Large IBD datasets showed higher C. perfringens prevalence in stool samples of IBD adults [18.7-27.1%] versus healthy controls [5.1%]. In vitro, C. perfringens supernatants were toxic to cell types beneath the intestinal epithelial barrier, including endothelial cells, neuroblasts, and neutrophils, while the impact on epithelial cells was less pronounced, suggesting C. perfringens may be particularly damaging when barrier integrity is compromised. Further characterization using purified toxins and genetic insertion mutants confirmed perfringolysin O [PFO] toxin was sufficient for toxicity. Toxin RNA signatures were found in the original patient biopsies by PCR, suggesting intestinal production. C. perfringens supernatants also induced activation of neuroblast and dorsal root ganglion neurons in vitro, suggesting C. perfringens in inflamed mucosal tissue may directly contribute to abdominal pain, a frequent IBD symptom. CONCLUSIONS: Gastrointestinal carriage of certain toxigenic C. perfringens may have an important pathogenic impact on IBD patients. These findings support routine monitoring of C. perfringens and PFO toxins and potential treatment in patients.
Asunto(s)
Toxinas Bacterianas , Clostridium perfringens , Heces , Enfermedades Inflamatorias del Intestino , Mucosa Intestinal , Humanos , Clostridium perfringens/aislamiento & purificación , Clostridium perfringens/genética , Clostridium perfringens/patogenicidad , Niño , Mucosa Intestinal/microbiología , Mucosa Intestinal/patología , Enfermedades Inflamatorias del Intestino/microbiología , Toxinas Bacterianas/genética , Heces/microbiología , Femenino , Masculino , Adolescente , Biopsia , Infecciones por Clostridium/microbiología , Proteínas HemolisinasRESUMEN
Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium ion channel highly expressed in the primary sensory neurons, functioning as a polymodal sensor for exogenous and endogenous stimuli, and has been implicated in neuropathic pain and respiratory disease. Herein, we describe the optimization of potent, selective, and orally bioavailable TRPA1 small molecule antagonists with strong in vivo target engagement in rodent models. Several lead molecules in preclinical single- and short-term repeat-dose toxicity studies exhibited profound prolongation of coagulation parameters. Based on a thorough investigative toxicology and clinical pathology analysis, anticoagulation effects in vivo are hypothesized to be manifested by a metaboliteâgenerated by aldehyde oxidase (AO)âpossessing a similar pharmacophore to known anticoagulants (i.e., coumarins, indandiones). Further optimization to block AO-mediated metabolism yielded compounds that ameliorated coagulation effects in vivo, resulting in the discovery and advancement of clinical candidate GDC-6599, currently in Phase II clinical trials for respiratory indications.
Asunto(s)
Enfermedades Respiratorias , Canales de Potencial de Receptor Transitorio , Humanos , Canales de Potencial de Receptor Transitorio/metabolismo , Canal Catiónico TRPA1 , Aldehído Oxidasa/metabolismo , Oxidorreductasas/metabolismo , Proteínas del Citoesqueleto/metabolismoRESUMEN
Sensory neurons of the dorsal root ganglion (DRG) are critical for maintaining tissue homeostasis by sensing and initiating responses to stimuli. While most preclinical studies of DRGs are conducted in rodents, much less is known about the mechanisms of sensory perception in primates. We generated a transcriptome atlas of mouse, guinea pig, cynomolgus monkey, and human DRGs by implementing a common laboratory workflow and multiple data-integration approaches to generate high-resolution cross-species mappings of sensory neuron subtypes. Using our atlas, we identified conserved core modules highlighting subtype-specific biological processes related to inflammatory response. We also identified divergent expression of key genes involved in DRG function, suggesting species-specific adaptations specifically in nociceptors that likely point to divergent function of nociceptors. Among these, we validated that TAFA4, a member of the druggable genome, was expressed in distinct populations of DRG neurons across species, highlighting species-specific programs that are critical for therapeutic development.
Asunto(s)
Ganglios Espinales , Transcriptoma , Ratones , Humanos , Animales , Cobayas , Ganglios Espinales/metabolismo , Macaca fascicularis , Nociceptores/metabolismo , Células Receptoras Sensoriales/metabolismo , Sensación , Citocinas/metabolismoRESUMEN
BACKGROUND: Dose-limiting toxicities significantly impact the benefit/risk profile of many drugs. Whole genome sequencing (WGS) in patients receiving drugs with dose-limiting toxicities can identify therapeutic hypotheses to prevent these toxicities. Chemotherapy-induced peripheral neuropathy (CIPN) is a common dose-limiting neurological toxicity of chemotherapies with no effective approach for prevention. METHODS: We conducted a genetic study of time-to-first peripheral neuropathy event using 30× germline WGS data from whole blood samples from 4900 European-ancestry cancer patients in 14 randomized controlled trials. A substantial number of patients in these trials received taxane and platinum-based chemotherapies as part of their treatment regimen, either standard of care or in combination with the PD-L1 inhibitor atezolizumab. The trials spanned several cancers including renal cell carcinoma, triple negative breast cancer, non-small cell lung cancer, small cell lung cancer, bladder cancer, ovarian cancer, and melanoma. RESULTS: We identified a locus consisting of low-frequency variants in intron 13 of GRID2 associated with time-to-onset of first peripheral neuropathy (PN) indexed by rs17020773 (p = 2.03 × 10-8, all patients, p = 6.36 × 10-9, taxane treated). Gene-level burden analysis identified rare coding variants associated with increased PN risk in the C-terminus of GPR68 (p = 1.59 × 10-6, all patients, p = 3.47 × 10-8, taxane treated), a pH-sensitive G-protein coupled receptor (GPCR). The variants driving this signal were found to alter predicted arrestin binding motifs in the C-terminus of GPR68. Analysis of snRNA-seq from human dorsal root ganglia (DRG) indicated that expression of GPR68 was highest in mechano-thermo-sensitive nociceptors. CONCLUSIONS: Our genetic study provides insight into the impact of low-frequency and rare coding genetic variation on PN risk and suggests that further study of GPR68 in sensory neurons may yield a therapeutic hypothesis for prevention of CIPN.
Asunto(s)
Antineoplásicos , Carcinoma de Pulmón de Células no Pequeñas , Neoplasias Pulmonares , Enfermedades del Sistema Nervioso Periférico , Femenino , Humanos , Antineoplásicos/efectos adversos , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Paclitaxel/efectos adversos , Enfermedades del Sistema Nervioso Periférico/inducido químicamente , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Ensayos Clínicos Controlados Aleatorios como Asunto , Receptores Acoplados a Proteínas G/genética , Taxoides/efectos adversosRESUMEN
The voltage-gated sodium (NaV) channel NaV1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available NaV channel-blocking drugs are not selective among the nine NaV channel subtypes, NaV1.1-NaV1.9. Moreover, the two currently known classes of NaV1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure-activity relationships of the acylsulfonamide class of NaV1.7 inhibitors, exemplified by the clinical development candidate GDC-0310, has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the NaV1.7 channel, we pursued high-resolution ligand-bound NaV1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that GDC-0310 engages the NaV1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in NaV channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective NaV channel modulators targeting VSD4.
Asunto(s)
Microscopía por Crioelectrón , Humanos , Ligandos , Dominios Proteicos , Sitios de Unión , Relación Estructura-ActividadRESUMEN
Loss-of-function mutations in Nav1.7, a voltage-gated sodium channel, cause congenital insensitivity to pain (CIP) in humans, demonstrating that Nav1.7 is essential for the perception of pain. However, the mechanism by which loss of Nav1.7 results in insensitivity to pain is not entirely clear. It has been suggested that loss of Nav1.7 induces overexpression of enkephalin, an endogenous opioid receptor agonist, leading to opioid-dependent analgesia. Using behavioral pharmacology and single-cell RNA-seq analysis, we find that overexpression of enkephalin occurs only in cLTMR neurons, a subclass of sensory neurons involved in low-threshold touch detection, and that this overexpression does not play a role in the analgesia observed following genetic removal of Nav1.7. Furthermore, we demonstrate using laser speckle contrast imaging (LSCI) and in vivo electrophysiology that Nav1.7 function is required for the initiation of C-fiber action potentials (APs), which explains the observed insensitivity to pain following genetic removal or inhibition of Nav1.7.
Asunto(s)
Analgésicos Opioides , Nociceptores , Ratones , Humanos , Animales , Analgésicos Opioides/farmacología , Potenciales de Acción , Canal de Sodio Activado por Voltaje NAV1.7/genética , Dolor/genética , Células Receptoras Sensoriales , Péptidos Opioides , Encefalinas , Ganglios EspinalesRESUMEN
Transient receptor potential ankyrin 1 (TRPA1) is a nonselective calcium-permeable ion channel highly expressed in the primary sensory neurons functioning as a polymodal sensor for exogenous and endogenous stimuli and has generated widespread interest as a target for inhibition due to its implication in neuropathic pain and respiratory disease. Herein, we describe the optimization of a series of potent, selective, and orally bioavailable TRPA1 small molecule antagonists, leading to the discovery of a novel tetrahydrofuran-based linker. Given the balance of physicochemical properties and strong in vivo target engagement in a rat AITC-induced pain assay, compound 20 was progressed into a guinea pig ovalbumin asthma model where it exhibited significant dose-dependent reduction of inflammatory response. Furthermore, the structure of the TRPA1 channel bound to compound 21 was determined via cryogenic electron microscopy to a resolution of 3 Å, revealing the binding site and mechanism of action for this class of antagonists.
Asunto(s)
Asma/tratamiento farmacológico , Furanos/uso terapéutico , Purinas/uso terapéutico , Canal Catiónico TRPA1/antagonistas & inhibidores , Animales , Asma/inducido químicamente , Asma/complicaciones , Células CHO , Cricetulus , Furanos/síntesis química , Furanos/metabolismo , Cobayas , Humanos , Inflamación/tratamiento farmacológico , Inflamación/etiología , Ligandos , Masculino , Estructura Molecular , Ovalbúmina , Oxadiazoles/síntesis química , Oxadiazoles/metabolismo , Oxadiazoles/uso terapéutico , Unión Proteica , Purinas/síntesis química , Purinas/metabolismo , Ratas Sprague-Dawley , Relación Estructura-Actividad , Canal Catiónico TRPA1/metabolismoRESUMEN
Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.
Asunto(s)
Azetidinas/farmacología , Benzamidas/farmacología , Descubrimiento de Drogas , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Sulfonamidas/farmacología , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Animales , Azetidinas/química , Azetidinas/farmacocinética , Benzamidas/química , Benzamidas/farmacocinética , Células Cultivadas , Células HEK293 , Humanos , Piperidinas/química , Piperidinas/farmacocinética , Piperidinas/farmacología , Ratas Sprague-Dawley , Sulfonamidas/química , Sulfonamidas/farmacocinética , Bloqueadores del Canal de Sodio Activado por Voltaje/química , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacocinéticaRESUMEN
The transient receptor potential (TRP) superfamily of ion channels has garnered significant attention by the pharmaceutical industry. In particular, TRP channels showing high levels of expression in sensory neurons such as TRPV1, TRPA1, and TRPM8, have been considered as targets for indications where sensory neurons play a fundamental role, such as pain, itch, and asthma. Modeling these indications in rodents is challenging, especially in mice. The rat is the preferred species for pharmacological studies in pain, itch, and asthma, but until recently, genetic manipulation of the rat has been technically challenging. Here, using CRISPR technology, we have generated a TRPA1 KO rat to enable more sophisticated modeling of pain, itch, and asthma. We present a detailed phenotyping of the TRPA1 KO rat in models of pain, itch, and asthma that have previously only been investigated in the mouse. With the exception of nociception induced by direct TRPA1 activation, we have found that the TRPA1 KO rat shows apparently normal behavioral responses in multiple models of pain and itch. Immune cell infiltration into the lung in the rat OVA model of asthma, on the other hand, appears to be dependent on TRPA1, similar to was has been observed in TRPA1 KO mice. Our hope is that the TRPA1 KO rat will become a useful tool in further studies of TRPA1 as a drug target.
Asunto(s)
Asma/genética , Conducta Animal/fisiología , Dolor/genética , Prurito/genética , Canal Catiónico TRPA1/genética , Animales , Asma/metabolismo , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Dolor/metabolismo , Fenotipo , Prurito/metabolismo , Ratas , Ratas Transgénicas , Canal Catiónico TRPA1/metabolismoRESUMEN
NMDA receptors (NMDARs) play subunit-specific roles in synaptic function and are implicated in neuropsychiatric and neurodegenerative disorders. However, the in vivo consequences and therapeutic potential of pharmacologically enhancing NMDAR function via allosteric modulation are largely unknown. We examine the in vivo effects of GNE-0723, a positive allosteric modulator of GluN2A-subunit-containing NMDARs, on brain network and cognitive functions in mouse models of Dravet syndrome (DS) and Alzheimer's disease (AD). GNE-0723 use dependently potentiates synaptic NMDA receptor currents and reduces brain oscillation power with a predominant effect on low-frequency (12-20 Hz) oscillations. Interestingly, DS and AD mouse models display aberrant low-frequency oscillatory power that is tightly correlated with network hypersynchrony. GNE-0723 treatment reduces aberrant low-frequency oscillations and epileptiform discharges and improves cognitive functions in DS and AD mouse models. GluN2A-subunit-containing NMDAR enhancers may have therapeutic benefits in brain disorders with network hypersynchrony and cognitive impairments.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Encéfalo/metabolismo , Cognición/efectos de los fármacos , Ciclopropanos/farmacología , Epilepsias Mioclónicas/tratamiento farmacológico , Nitrilos/farmacología , Receptores de N-Metil-D-Aspartato/metabolismo , Tiazoles/farmacología , Regulación Alostérica/efectos de los fármacos , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Encéfalo/efectos de los fármacos , Células CHO , Cricetulus , Modelos Animales de Enfermedad , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/metabolismo , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Pirazoles/farmacología , Receptores de N-Metil-D-Aspartato/agonistasRESUMEN
The original version of this article unfortunately contained a mistake. A few entries were incorrect in Table 2.