Discovery of small molecule antagonists of TRPV1

Small molecule antagonists of the vanilloid receptor 1 (TRPV1, also known as VR1) are disclosed. Ureas such as 5 (SB-452533) were used to explore the structure activity relationship with several potent analogues identified. Pharmacological studies using electrophysiological and FLIPR Ca(2+) based assays showed compound 5 was an antagonist versus capsaicin, noxious heat and acid mediated activation of TRPV1. Study of a quaternary salt of 5 supports a mode of action in which compounds from this series cause inhibition via an extracellularly accessible binding site on the TRPV1 receptor.     

Do TRPV1 antagonists increase the risk for skin tumourigenesis? A collaborative in vitro and in vivo assessment

A recent hypothesis suggesting that the pharmacological target TRPV1 (transient receptor potential vanilloid subfamily, member 1) may function as a tumour suppressor, which potentially impacts the development of TRPV1 antagonist therapeutics for a range of conditions. However, little is known about the long-term physiologic effects of TRPV1 blockade in the skin. In vitro and in vivo studies suggested that the potent TRPV1 competitive antagonist AMG-9810 promoted proliferation in N/TERT1 cells (telomerase-immortalised primary human keratinocytes 1) and tumour development in mouse skin that was mediated through EGFR/Akt/mTOR signalling. We attempted to reproduce the reported in vitro and in vivo findings to further explore this hypothesis to understand the underlying mechanism and the risk associated with TRPV1 antagonism in the skin. In vitro proliferation studies using multiple methods and topical application with AMG-9810 and structurally similar TRPV1 antagonists such as SB-705498 and PAC-14028 were performed. Although we confirmed expression of TRPV1 in primary human epidermal keratinocytes (HEKn) and spontaneously immortalised human keratinocytes (HaCaT), we were unable to demonstrate cell proliferation in either cell type or any clear evidence of increased expression of proteins in the EGFR/Akt/mTOR signalling pathway with these molecules. We were also unable to demonstrate skin tumour promotion or underlying molecular mechanisms involved in the EGFR/Akt/mTOR signalling pathway in a single-dose and two-stage carcinogenesis mouse study treated with TRPV1 antagonists. In conclusion, our data suggest that inhibiting the pharmacological function of TRPV1 in skin by specific antagonists has not been considered to be indicative of skin tumour development.     

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.     

Non-Competitive but pH-Dependent Action of SB-366791 on Proton-Induced Activation of TRPV1 Receptors

The transient receptor potential vanilloid type 1 (TRPV1) is a nonselective cation channel gated by numerous chemical and physical stimuli (protons, capsaicin, heat, etc). TRPV1 receptors are important integrators of multiple noxious and inflammatory signals in vertebrates. Modulation of TRPV1 receptors activity is considered to be a promising strategy for pain treatment. SB-366791 is a TRPV1 antagonist that demonstrates good analgesic effects in various models of pain. Molecular mechanisms of the SB-366791 action on TRPV1 are not clear. It antagonizes capsaicin activation in a competitive manner, but the data on its action in the case of activation by protons are controversial. Here we studied effects of SB-366791 when TRPV1 receptors are activated by acidification. We carried out patch-clamp experiments (voltage-clamp mode) on cultured CHO cells stably expressing rat TRPV1 receptors. The whole-cell proton-evoked currents were reduced in the presence of SB-366791. Concentration dependencies of the inhibitory effect of SB-366791 were studied at different pH values. Stronger acidification reduced the maximum effect of SB-366791, while the IC₅₀ values were virtually unaffected. Thus, SB-366791 acts in a non-competitive but pH-dependent way. Probably, there is an allosteric interplay between proton- and capsaicin-binding sites.     

Alpha-methylation at benzylic fragment of N-aryl-N'-benzyl ureas provides TRPV1 antagonists with better pharmacokinetic properties and higher efficacy in inflammatory pain model

SAR studies for N-aryl-N'-benzyl urea class of TRPV1 antagonists have been extended to cover alpha-benzyl alkylation. Alkylated compounds showed weaker in vitro potencies in blocking capsaicin activation of TRPV1 receptor, but possessed improved pharmacokinetic properties. Further structural manipulations that included replacement of isoquinoline core with indazole and isolation of single enantiomer led to TRPV1 antagonists like (R)-16a with superior pharmacokinetic properties and greater potency in animal model of inflammatory pain.     

Identification and characterization of novel TRPV4 modulators

TRPV4, a close relative of the vanilloid receptor TRPV1, is activated by diverse modalities such as endogenous lipid ligands, hypotonicity, protein kinases and, possibly, mechanical inputs. While its multiple roles in vivo are being explored with KO mice and selective agonists, there is a dearth of selective antagonists available to examine TRPV4 function. Herein we detail the use of a focused library of commercial compounds in order to identify RN-1747 and RN-1734, a pair of structurally related small molecules endowed with TRPV4 agonist and antagonist properties, respectively. Their activities against human, rat and mouse TRPV4 were characterized using electrophysiology and intracellular calcium influx. Significantly, antagonist RN-1734 was observed to completely inhibit both ligand- and hypotonicity-activated TRPV4. In addition, RN-1734 was found to be selective for TRPV4 in a TRP selectivity panel including TRPV1, TRPV3 and TRPM8, and could thus be a valuable pharmacological probe for TRPV4 studies.     

Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): effect of cAMP, cannabinoid CB(1) receptors and endovanilloids

The transient receptor potential channel of melastatin type 8 (TRPM8), which is gated by low (<25 degrees C) temperature and chemical compounds, is regulated by protein kinase C-mediated phosphorylation in a way opposite to that observed with the transient receptor potential channel of vanilloid type 1 (TRPV1), i.e. by being desensitized and not sensitized. As TRPV1 is sensitized also by protein kinase A (PKA)-mediated phosphorylation, we investigated the effect of two activators of the PKA pathway, 8-Br-cAMP and forskolin, on the activity of menthol and icilin at TRPM8 in HEK-293 cells stably overexpressing the channel (TRPM8-HEK-293 cells). We also studied the effect on TRPM8 of: (1) a series of compounds previously shown to activate or antagonize TRPV1, and (2) co-stimulation of transiently co-expressed cannabinoid CB(1) receptors. Both 8-Br-cAMP (100 microM) and forskolin (10 microM) right-shifted the dose-response curves for the TRPM8-mediated effect of icilin and menthol on intracellular Ca(2+). The inhibitory effects of 8-Br-cAMP and forskolin were attenuated by the selective PKA inhibitor Rp-cAMP-S. Stimulation of human CB(1) receptors transiently co-expressed in TRPM8-HEK-293 cells also inhibited TRPM8 response to icilin. Finally, some TRPV1 agonists and antagonists, but not iodinated antagonists, antagonized icilin- and much less so menthol-, induced TRPM8 activation. Importantly, the endovanilloids/endocannabinoids, anandamide and NADA, also antagonized TRPM8 at submicromolar concentrations. Although these findings need to be confirmed by experiments directly measuring TRPM8 activity in natively TRPM8-expressing cells, they support the notion that the same regulatory events have opposing actions on TRPM8 and TRPV1 receptors and identify anandamide and NADA as the first potential endogenous functional antagonists of TRPM8 channels.     

2-Alkyl/alkenyl substituted pyridine C-region analogues of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides as highly potent TRPV1 antagonists

A series of 2-alkyl/alkenyl pyridine C-region derivatives of 2-(3-fluoro-4-methylsulfonylaminophenyl)propanamides were investigated as hTRPV1 antagonists. Multiple compounds showed excellent and stereospecific TRPV1 antagonism with better potency than previous lead 2. Among them, compound 15f demonstrated a strong analgesic profile in a rat neuropathic pain model and blocked capsaicin-induced hypothermia in a dose-dependent manner. Docking analysis of (S)-15f with our hTRPV1 homology model provided insight into its specific binding mode.     

Chroman and tetrahydroquinoline ureas as potent TRPV1 antagonists

Novel chroman and tetrahydroquinoline ureas were synthesized and evaluated for their activity as TRPV1 antagonists. It was found that aryl substituents on the 7- or 8-position of both bicyclic scaffolds imparted the best in vitro potency at TRPV1. The most potent chroman ureas were assessed in chronic and acute pain models, and compounds with the ability to cross the blood-brain barrier were shown to be highly efficacious. The tetrahydroquinoline ureas were found to be potent CYP3A4 inhibitors, but replacement of bulky substituents at the nitrogen atom of the tetrahydroisoquinoline moiety with small groups such as methyl can minimize the inhibition.     

Potent and orally efficacious benzothiazole amides as TRPV1 antagonists

Benzothiazole amides were identified as TRPV1 antagonists from high throughput screening using recombinant human TRPV1 receptor and structure-activity relationships were explored to pinpoint key pharmacophore interactions. By increasing aqueous solubility, through the attachment of polar groups to the benzothiazole core, and enhancing metabolic stability, by blocking metabolic sites, the drug-like properties and pharmokinetic profiles of benzothiazole compounds were sufficiently optimized such that their therapeutic potential could be verified in rat pharmacological models of pain.     

Tetrahydro-naphthols as orally available TRPV1 inhibitors

Starting from a naphthol-based lead series with low oral bioavailability, we have identified potent TRPV1 antagonists with oral bioavailability in rats. These compounds emerged from SAR studies aimed at replacing the lead's phenol structure whilst maintaining potency. Compound rac-6a is an orally available TRPV1 antagonist with single-digit nanomolar activity. The enantiomers show a low eudismic ratio at the receptor level.     

A new era for the design of TRPV1 antagonists and agonists with the use of structural information and molecular docking of capsaicin-like compounds

The design of TRPV1 antagonists and agonists has reached a new era since TRPV1 structures at near-atomic resolution are available. Today, the ligand-binding forms of several classical antagonists and agonists are known; therefore, the specific role of key TRPV1’s residues in binding of ligands can be elucidated. It is possible to place the well-defined pharmacophore of TRPV1 ligands, conformed by head, neck, and tail groups, in the right pocket regions of TRPV1. It will allow a more thorough use of molecular modelling methods to conduct more effective rational drug design protocols. In this work, important points about the interactions between TRPV1 and capsaicin-like compounds are spelled out, based on the known pharmacophore of the ligands and the already available TRPV1 structures. These points must be addressed to generate reliable poses of novel candidates and should be considered during the design of novel TRPV1 antagonists and agonists.     

The chimeric approach reveals that differences in the TRPV1 pore domain determine species-specific sensitivity to block of heat activation

The capsaicin-, heat-, and proton-activated ion channel TRPV1, a member of the transient receptor potential cation channel family is a polymodal nociceptor. For almost a decade, TRPV1 has been explored by the pharmaceutical industry as a potential target for example for pain conditions. Antagonists which block TRPV1 activation by capsaicin, heat, and protons were developed by a number of pharmaceutical companies. The unexpected finding of hyperthermia as an on-target side effect in clinical studies using polymodal TRPV1 antagonists has prompted companies to search for ways to circumvent hyperthermia, for example by the development of modality-selective antagonists. The significant lack of consistency of the pharmacology of many TRPV1 antagonists across different species has been a further obstacle. JYL-1421 for example was shown to block capsaicin and heat responses in human and monkey TRPV1 while it was largely ineffective in blocking heat responses in rat TRPV1. These findings suggested structural dissimilarities between different TRPV1 species relevant for small compound antagonism for example of heat activation. Using a chimeric approach (human and rat TRPV1) in combination with a novel FLIPR-based heat activation assay and patch-clamp electrophysiology we have identified the pore region as being strongly linked to the observed species differences. We demonstrate that by exchanging the pore domains JYL-1421, which is modality-selective in rat can be made modality-selective in human TRPV1 and vice-versa.     

4-(2-Pyridyl)piperazine-1-benzimidazoles as potent TRPV1 antagonists

A series of 4-(2-pyridyl)piperazine-1-benzimidazole analogues based on compound 1 was synthesized and evaluated for TRPV1 antagonist activity in capsaicin-induced (CAP) and pH5.5-induced (pH) FLIPR assays in a human TRPV1-expressing HEK293 cell line. Potent TRPV1 antagonists were identified through SAR studies. From these studies, several antagonists were found, with IC(50) values ranging from 32 nM to approximately 5000 nM. Among these, 11 [IC(50)=90 nM (CAP) and 104 nM (pH)] was further evaluated and found to be orally available in rats (F%=19.7).     

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.     

Procognitive 5-HT6 antagonists in the rat forced swimming test: Potential therapeutic utility in mood disorders associated with Alzheimer's disease

Aims5-HT₆ receptor subtype is predominantly expressed in the brain, and preclinical evidence suggests its potential role in the cognitive function. Brain microdialysis studies demonstrated that 5-HT₆ antagonists enhance not only cholinergic but also monoaminergic neurotransmission, a property that may differentiate from acetylcholine esterase (AChE) inhibitors such as donepezil. In this study we compared the antidepressant-like effects of 5-HT₆ antagonists with donepezil to determine whether their different effects on monoamines are behaviorally relevant.Main methodsSelective 5-HT₆ antagonists (SB-399885 and SB-271046) and donepezil were evaluated in the rat forced swimming test since this is known to identify drugs such as antidepressants which can increase brain monoamine levels. Binding assay was undertaken by using [¹²⁵I]SB-258585 to measure brain 5-HT₆ receptor occupancy.Key findingsSystemic administration of SB-399885 (3 and 10 mg/kg, i.p.) and SB-271046 (10 and 30 mg/kg, i.p.) produced a significant reduction of immobility time in the rat forced swimming test with a similar profile in terms of 5-HT₆ receptor occupancy (62 and 96% for 3 and 10 mg/kg SB-399885 respectively; 56 and 84% for 10 and 30 mg/kg SB-271046 respectively). In contrast, donepezil (0.5 and 1 mg/kg i.p.) did not show any effects in this model.SignificanceThese data suggest that 5-HT₆ antagonists, at doses corresponding to those occupy central 5-HT₆ receptors, could have an antidepressive effect in humans. This may differentiate 5-HT₆ antagonists from AChE inhibitors with respect to the mood control in the symptomatic treatment of Alzheimer's disease.     

Alpha-substituted N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues as potent and stereospecific TRPV1 antagonists

A series of alpha-substituted N-(4-tert-butylbenzyl)-N'-[4-(methylsulfonylamino)benzyl]thiourea analogues have been investigated as TRPV1 receptor antagonists. alpha-Methyl substituted analogues showed potent and stereospecific antagonism to the action of capsaicin on rat TRPV1 heterologously expressed in Chinese hamster ovary cells. In particular, compounds 14 and 18, which possess the R-configuration, exhibited excellent potencies (respectively, K(i)=41 and 39.2 nM and K(i(ant))=4.5 and 37 nM).     

Role of TRPV receptors in respiratory diseases

Transient receptor potential vanilloid type channels (TRPVs) are expressed in several cell types in human and animal lungs. Increasing evidence has demonstrated important roles of these cation channels, particularly TRPV1 and TRPV4, in the regulation of airway function. These TRPVs can be activated by a number of endogenous substances (hydrogen ion, certain lipoxygenase products, etc.) and changes in physiological conditions (e.g., temperature, osmolarity, etc.). Activation of these channels can evoke Ca(2+) influx and excitation of the neuron. TRPV1 channels are generally expressed in non-myelinated afferents innervating the airways and lungs, which also contain sensory neuropeptides such as tachykinins. Upon stimulation, these sensory nerves elicit centrally-mediated reflex responses as well as local release of tachykinins, and result in cough, airway irritation, reflex bronchoconstriction and neurogenic inflammation in the airways. Recent studies clearly demonstrated that the excitability of TRPV1 channels is up-regulated by certain autacoids (e.g., prostaglandin E(2), bradykinin) released during airway inflammatory reaction. Under these conditions, the TRPV1 can be activated by a slight increase in airway temperature or tissue acidity. Indirect evidence also suggests that TRPV channels may play a part in the pathogenesis of certain respiratory diseases such as asthma and chronic cough. Therefore, the potential use of TRPV antagonists as a novel therapy for these diseases certainly merits further investigation.     

Diaryl urea analogues of SB-334867 as orexin-1 receptor antagonists

As a part of our program to develop OX1-CB1 bivalent ligands, we required a better understanding of the basic structure-activity relationships (SARs) of orexin antagonists. A series of SB-334867 analogues were synthesized and evaluated in calcium mobilization assays. SAR results suggest that the 2-methylbenzoxazole moiety may be replaced with a disubstituted 4-aminophenyl group without loss of activity and an electron-deficient system is generally preferred at the 1,5-naphthyridine moiety for OX1 antagonist activity. In particular, substitution of larger potential linkers such as n-hexyl provided compound 33 with equivalent activity at the OX1 receptor compared to the lead compound SB-334867. These compounds should be of value in the development of ligands targeting the orexin-1 receptor and its potential heterodimers.     

TRP channels and analgesia

Since cloning and characterizing the first nociceptive ion channel Transient Receptor Potential (TRP) Vanilloid 1 (TRPV1), other TRP channels involved in nociception have been cloned and characterized, which include TRP Vanilloid 2 (TRPV2), TRP Vanilloid 3 (TRPV3), TRP Vanilloid 4 (TRPV4), TRP Ankyrin 1 (TRPA1) and TRP Melastatin 8 (TRPM8), more recently TRP Canonical 1, 5, 6 (TRPC1, 5, 6), TRP Melastatin 2 (TRPM2) and TRP Melastatin 3 (TRPM3). These channels are predominantly expressed in C and Aδ nociceptors and transmit noxious thermal, mechanical and chemical sensitivities. TRP channels are modulated by pro-inflammatory mediators, neuropeptides and cytokines. Significant advances have been made targeting these receptors either by antagonists or agonists to treat painful conditions. In this review, we will discuss TRP channels as targets for next generation analgesics and the side effects that may ensue as a result of blocking/activating these receptors, because they are also involved in physiological functions such as release of vasoactive neuropeptides and regulation of vascular tone, maintenance of the body temperature, gastrointestinal motility, urinary bladder control, etc.