Discovery of piperidine carboxamide TRPV1 antagonists

A series of piperidine carboxamides were developed as potent antagonists of the transient receptor potential vanilloid-1 (TRPV1), an emerging target for the treatment of pain. A focused library of polar head groups led to the identification of a benzoxazinone amide that afforded good potency in cell-based assays. Synthesis and a QSAR model will be presented.     

Chili und der Capsaicinrezeptor TRPV1

Some like it hot – and spicy: Chili and the capsaicin receptor TRPV1 Since many hundred years, many people like to eat chili pepper containing the pungent ingredient capsaicin that is responsible for making the food hot and spicy. Capsaicin activates transient receptor potential TRPV1 channels that are predominantly expressed in sensory neurons involved in pain sensation. TRPV1 is a noxious heat sensor and can also be activated by protons and several animal toxins. Thus, TRPV1 is a polymodal sensor of multiple noxious stimuli that cause pain. TRPV1 functions as a nocisensor that detects chemical and thermal stimuli and transduces this stimulation into sensory nerve impulses which leads to the perception of pain. Inhibition of TRPV1 reduces or abolishes pain sensation. A strong activation of TRPV1 induces a long-lasting refractory period of the pain-detecting system (desensitization) and may even lead to an irreversible loss of TRPV1-expressing sensory neurons. It still remains unclear why many people love hot and spicy food, accompanied by a burning sensation in the mouth.     

Modulation of neuropathic-pain-related behaviour by the spinal endocannabinoid/endovanilloid system

Neuropathic pain refers to chronic pain that results from injury to the nervous system. The mechanisms involved in neuropathic pain are complex and involve both peripheral and central phenomena. Although numerous pharmacological agents are available for the treatment of neuropathic pain, definitive drug therapy has remained elusive. Recent drug discovery efforts have identified an original neurobiological approach to the pathophysiology of neuropathic pain. The development of innovative pharmacological strategies has led to the identification of new promising pharmacological targets, including glutamate antagonists, microglia inhibitors and, interestingly, endogenous ligands of cannabinoids and the transient receptor potential vanilloid type 1 (TRPV1). Endocannabinoids (ECs), endovanilloids and the enzymes that regulate their metabolism represent promising pharmacological targets for the development of a successful pain treatment. This review is an update of the relationship between cannabinoid receptors (CB1) and TRPV1 channels and their possible implications for neuropathic pain. The data are focused on endogenous spinal mechanisms of pain control by anandamide, and the current and emerging pharmacotherapeutic approaches that benefit from the pharmacological modulation of spinal EC and/or endovanilloid systems under chronic pain conditions will be discussed.     

TRPV1: a therapeutic target for novel analgesic drugs?

The vanilloid receptor TRPV1 is now recognized as a molecular integrator of painful stimuli ranging from noxious heat to endovanilloids in inflammation. Pharmacological blockade of TRPV1 represents a new strategy in pain relief. TRPV1 antagonists are expected to prevent pain by silencing receptors where pain is generated rather than stopping the propagation of pain, as most-traditional pain killers do. This hypothesis has already being tested in the clinic by administering small molecule TRPV1 antagonists (e.g. GlaxoSmithKline SB-705498) for migraine and dental pain. Paradoxically, in some murine models of chronic pain, TRPV1-deficient mice exhibit more pain-related behavior than their wild-type littermates, indicating that the understanding of TRPV1 in pain is still incomplete. Moreover, there is mounting evidence to suggest the existence of functional TRPV1 both in the brain and in various non-neuronal tissues. The biological role of these receptors remains elusive, but their tissue distribution clearly indicates that they are involved in many more functions than just pain perception. Here, we review the potential therapeutic indications and adverse effects of TRPV1 antagonists.     

瞬时受体电位香草酸亚型1(TRPV1)与炎性痛

瞬时受体电位香草酸亚型1(transient receptor potential vanilloid 1,TRPV1)是TRP超家族的成员之一,是一种非选择性的阳离子通道。TRPV1广泛分布于伤害性感受器上,并且在伤害性感受器中起重要作用。TRPV1能够感受伤害性刺激,将之转化为动作电位,传至中枢形成痛觉。炎症时释放的许多炎症介质都能够与TRPV1发生相互作用,产生疼痛或痛觉过敏,并且通过各种不同的信号通路来调制TRPV1的活性。深入研究TRPV1的作用机制,有助于理解痛觉生理和开发新型镇痛药物。     

Silencing of vanilloid receptor TRPV1 by RNAi reduces neuropathic and visceral pain in vivo

RNA interference (RNAi) has proven to be a powerful technique to study the function of genes by producing knock-down phenotypes. Here, we report that intrathecal injection of an siRNA against the transient receptor potential vanilloid receptor 1 (TRPV1) reduced cold allodynia of mononeuropathic rats by more than 50% over a time period of approximately 5 days. A second siRNA targeted to a different region of the TRPV1 gene was employed and confirmed the analgesic action of a TRPV1 knock-down. Furthermore, siRNA treatment diminished spontaneous visceral pain behavior induced by capsaicin application to the rectum of mice. The analgesic effect of siRNA-mediated knockdown of TRPV1 in the visceral pain model was comparable to that of the low-molecular weight receptor antagonist BCTC. Our data demonstrate that TRPV1 antagonists, including TRPV1 siRNAs, have potential in the treatment of both, neuropathic and visceral pain.     

Antinociceptive effect of antisense oligonucleotides against the vanilloid receptor VR1/TRPV1

To examine the role of the vanilloid receptor TRPV1 in neuropathic pain, we assessed the effects of the receptor antagonist thioxo-BCTC and antisense oligonucleotides against the TRPV1 mRNA in a rat model of spinal nerve ligation. In order to identify accessible sites on the mRNA of TRPV1, the RNase H assay was used, leading to the successful identification of binding sites for antisense oligonucleotides. Cotransfection studies using Cos-7 cells were employed to identify the most effective antisense oligonucleotide efficiently inhibiting the expression of a fusion protein consisting of TRPV1 and the green fluorescent protein in a specific and concentration-dependent manner. In an in vivo rat model of spinal nerve ligation, intravenous application of the TRPV1 antagonist thioxo-BCTC reduced mechanical hypersensitivity yielding an ED(50) value of 10.6mg/kg. Intrathecal administration of the antisense oligonucleotide against TRPV1, but not the mismatch oligonucleotide or a vehicle control, reduced mechanical hypersensitivity in rats with spinal nerve ligation in a similar manner. Immunohistochemical analysis revealed neuropathy- and antisense-associated regulation of TRPV1 protein expression in spinal cord and dorsal root ganglia. Our data demonstrate comparative analgesic effects of a TRPV1 anatagonist and a rationally designed TRPV1 antisense oligonucleotide in a spinal nerve ligation model of neuropathic pain and thus, lend support to the validation of TRPV1 as a promising target for the treatment of neuropathic pain.     

TRPV1通道在中枢神经系统中的功能

TRPV1（transient receptor potential vanilloid 1）是在机体广泛分布的非选择性阳离子通道,能被氢离子、高温以及其它内源性和外源性配体激活.其在外周神经系统中主要参与伤害性高温的感受以及痛觉过敏等生理机制.TRPV1在中枢神经系统中功能的研究进展主要体现在突触传递,体温调节,痛觉的调制和细胞凋亡等方面.TRPV1的激活降低突触前谷氨酸的释放及增强已存在的突触后AMPA受体的作用,从而增强了突触传递效能.外周的TRPV1通过激活能够抑制血管的收缩和生热作用,从而抑制体温的升高,当TRPV1被阻断时就发生体温过高,而TRPV1体温调节的中枢作用机制可能是通过直接作用于体温调节中枢.脑干的痛觉调制环路的激活TRPV1可以引起谷氨酸盐的释放,进而激活突触后I类mGlu受体以及NMDA受体,从而起到镇痛的功能.另外近年发现TRPV1在中枢也参与呕吐、呼吸、心率及血压的调节.     

Mice lacking functional TRPV1 are protected from pressure overload cardiac hypertrophy

TRPV1 (transient receptor potential cation channel, subfamily V, member 1) is best studied in peripheral sensory neurons as a pain receptor; however TRPV1 is expressed in numerous tissues and cell types including those of the cardiovascular system. TRPV1 expression is upregulated in the hypertrophic heart, and the channel is positioned to receive stimulatory signals in the hypertrophic heart. We hypothesized that TRPV1 has a role in regulating cardiac hypertrophy. Using transverse aortic constriction to model pressure overload cardiac hypertrophy we show that mice lacking functional TRPV1, compared to wild type, have improved heart function, and reduced hypertrophic, fibrotic and apoptotic markers. This suggests that TRPV1 plays a role in the progression of cardiac hypertrophy, and presents a possible therapeutic target for the treatment of cardiac hypertrophy and heart failure.     

Co-activation of P2Y2 Receptor and TRPV Channel by ATP: Implications for ATP Induced Pain

1. Extracellular ATP is recognized as a peripheral modulator of pain. Activation of ionotropic P2X receptors in sensory neurons has been implicated in induction of pain, whereas metabotropic P2Y receptors in potentiation of pain induced by chemical or physical stimuli via capsaicin sensitive TRPV1 channel. Here we report that P2Y2 receptor activation by ATP can activate the TRPV1 channel in absence of any other stimuli. 2. ATP-induced Ca2+ signaling was studied in Neuro2a cells. ATP evoked release of intracellular Ca2+ from ER and Ca2+ influx through a fast inactivating channel. The Ca2+ response was induced by P2Y receptor agonists in the order of potency ATP>or=UTP>or=ATPgammaS>ADP and was inhibited by suramin and PPADS. The P2X receptor agonist alpha beta methyl ATP was ineffective. 3. The Ca2+ influx was blocked by ruthenium red, an inhibitor of TRPV1 channel. Capsaicin, the most potent activator of the TRPV1 channel, evoked a fast inactivating Ca2+ transient suggesting the presence of endogenous TRPV1 channels in Neuro2a cells. NMS and PDBu, repressors of IP3 formation, drastically inhibited both the components of Ca2+ response. 4. Our data show co-activation of the P2Y2 receptor and capsaicin sensitive TRPV1 channel by ATP. Such functional interaction between endogenous P2Y2 receptor and TRPV1 channels could explain the ATP-induced pain.     

Mice lacking functional TRPV1 are protected from pressure overload cardiac hypertrophy

TRPV1 (transient receptor potential cation channel, subfamily V, member 1) is best studied in peripheral sensory neurons as a pain receptor; however TRPV1 is expressed in numerous tissues and cell types including those of the cardiovascular system. TRPV1 expression is upregulated in the hypertrophic heart, and the channel is positioned to receive stimulatory signals in the hypertrophic heart. We hypothesized that TRPV1 has a role in regulating cardiac hypertrophy. Using transverse aortic constriction to model pressure overload cardiac hypertrophy we show that mice lacking functional TRPV1, compared to wild type, have improved heart function, and reduced hypertrophic, fibrotic and apoptotic markers. This suggests that TRPV1 plays a role in the progression of cardiac hypertrophy, and presents a possible therapeutic target for the treatment of cardiac hypertrophy and heart failure.     

Modulation of TRPV4 by diverse mechanisms

Transient receptor potential ion channels (TRP) are a superfamily of non-selective ion channels which are opened in response to a diverse range of stimuli. The TRP vanilloid 4 (TRPV4) ion channel is opened in response to heat, mechanical stimuli, hypo-osmolarity and arachidonic acid metabolites. However, recently TRPV4 has been identified as an ion channel that is modulated by, and opened by intracellular signalling cascades from other receptors and signalling pathways. Although TRPV4 knockout mice show relatively mild phenotypes, some mutations in TRPV4 cause severe developmental abnormalities, such as the skeletal dyplasia and arthropathy. Regulated TRPV4 function is also essential for healthy cardiovascular system function as a potent agonist compromises endothelial cell function, leading to vascular collapse. A better understanding of the signalling mechanisms that modulate TRPV4 function is necessary to understand its physiological roles. Post translational modification of TRPV4 by kinases and other signalling molecules can modulate TRPV4 opening in response to stimuli such as mechanical and hyposmolarity and there is an emerging area of research implicating TRPV4 as a transducer of these signals as opposed to a direct sensor of the stimuli. Due to its wide expression profile, TRPV4 is implicated in multiple pathophysiological states. TRPV4 contributes to the sensation of pain due to hypo-osmotic stimuli and inflammatory mechanical hyperalsgesia, where TRPV4 sensitizaton by intracellular signalling leads to pain behaviors in mice. In the vasculature, TRPV4 is a regulator of vessel tone and is implicated in hypertension and diabetes due to endothelial dysfunction. TRPV4 is a key regulator of epithelial and endothelial barrier function and signalling to and opening of TRPV4 can disrupt these critical protective barriers. In respiratory function, TRPV4 is involved in cystic fibrosis, cilary beat frequency, bronchoconstriction, chronic obstructive pulmonary disease, pulmonary hypertension, acute lung injury, acute respiratory distress syndrome and cough.In this review we highlight how modulation of TRPV4 opening is a vital signalling component in a range of tissues and why understanding of TRPV4 regulation in the body may lead to novel therapeutic approaches to treating a range of disease states.     

Analgesic Effect of Electroacupuncture in a Mouse Fibromyalgia Model: Roles of TRPV1, TRPV4, and pERK

Fibromyalgia (FM) is among the most common chronic pain syndromes encountered in clinical practice, but there is limited understanding of FM pathogenesis. We examined the contribution of transient receptor potential vanilloid 1 (TRPV1) and TRPV4 channels to chronic pain in the repeated acid injection mouse model of FM and the potential therapeutic efficacy of electroacupuncture. Electroacupuncture (EA) at the bilateral Zusanli (ST36) acupoint reduced the long-lasting mechanical hyperalgesia induced by repeated acid saline (pH 4) injection in mouse hindpaw. Isolated L5 dorsal root ganglion (DRG) neurons from FM model mice (FM group) were hyperexcitable, an effect reversed by EA pretreatment (FM + EA group). The increase in mechanical hyperalgesia was also accompanied by upregulation of TRPV1 expression and phosphoactivation of extracellular signal regulated kinase (pERK) in the DRG, whereas DRG expression levels of TRPV4, p-p38, and p-JNK were unaltered. Blockade of TRPV1, which was achieved using TRPV1 knockout mice or via antagonist injection, and pERK suppressed development of FM-like pain. Both TRPV1 and TRPV4 protein expression levels were increased in the spinal cord (SC) of model mice, and EA at the ST36 acupoint decreased overexpression. This study strongly suggests that DRG TRPV1 overexpression and pERK signaling, as well as SC TRPV1 and TRPV4 overexpression, mediate hyperalgesia in a mouse FM pain model. The therapeutic efficacy of EA may result from the reversal of these changes in pain transmission pathways.     

Intact microtubules preserve transient receptor potential vanilloid 1 (TRPV1) functionality through receptor binding

The transient receptor potential cation channel subfamily V member 1 (TRPV1) is a protein currently under scrutiny as a pharmacological target for pain management therapies. Recently, the role of TRPV1-microtubule interaction in transducing nociception stimuli to cells by cytoskeletal rearrangement was proposed. In this work, we investigate TRPV1-microtubule interaction in living cells under the resting or activated state of TRPV1, as well as in presence of structurally intact or depolymerized cytoskeletal microtubules. We combined a toolbox of high resolution/high sensitivity fluorescence imaging techniques (such as FRET, correlation spectroscopy, and fluorescence anisotropy) to monitor TRPV1 aggregation status, membrane mobility, and interaction with microtubules. We found that TRPV1 is a dimeric membrane protein characterized by two populations with different diffusion properties in basal condition. After stimulation with resiniferatoxin, TRPV1 dimers tetramerize. The tetramers and the slower population of TRPV1 dimers bind dynamically to intact microtubules but not to tubulin dimers. Upon microtubule disassembly, the interaction with TRPV1 is lost thereby inducing receptor self-aggregation with partial loss of functionality. Intact microtubules play an essential role in maintaining TRPV1 functionality toward activation stimuli. This previously undisclosed property mirrors the recently reported role of TRPV1 in modulating microtubule assembly/disassembly and suggests the participation of these two players in a feedback cycle linking nociception and cytoskeletal remodeling.     

Targeting the Transient Receptor Potential Vanilloid Type 1 (TRPV1) Assembly Domain Attenuates Inflammation-induced Hypersensitivity

The transient receptor potential channel vanilloid type 1 (TRPV1) is a non-selective cation channel expressed in sensory neurons of the dorsal root and trigeminal ganglia. TRPV1 is a polymodal channel activated by noxious heat, capsaicin, and protons. As a sensor for noxious stimuli, TRPV1 channel has been described as a key contributor to pain signaling. To form a functional channel, TRPV1 subunits must assemble into tetramers, and several studies have identified the TRPV1 C terminus as an essential element in subunit association. Here we combined biochemical assays with electrophysiology and imaging-based bimolecular fluorescence complementation (BiFC) and bioluminescence resonance energy transfer (BRET) in live cells to identify a short motif in the C-terminal tail of the TRPV1 subunit that governs channel assembly. Removing this region through early truncation or targeted deletion results in loss of subunit association and channel function. Importantly, we found that interfering with TRPV1 subunit association using a plasma membrane-tethered peptide attenuated mechanical and thermal hypersensitivity in two mouse models of inflammatory hyperalgesia. This represents a novel mechanism to disrupt TRPV1 subunit assembly and hence may offer a new analgesic tool for pain relief.     

The role of spinal cord vanilloid (TRPV1) receptors in pain modulation

Transient receptor potential vanilloid 1 (TRPV1) receptor is a nonselective cation channel activated by capsaicin, a pungent substance from chili peppers. It is considered to act as an integrator of various physical and chemical nociceptive stimuli, as it can be gated by noxious heat (>43 oC), low pH (protons) and also by recently described endogenous lipids. The structure and function of TRPV1 receptors was vigorously studied, especially since its cloning in 1997. However, most of the research was pointed towards the role of TRPV1 receptors in the peripheral tissues. Mounting evidence now suggests that TRPV1 receptors on the central branches of dorsal root ganglion neurons in the spinal cord may play an important role in modulation of pain and nociceptive transmission. The aim of this short review was to summarize the knowledge about TRPV1 receptors in the spinal cord dorsal horn, preferentially from morphological and electrophysiological studies on spinal cord slices and from in vivo experiments.     

The mechanism of μ-opioid receptor (MOR)-TRPV1 crosstalk in TRPV1 activation involves morphine anti-nociception,tolerance and dependence

Initiated by the activation of various nociceptors, pain is a reaction to specific stimulus modalities. The μ-opioid receptor (MOR) agonists, including morphine, remain the most potent analgesics to treat patients with moderate to severe pain. However, the utility of MOR agonists is limited by the adverse effects associated with the use of these drugs, including analgesic tolerance and physical dependence. A strong connection has been suggested between the expression of the transient receptor potential vanilloid type 1 (TRPV1) ion channel and the development of inflammatory hyperalgesia. TRPV1 is important for thermal nociception induction, and is mainly expressed on sensory neurons. Recent reports suggest that opioid or TRPV1 receptor agonist exposure has contrasting consequences for anti-nociception, tolerance and dependence. Chronic morphine exposure modulates TRPV1 activation and induces the anti-nociception effects of morphine. The regulation of many downstream targets of TRPV1 plays a critical role in this process, including calcitonin gene-related peptide (CGRP) and substance P (SP). Additional factors also include capsaicin treatment blocking the anti-nociception effects of morphine in rats, as well as opioid modulation of TRPV1 responses through the cAMP-dependent PKA pathway and MAPK signaling pathways. Here, we review new insights concerning the mechanism underlying MOR-TRPV1 crosstalk and signaling pathways and discuss the potential mechanisms of morphine-induced anti-nociception, tolerance and dependence associated with the TRPV1 signaling pathway and highlight how understanding these mechanisms might help find therapeutic targets for the treatment of morphine induced antinociception, tolerance and dependence.     

The Impact of TRPV1 on Cancer Pathogenesis and Therapy: A Systematic Review

The transient receptor potential cation channel subfamily V member 1 (TRPV1) is a transmembrane protein that can be activated by various physical and chemical stimuli and is associated with pain transduction. In recent years, TRPV1 was discovered to play essential roles in cancer tumorigenesis and development, as TRPV1 expression levels are altered in numerous cancer cell types. Several investigations have discovered direct associations between TRPV1 and cancer cell proliferation, cell death, and metastasis. Furthermore, about two dozen TRPV1 agonists/antagonists are under clinical trial, as TRPV1 is a potential drug target for treating various diseases. Hence, more researchers are focusing on the effects of TRPV1 agonists or antagonists on cancer tumorigenesis and development. However, both agonists and antagonists may reveal anti-cancer effects, and the effect may function via or be independent of TRPV1. In this review, we provide an overview of the impact of TRPV1 on cancer cell proliferation, cell death, and metastasis, as well as on cancer therapy and the tumor microenvironment, and consider the implications of using TRPV1 agonists and antagonists for future research and potential therapeutic approaches.     

Type III Nrg1 back signaling enhances functional TRPV1 along sensory axons contributing to basal and inflammatory thermal pain sensation

Type III Nrg1, a member of the Nrg1 family of signaling proteins, is expressed in sensory neurons, where it can signal in a bi-directional manner via interactions with the ErbB family of receptor tyrosine kinases (ErbB RTKs). Type III Nrg1 signaling as a receptor (Type III Nrg1 back signaling) can acutely activate phosphatidylinositol-3-kinase (PtdIns3K) signaling, as well as regulate levels of α7* nicotinic acetylcholine receptors, along sensory axons. Transient receptor potential vanilloid 1 (TRPV1) is a cation-permeable ion channel found in primary sensory neurons that is necessary for the detection of thermal pain and for the development of thermal hypersensitivity to pain under inflammatory conditions. Cell surface expression of TRPV1 can be enhanced by activation of PtdIns3K, making it a potential target for regulation by Type III Nrg1. We now show that Type III Nrg1 signaling in sensory neurons affects functional axonal TRPV1 in a PtdIns3K-dependent manner. Furthermore, mice heterozygous for Type III Nrg1 have specific deficits in their ability to respond to noxious thermal stimuli and to develop capsaicin-induced thermal hypersensitivity to pain. Cumulatively, these results implicate Type III Nrg1 as a novel regulator of TRPV1 and a molecular mediator of nociceptive function.     

Sensation of TRPV1 via 5-hydroxytryptamine signaling modulates pain hypersensitivity in a 6-hydroxydopamine induced mice model of Parkinson’s disease

Parkinson’s disease (PD) related pain can be assigned to either nociceptive pain or neuropathic pain, in which Transient receptor potential vanilloid 1 (TRPV1) has been demonstrated to play a pivotal role. Yet little research has examined possible involvement of TRPV1 in pain in PD. Here, we show that TRPV1 is highly expressed in PD and blocking TRPV1 can alleviate pain in PD. The level of TRPV1 in 6-OHDA induced semi mice model of PD was evaluated. The effect of TRPV1 and involved serotonin (5-HT) was also examined in the model. Unilateral injection of 6-OHDA in striatum significantly decreased thermal pain threshold and induced mechanical allodynia without changes in conditioned place preference. Immunostaining revealed that great increased expression in TRPV1 in the Vc of 6-OHDA lesioned mice compared with sham mice. TRPV1 sensitization was maintained by 5-HT/5-HT3A. In 6-OHDA-lesioned mice model of PD, TRPV1 sensitization might be implicated in the maintenance of behavioral hypersensitivity by enhanced descending 5-HT pain facilitation and dorsal horn 5-HT3AR mechanism.