RESUMO
Discovery and development of new molecules directed against validated pain targets is required to advance the treatment of pain disorders. Voltage-gated sodium channels (NaVs) are responsible for action potential initiation and transmission of pain signals. NaV1.8 is specifically expressed in peripheral nociceptors and has been genetically and pharmacologically validated as a human pain target. Selective inhibition of NaV1.8 can ameliorate pain while minimizing effects on other NaV isoforms essential for cardiac, respiratory, and central nervous system physiology. Here we present the pharmacology, interaction site, and mechanism of action of LTGO-33, a novel NaV1.8 small molecule inhibitor. LTGO-33 inhibited NaV1.8 in the nM potency range and exhibited over 600-fold selectivity against human NaV1.1-NaV1.7 and NaV1.9. Unlike prior reported NaV1.8 inhibitors that preferentially interacted with an inactivated state via the pore region, LTGO-33 was state-independent with similar potencies against closed and inactivated channels. LTGO-33 displayed species specificity for primate NaV1.8 over dog and rodent NaV1.8 and inhibited action potential firing in human dorsal root ganglia neurons. Using chimeras combined with mutagenesis, the extracellular cleft of the second voltage-sensing domain was identified as the key site required for channel inhibition. Biophysical mechanism of action studies demonstrated that LTGO-33 inhibition was relieved by membrane depolarization, suggesting the molecule stabilized the deactivated state to prevent channel opening. LTGO-33 equally inhibited wild-type and multiple NaV1.8 variants associated with human pain disorders. These collective results illustrate LTGO-33 inhibition via both a novel interaction site and mechanism of action previously undescribed in NaV1.8 small molecule pharmacologic space. SIGNIFICANCE STATEMENT: NaV1.8 sodium channels primarily expressed in peripheral pain-sensing neurons represent a validated target for the development of novel analgesics. Here we present the selective small molecule NaV1.8 inhibitor LTGO-33 that interdicts a distinct site in a voltage-sensor domain to inhibit channel opening. These results inform the development of new analgesics for pain disorders.
Assuntos
Canais de Sódio Disparados por Voltagem , Humanos , Animais , Cães , Dor/tratamento farmacológico , Analgésicos/farmacologia , Neurônios , Potenciais de Ação , Gânglios Espinais , Canal de Sódio Disparado por Voltagem NAV1.7 , Bloqueadores dos Canais de Sódio/farmacologiaRESUMO
NaV1.7 is an actively pursued, genetically validated, target for pain. Recently reported quinolinone sulfonamide inhibitors displayed promising selectivity profiles as well as efficacy in preclinical pain models; however, concerns about off-target liabilities associated with this series resulted in an effort to reduce the lipophilicity of these compounds. Successful prosecution of this strategy was challenging due to the opposing requirement for lipophilic inhibitors for NaV1.7 potency and in vivo clearance (CL). Deconstruction of the heterocyclic core of the quinolinone series and utilization of an intramolecular hydrogen bond to mimic the requisite pharmacophore enabled the introduction of polarity without adversely impacting CL. Ultimately, this strategy led to the identification of compound 29, which demonstrated favorable ADME and was efficacious in pre-clinical models of pain.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7 , Quinolonas , Humanos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Dor/tratamento farmacológico , Relação Estrutura-Atividade , Sulfanilamida , Sulfonamidas/química , Sulfonamidas/farmacologia , Ureia/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/químicaRESUMO
Recently, the identification of several classes of aryl sulfonamides and acyl sulfonamides that potently inhibit NaV1.7 and demonstrate high levels of selectivity over other NaV isoforms have been reported. The fully ionizable nature of these inhibitors has been shown to be an important part of the pharmacophore for the observed potency and isoform selectivity. The requirement of this functionality, however, has presented challenges associated with optimization toward inhibitors with drug-like properties and minimal off-target activity. In an effort to obviate these challenges, we set out to develop an orally bioavailable, selective NaV1.7 inhibitor, lacking these acidic functional groups. Herein, we report the discovery of a novel series of inhibitors wherein a triazolesulfone has been designed to serve as a bioisostere for the acyl sulfonamide. This work culminated in the delivery of a potent series of inhibitors which demonstrated good levels of selectivity over NaV1.5 and favorable pharmacokinetics in rodents.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sulfonamidas/farmacologia , Animais , Relação Dose-Resposta a Droga , Humanos , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Estrutura Molecular , Ratos , Relação Estrutura-Atividade , Sulfonamidas/químicaRESUMO
Potent and selective antagonists of the voltage-gated sodium channel NaV1.7 represent a promising avenue for the development of new chronic pain therapies. We generated a small molecule atropisomer quinolone sulfonamide antagonist AMG8379 and a less active enantiomer AMG8380. Here we show that AMG8379 potently blocks human NaV1.7 channels with an IC50 of 8.5 nM and endogenous tetrodotoxin (TTX)-sensitive sodium channels in dorsal root ganglion (DRG) neurons with an IC50 of 3.1 nM in whole-cell patch clamp electrophysiology assays using a voltage protocol that interrogates channels in a partially inactivated state. AMG8379 was 100- to 1000-fold selective over other NaV family members, including NaV1.4 expressed in muscle and NaV1.5 expressed in the heart, as well as TTX-resistant NaV channels in DRG neurons. Using an ex vivo mouse skin-nerve preparation, AMG8379 blocked mechanically induced action potential firing in C-fibers in both a time-dependent and dose-dependent manner. AMG8379 similarly reduced the frequency of thermally induced C-fiber spiking, whereas AMG8380 affected neither mechanical nor thermal responses. In vivo target engagement of AMG8379 in mice was evaluated in multiple NaV1.7-dependent behavioral endpoints. AMG8379 dose-dependently inhibited intradermal histamine-induced scratching and intraplantar capsaicin-induced licking, and reversed UVB radiation skin burn-induced thermal hyperalgesia; notably, behavioral effects were not observed with AMG8380 at similar plasma exposure levels. AMG8379 is a potent and selective NaV1.7 inhibitor that blocks sodium current in heterologous cells as well as DRG neurons, inhibits action potential firing in peripheral nerve fibers, and exhibits pharmacodynamic effects in translatable models of both itch and pain.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Potenciais de Ação/efeitos dos fármacos , Animais , Comportamento Animal/efeitos dos fármacos , Relação Dose-Resposta a Droga , Gânglios Espinais/citologia , Gânglios Espinais/efeitos dos fármacos , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Neurônios/efeitos dos fármacos , Dor/prevenção & controle , Dor/psicologia , Técnicas de Patch-Clamp , Prurido/prevenção & controle , Prurido/psicologia , Quinolonas/farmacologia , Bibliotecas de Moléculas Pequenas , Estereoisomerismo , Sulfonamidas/farmacologiaRESUMO
The NaV1.7 ion channel has garnered considerable attention as a target for the treatment of pain. Herein we detail the discovery and structure-activity relationships of a novel series of biaryl amides. Optimization led to the identification of several state-dependent, potent and metabolically stable inhibitors which demonstrated promising levels of selectivity over NaV1.5 and good rat pharmacokinetics. Compound 18, which demonstrated preferential inhibition of a slow inactivated state of NaV1.7, was advanced into a rat formalin study where upon reaching unbound drug levels several fold over the rat NaV1.7 IC50 it failed to demonstrate a robust reduction in nociceptive behavior.
Assuntos
Amidas/farmacologia , Descoberta de Drogas , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Amidas/síntese química , Amidas/química , Animais , Relação Dose-Resposta a Droga , Humanos , Camundongos , Estrutura Molecular , Ratos , Relação Estrutura-AtividadeRESUMO
The voltage-gated sodium channel NaV1.7 has received much attention from the scientific community due to compelling human genetic data linking gain- and loss-of-function mutations to pain phenotypes. Despite this genetic validation of NaV1.7 as a target for pain, high quality pharmacological tools facilitate further understanding of target biology, establishment of target coverage requirements and subsequent progression into the clinic. Within the sulfonamide class of inhibitors, reduced potency on rat NaV1.7 versus human NaV1.7 was observed, rendering in vivo rat pharmacology studies challenging. Herein, we report the discovery and optimization of novel benzoxazine sulfonamide inhibitors of human, rat and mouse NaV1.7 which enabled pharmacological assessment in traditional behavioral rodent models of pain and in turn, established a connection between formalin-induced pain and histamine-induced pruritus in mice. The latter represents a simple and efficient means of measuring target engagement.
Assuntos
Benzoxazinas/química , Benzoxazinas/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sulfonamidas/química , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacologia , Analgésicos/uso terapêutico , Animais , Benzoxazinas/farmacocinética , Benzoxazinas/uso terapêutico , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Dor/tratamento farmacológico , Dor/metabolismo , Ratos , Ratos Sprague-Dawley , Sulfonamidas/farmacocinética , Sulfonamidas/uso terapêutico , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
The transient receptor potential ankyrin 1 (TRPA1) channel has been implicated in pathophysiological processes that include asthma, cough, and inflammatory pain. Agonists of TRPA1 such as mustard oil and its key component allyl isothiocyanate (AITC) cause pain and neurogenic inflammation in humans and rodents, and TRPA1 antagonists have been reported to be effective in rodent models of pain. In our pursuit of TRPA1 antagonists as potential therapeutics, we generated AMG0902, a potent (IC90 of 300 nM against rat TRPA1), selective, brain penetrant (brain to plasma ratio of 0.2), and orally bioavailable small molecule TRPA1 antagonist. AMG0902 reduced mechanically evoked C-fiber action potential firing in a skin-nerve preparation from mice previously injected with complete Freund's adjuvant, supporting the role of TRPA1 in inflammatory mechanosensation. In vivo target coverage of TRPA1 by AMG0902 was demonstrated by the prevention of AITC-induced flinching/licking in rats. However, oral administration of AMG0902 to rats resulted in little to no efficacy in models of inflammatory, mechanically evoked hypersensitivity; and no efficacy was observed in a neuropathic pain model. Unbound plasma concentrations achieved in pain models were about 4-fold higher than the IC90 concentration in the AITC target coverage model, suggesting that either greater target coverage is required for efficacy in the pain models studied or TRPA1 may not contribute significantly to the underlying mechanisms.
Assuntos
Hiperalgesia/metabolismo , Inflamação/complicações , Ciática/complicações , Canais de Cátion TRPC/metabolismo , Potenciais de Ação/efeitos dos fármacos , Potenciais de Ação/genética , Aminas/uso terapêutico , Analgésicos/uso terapêutico , Animais , Anti-Inflamatórios não Esteroides/farmacologia , Células CHO , Cricetulus , Ácidos Cicloexanocarboxílicos/uso terapêutico , Comportamento Exploratório/efeitos dos fármacos , Adjuvante de Freund/toxicidade , Gabapentina , Hiperalgesia/tratamento farmacológico , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Naproxeno/farmacologia , Fibras Nervosas Amielínicas/efeitos dos fármacos , Fibras Nervosas Amielínicas/fisiologia , Limiar da Dor/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Ciática/tratamento farmacológico , Canal de Cátion TRPA1 , Canais de Cátion TRPC/antagonistas & inibidores , Canais de Cátion TRPC/genética , Ácido gama-Aminobutírico/uso terapêuticoRESUMO
The nine members of the voltage-gated sodium channel (Nav) family mediate inward sodium currents that depolarize neurons and lead to action potential firing. Increased Nav expression and function in sensory ganglia may drive ectopic action potentials and result in neuropathic pain. Using patch-clamp electrophysiology and molecular biology techniques, experiments were performed to elucidate the contribution of Nav channels to sodium currents in rat dorsal root ganglion (DRG) neurons following the L5/L6 spinal nerve ligation (SNL) model of neuropathic pain. The abundance of DRG neurons with fast, tetrodotoxin sensitive (TTX-S) currents was seven-fold higher whereas the abundance of DRG neurons with slow, tetrodotoxin resistant (TTX-R) currents was nearly thirty-fold lower when comparing ipsilateral (injured) to contralateral (uninjured) neurons. TTX-S currents were elevated in larger neurons while TTX-R currents were reduced in both small and large neurons. Among Nav transcripts encoding TTX-R channels, Scn10a (Nav1.8) and Scn11a (Nav1.9) expression was twenty- to thirty-fold lower, while among Nav transcripts encoding TTX-S channels, Scn3a (Nav1.3) expression was four-fold higher in injured compared to uninjured DRG by qRT-PCR analysis. In summary, the SNL model of neuropathic pain induced a phenotypic switch in Nav expression from TTX-R to TTX-S channels in injured DRG neurons. Transcriptional reprogramming of Nav genes may drive ectopic action potential firing and contribute to neuropathic pain.
Assuntos
Lateralidade Funcional/fisiologia , Gânglios Espinais/patologia , Neurônios/fisiologia , Traumatismos dos Nervos Periféricos/patologia , Canais de Sódio Disparados por Voltagem/metabolismo , Animais , Fenômenos Biofísicos/efeitos dos fármacos , Estimulação Elétrica , Regulação da Expressão Gênica/efeitos dos fármacos , Hiperalgesia/etiologia , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/fisiologia , Neurônios/efeitos dos fármacos , Técnicas de Patch-Clamp , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia , Canais de Sódio Disparados por Voltagem/genéticaRESUMO
Many efforts are underway to develop selective inhibitors of the voltage-gated sodium channel NaV1.7 as new analgesics. Thus far, however, in vitro selectivity has proved difficult for small molecules, and peptides generally lack appropriate pharmacokinetic properties. We previously identified the NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity via structure-guided analoging. To further understand GpTx-1 binding to NaV1.7, we have mapped the binding site to transmembrane segments 1-4 of the second pseudosubunit internal repeat (commonly referred to as Site 4) using NaV1.5/NaV1.7 chimeric protein constructs. We also report that select GpTx-1 amino acid residues apparently not contacting NaV1.7 can be derivatized with a hydrophilic polymer without adversely affecting peptide potency. Homodimerization of GpTx-1 with a bifunctional polyethylene glycol (PEG) linker resulted in a compound with increased potency and a significantly reduced off-rate, demonstrating the ability to modulate the function and properties of GpTx-1 by linking to additional molecules.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Engenharia de Proteínas , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Dimerização , Relação Dose-Resposta a Droga , Humanos , Conformação Molecular , Ligação Proteica , Estrutura Terciária de Proteína , Relação Estrutura-Atividade , Bloqueadores do Canal de Sódio Disparado por Voltagem/químicaRESUMO
The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.
Assuntos
Ensaios de Triagem em Larga Escala , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Técnicas de Patch-Clamp , Bloqueadores do Canal de Sódio Disparado por Voltagem/isolamento & purificação , Animais , Células CHO , Cricetulus , Fenômenos Eletrofisiológicos , Eletrofisiologia , Células HEK293 , Humanos , Canal de Sódio Disparado por Voltagem NAV1.7/genéticaRESUMO
Pituitary adenylate cyclase activating polypeptide-38 (PACAP38) may play an important role in primary headaches. Preclinical evidence suggests that PACAP38 modulates trigeminal nociceptive activity mainly through PAC1 receptors while clinical studies report that plasma concentrations of PACAP38 are elevated in spontaneous attacks of cluster headache and migraine and normalize after treatment with sumatriptan. Intravenous infusion of PACAP38 induces migraine-like attacks in migraineurs and cluster-like attacks in cluster headache patients. A rodent-specific PAC1 receptor antibody Ab181 was developed, and its effect on nociceptive neuronal activity in the trigeminocervical complex was investigated in vivo in an electrophysiological model relevant to primary headaches. Ab181 is potent and selective at the rat PAC1 receptor and provides near-maximum target coverage at 10 mg/kg for more than 48 hours. Without affecting spontaneous neuronal activity, Ab181 effectively inhibits stimulus-evoked activity in the trigeminocervical complex. Immunohistochemical analysis revealed its binding in the trigeminal ganglion and sphenopalatine ganglion but not within the central nervous system suggesting a peripheral site of action. The pharmacological approach using a specific PAC1 receptor antibody could provide a novel mechanism with a potential clinical efficacy in the treatment of primary headaches.
Assuntos
Transtornos de Enxaqueca , Receptores de Polipeptídeo Hipofisário Ativador de Adenilato Ciclase , Animais , Cefaleia/induzido quimicamente , Cefaleia/tratamento farmacológico , Humanos , Nociceptividade , Polipeptídeo Hipofisário Ativador de Adenilato Ciclase/uso terapêutico , RatosRESUMO
BACKGROUND: Taste bud cells transmit information regarding the contents of food from taste receptors embedded in apical microvilli to gustatory nerve fibers innervating basolateral membranes. In particular, taste cells depolarize, activate voltage-gated sodium channels, and fire action potentials in response to tastants. Initial cell depolarization is attributable to sodium influx through TRPM5 in sweet, bitter, and umami cells and an undetermined cation influx through an ion channel in sour cells expressing PKD2L1, a candidate sour taste receptor. The molecular identity of the voltage-gated sodium channels that sense depolarizing signals and subsequently initiate action potentials coding taste information to gustatory nerve fibers is unknown. RESULTS: We describe the molecular and histological expression profiles of cation channels involved in electrical signal transmission from apical to basolateral membrane domains. TRPM5 was positioned immediately beneath tight junctions to receive calcium signals originating from sweet, bitter, and umami receptor activation, while PKD2L1 was positioned at the taste pore. Using mouse taste bud and lingual epithelial cells collected by laser capture microdissection, SCN2A, SCN3A, and SCN9A voltage-gated sodium channel transcripts were expressed in taste tissue. SCN2A, SCN3A, and SCN9A were expressed beneath tight junctions in subsets of taste cells. SCN3A and SCN9A were expressed in TRPM5 cells, while SCN2A was expressed in TRPM5 and PKD2L1 cells. HCN4, a gene previously implicated in sour taste, was expressed in PKD2L1 cells and localized to cell processes beneath the taste pore. CONCLUSION: SCN2A, SCN3A and SCN9A voltage-gated sodium channels are positioned to sense initial depolarizing signals stemming from taste receptor activation and initiate taste cell action potentials. SCN2A, SCN3A and SCN9A gene products likely account for the tetrodotoxin-sensitive sodium currents in taste receptor cells.
Assuntos
Células Epiteliais/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Canais de Sódio/metabolismo , Papilas Gustativas/metabolismo , Animais , Canais de Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização , Imuno-Histoquímica , Hibridização In Situ , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Canal de Sódio Disparado por Voltagem NAV1.2 , Canal de Sódio Disparado por Voltagem NAV1.3 , Canal de Sódio Disparado por Voltagem NAV1.7 , Proteínas do Tecido Nervoso/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Sódio/genética , Canais de Cátion TRPM/metabolismo , Paladar/fisiologia , Língua/metabolismoRESUMO
Drug discovery research on new pain targets with human genetic validation, including the voltage-gated sodium channel NaV1.7, is being pursued to address the unmet medical need with respect to chronic pain and the rising opioid epidemic. As part of early research efforts on this front, we have previously developed NaV1.7 inhibitory peptide-antibody conjugates with tarantula venom-derived GpTx-1 toxin peptides with an extended half-life (80 h) in rodents but only moderate in vitro activity (hNaV1.7 IC50 = 250 nM) and without in vivo activity. We identified the more potent peptide JzTx-V from our natural peptide collection and improved its selectivity against other sodium channel isoforms through positional analogueing. Here we report utilization of the JzTx-V scaffold in a peptide-antibody conjugate and architectural variations in the linker, peptide loading, and antibody attachment site. We found conjugates with 100-fold improved in vitro potency relative to those of complementary GpTx-1 analogues, but pharmacokinetic and bioimaging analyses of these JzTx-V conjugates revealed a shorter than expected plasma half-life in vivo with accumulation in the liver. In an attempt to increase circulatory serum levels, we sought the reduction of the net +6 charge of the JzTx-V scaffold while retaining a desirable NaV in vitro activity profile. The conjugate of a JzTx-V peptide analogue with a +2 formal charge maintained NaV1.7 potency with 18-fold improved plasma exposure in rodents. Balancing the loss of peptide and conjugate potency associated with the reduction of net charge necessary for improved target exposure resulted in a compound with moderate activity in a NaV1.7-dependent pharmacodynamic model but requires further optimization to identify a conjugate that can fully engage NaV1.7 in vivo.
Assuntos
Imunoconjugados , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/química , Venenos de Aranha/química , Bloqueadores do Canal de Sódio Disparado por Voltagem , Animais , Anticorpos/química , Descoberta de Drogas , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Masculino , Camundongos , Terapia de Alvo Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/imunologia , Peptídeos/farmacocinética , Venenos de Aranha/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinéticaRESUMO
Inhibitors of the voltage-gated sodium channel NaV1.7 are being investigated as pain therapeutics due to compelling human genetics. We previously identified NaV1.7-inhibitory peptides GpTx-1 and JzTx-V from tarantula venom screens. Potency and selectivity were modulated through attribute-based positional scans of native residues via chemical synthesis. Herein, we report JzTx-V lead optimization to identify a pharmacodynamically active peptide variant. Molecular docking of peptide ensembles from NMR into a homology model-derived NaV1.7 structure supported prioritization of key residues clustered on a hydrophobic face of the disulfide-rich folded peptide for derivatization. Replacing Trp24 with 5-Br-Trp24 identified lead peptides with activity in electrophysiology assays in engineered and neuronal cells. 5-Br-Trp24 containing peptide AM-6120 was characterized in X-ray crystallography and pharmacokinetic studies and blocked histamine-induced pruritis in mice after subcutaneous administration, demonstrating systemic NaV1.7-dependent pharmacodynamics. Our data suggests a need for high target coverage based on plasma exposure for impacting in vivo end points with selectivity-optimized peptidic NaV1.7 inhibitors.
Assuntos
Descoberta de Drogas , Histamina/efeitos adversos , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Prurido/tratamento farmacológico , Venenos de Aranha/química , Animais , Células HEK293 , Humanos , Camundongos , Simulação de Acoplamento Molecular , Canal de Sódio Disparado por Voltagem NAV1.7/química , Peptídeos/farmacocinética , Peptídeos/uso terapêutico , Conformação Proteica , Dobramento de Proteína , Prurido/induzido quimicamente , Relação Estrutura-Atividade , Distribuição Tecidual , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/uso terapêuticoRESUMO
Identification of voltage-gated sodium channel NaV1.7 inhibitors for chronic pain therapeutic development is an area of vigorous pursuit. In an effort to identify more potent leads compared to our previously reported GpTx-1 peptide series, electrophysiology screening of fractionated tarantula venom discovered the NaV1.7 inhibitory peptide JzTx-V from the Chinese earth tiger tarantula Chilobrachys jingzhao. The parent peptide displayed nominal selectivity over the skeletal muscle NaV1.4 channel. Attribute-based positional scan analoging identified a key Ile28Glu mutation that improved NaV1.4 selectivity over 100-fold, and further optimization yielded the potent and selective peptide leads AM-8145 and AM-0422. NMR analyses revealed that the Ile28Glu substitution changed peptide conformation, pointing to a structural rationale for the selectivity gains. AM-8145 and AM-0422 as well as GpTx-1 and HwTx-IV competed for ProTx-II binding in HEK293 cells expressing human NaV1.7, suggesting that these NaV1.7 inhibitory peptides interact with a similar binding site. AM-8145 potently blocked native tetrodotoxin-sensitive (TTX-S) channels in mouse dorsal root ganglia (DRG) neurons, exhibited 30- to 120-fold selectivity over other human TTX-S channels and exhibited over 1,000-fold selectivity over other human tetrodotoxin-resistant (TTX-R) channels. Leveraging NaV1.7-NaV1.5 chimeras containing various voltage-sensor and pore regions, AM-8145 mapped to the second voltage-sensor domain of NaV1.7. AM-0422, but not the inactive peptide analog AM-8374, dose-dependently blocked capsaicin-induced DRG neuron action potential firing using a multi-electrode array readout and mechanically-induced C-fiber spiking in a saphenous skin-nerve preparation. Collectively, AM-8145 and AM-0422 represent potent, new engineered NaV1.7 inhibitory peptides derived from the JzTx-V scaffold with improved NaV selectivity and biological activity in blocking action potential firing in both DRG neurons and C-fibers.
Assuntos
Analgésicos/isolamento & purificação , Canal de Sódio Disparado por Voltagem NAV1.7/efeitos dos fármacos , Peptídeos/química , Bloqueadores dos Canais de Sódio/isolamento & purificação , Venenos de Aranha/química , Potenciais de Ação/efeitos dos fármacos , Substituição de Aminoácidos , Analgésicos/farmacologia , Animais , Capsaicina/farmacologia , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Gânglios Espinais/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Mutagênese Sítio-Dirigida , Fibras Nervosas Amielínicas/efeitos dos fármacos , Ressonância Magnética Nuclear Biomolecular , Técnicas de Patch-Clamp , Estimulação Física , Engenharia de Proteínas , Proteínas Recombinantes/efeitos dos fármacos , Bloqueadores dos Canais de Sódio/farmacologia , Relação Estrutura-Atividade , Tetrodotoxina/farmacologiaRESUMO
The voltage-gated sodium channel NaV1.7 is a genetically validated pain target under investigation for the development of analgesics. A therapeutic with a less frequent dosing regimen would be of value for treating chronic pain; however functional NaV1.7 targeting antibodies are not known. In this report, we describe NaV1.7 inhibitory peptide-antibody conjugates as an alternate construct for potential prolonged channel blockade through chemical derivatization of engineered antibodies. We previously identified NaV1.7 inhibitory peptide GpTx-1 from tarantula venom and optimized its potency and selectivity. Tethering GpTx-1 peptides to antibodies bifunctionally couples FcRn-based antibody recycling attributes to the NaV1.7 targeting function of the peptide warhead. Herein, we conjugated a GpTx-1 peptide to specific engineered cysteines in a carrier anti-2,4-dinitrophenol monoclonal antibody using polyethylene glycol linkers. The reactivity of 13 potential cysteine conjugation sites in the antibody scaffold was tuned using a model alkylating agent. Subsequent reactions with the peptide identified cysteine locations with the highest conversion to desired conjugates, which blocked NaV1.7 currents in whole cell electrophysiology. Variations in attachment site, linker, and peptide loading established design parameters for potency optimization. Antibody conjugation led to in vivo half-life extension by 130-fold relative to a nonconjugated GpTx-1 peptide and differential biodistribution to nerve fibers in wild-type but not NaV1.7 knockout mice. This study describes the optimization and application of antibody derivatization technology to functionally inhibit NaV1.7 in engineered and neuronal cells.
Assuntos
Imunoconjugados/farmacologia , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Peptídeos/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologia , Animais , Células HEK293 , Humanos , Imunoconjugados/química , Imunoconjugados/farmacocinética , Masculino , Camundongos , Modelos Moleculares , Peptídeos/química , Peptídeos/farmacocinética , Distribuição Tecidual , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinéticaRESUMO
Several reports have recently emerged regarding the identification of heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. The optimization of a series of internal NaV1.7 leads that address a number of metabolic liabilities including bioactivation, PXR activation, as well as CYP3A4 induction and inhibition led to the identification of potent and selective inhibitors that demonstrated favorable pharmacokinetic profiles and were devoid of the aforementioned liabilities. The key to achieving this within a series prone to transporter-mediated clearance was the identification of a small range of optimal cLogD values and the discovery of subtle PXR SAR that was not lipophilicity dependent. This enabled the identification of compound 20, which was advanced into a target engagement pharmacodynamic model where it exhibited robust reversal of histamine-induced scratching bouts in mice.
Assuntos
Isoquinolinas/química , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Sulfonamidas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Animais , Linhagem Celular , Citocromo P-450 CYP3A/biossíntese , Inibidores do Citocromo P-450 CYP3A/química , Inibidores do Citocromo P-450 CYP3A/farmacocinética , Inibidores do Citocromo P-450 CYP3A/farmacologia , Cães , Indução Enzimática , Histamina , Humanos , Isoquinolinas/administração & dosagem , Isoquinolinas/farmacocinética , Masculino , Camundongos Endogâmicos C57BL , Receptor de Pregnano X , Prurido/induzido quimicamente , Prurido/prevenção & controle , Ratos , Receptores de Esteroides/agonistas , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
Because of its strong genetic validation, NaV1.7 has attracted significant interest as a target for the treatment of pain. We have previously reported on a number of structurally distinct bicyclic heteroarylsulfonamides as NaV1.7 inhibitors that demonstrate high levels of selectivity over other NaV isoforms. Herein, we report the discovery and optimization of a series of atropisomeric quinolinone sulfonamide inhibitors [ Bicyclic sulfonamide compounds as sodium channel inhibitors and their preparation . WO 2014201206, 2014 ] of NaV1.7, which demonstrate nanomolar inhibition of NaV1.7 and exhibit high levels of selectivity over other sodium channel isoforms. After optimization of metabolic and pharmacokinetic properties, including PXR activation, CYP2C9 inhibition, and CYP3A4 TDI, several compounds were advanced into in vivo target engagement and efficacy models. When tested in mice, compound 39 (AM-0466) demonstrated robust pharmacodynamic activity in a NaV1.7-dependent model of histamine-induced pruritus (itch) and additionally in a capsaicin-induced nociception model of pain without any confounding effect in open-field activity.
Assuntos
Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Quinolonas/química , Sulfonamidas/química , Bloqueadores do Canal de Sódio Disparado por Voltagem/química , Analgésicos/química , Analgésicos/farmacocinética , Analgésicos/farmacologia , Animais , Capsaicina , Linhagem Celular , Cães , Histamina , Camundongos Endogâmicos C57BL , Simulação de Acoplamento Molecular , Dor/induzido quimicamente , Dor/prevenção & controle , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Prurido/induzido quimicamente , Prurido/prevenção & controle , Quinolonas/administração & dosagem , Quinolonas/síntese química , Quinolonas/farmacocinética , Quinolonas/farmacologia , Ratos , Relação Estrutura-Atividade , Sulfonamidas/administração & dosagem , Sulfonamidas/síntese química , Sulfonamidas/farmacocinética , Sulfonamidas/farmacologia , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacocinética , Bloqueadores do Canal de Sódio Disparado por Voltagem/farmacologiaRESUMO
[This corrects the article DOI: 10.1021/acsmedchemlett.6b00243.].
RESUMO
There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit NaV1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with NaV1.7 and related NaV subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on NaV1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a NaV1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work.