Vanilloid Compounds Research Articles

Role of phosphorylation and vanilloid ligand structure in ligand-dependent differential activations of transient receptor potential vanilloid 1.

Vanilloid analogs, which can activate transient receptor potential vanilloid 1 (TRPV1), have been classified into two types based on susceptibility to forskolin (FSK). Treatment of cells expressing TRPV1 with FSK enhances TRPV1 responses to capsaicin-type ligands while diminishing the responses to eugenol-type ligands. In this study, we determined the effect of FSK on the activation of TRPV1 stimulated with vanilloid ligands, through the influx of Ca2+ in HEK293T cells expressing TRPV1. Our findings suggest that the effects of FSK can be attributed to the phosphorylation of TRPV1, as evidenced by using a protein kinase A inhibitor and TRPV1 mutants at potential phosphorylation sites. Furthermore, we examined the structure-activity relationship of 13 vanilloid analogs. Our results indicated that vanilloid compounds could be classified into three types, that is the previously reported two types and a novel type of 10-shogaol, by which TRPV1 activation was insusceptible to the FSK treatment.

Curcumin Stereoisomer, Cis-Trans Curcumin, as a Novel Ligand to A1 and A3 Adenosine Receptors.

Adenosine receptors (ARs) are being explored to generate non-opioid pain therapeutics. Vanilloid compounds, curcumin, capsaicin, and vanillin possess antinociceptive properties through their interactions with the transient receptor potential channel family. However, their binding with adenosine receptors has not been well studied. The hypothesis in this study was that a vanilloid compound, cis-trans curcumin (CTCUR), binds to each of the two Gi-linked AR subtypes (A1AR and A3AR). CTCUR was synthesized from curcumin (CUR) using the cavitand-mediated photoisomerization technique. The cell lines transfected with the specific receptor (A1AR or A3AR) were treated with CTCUR or CUR and the binding was analyzed using competitive assays, confocal microscopy, and docking. The binding assays and molecular docking indicated that CTCUR had Ki values of 306 nM (A1AR) and 400 nM (A3AR). These values suggest that CTCUR is selective for Gi-linked ARs (A1AR or A3AR) over Gs-linked ARs (A2AAR or A2BAR), based on our previous published research. In addition, the docking showed that CTCUR binds to the toggle switch domain of ARs. Curcumin (CUR) did not exhibit binding at any of these receptors. In summary, CTCUR and other modifications of CUR can be developed as novel therapeutic ligands for the Gi-linked ARs (A1AR and A3AR) involved with pain and cancer.

Anticancer Activity of Region B Capsaicin Analogs.

The heterocyclic vanilloid compound capsaicin is responsible for the spicy and pungent flavor of chili peppers. Several convergent studies have shown that capsaicin suppresses the growth of multiple human cancers. Apart from capsaicin, natural and synthetic capsaicin-like compounds display growth suppressive activity in human cancers. The pharmacophore of capsaicin is comprised of three regions, namely region A (the aromatic ring), region B (the amide bond), and region C (the side chain). The present manuscript describes the isolation and synthesis of capsaicin analogs which have structural modifications in region B of the molecule. Furthermore, the pharmacokinetic properties, anticancer activity of region B capsaicin analogs, as well as the signaling pathways (underlying the growth-inhibitory effects of region B capsaicin analogs) have also been described. The discovery of novel, second-generation region B capsaicin analogs may foster the hope of innovative nutrition-based combination therapies in human cancers.

Reusable Electronic Tongue Based on Transient Receptor Potential Vanilloid 1 Nanodisc-Conjugated Graphene Field-Effect Transistor for a Spiciness-Related Pain Evaluation.

The sense of spiciness is related to the stimulation of vanilloid compounds contained in the foods. Although, the spiciness is commonly considered as the part of taste, it is more classified to the sense of pain stimulated on a tongue, namely, pungency, which is described as a tingling or burning on the tongue. Herein, first, a reusable electronic tongue based on a transient receptor potential vanilloid 1 (TRPV1) nanodisc conjugated graphene field-effect transistor is fabricated and spiciness-related pain evaluation with reusable electrode is demonstrated. The pungent compound reactive receptor TRPV1 is synthesized in the form of nanodiscs to maintain stability and reusability. The newly developed platform shows highly selective and sensitive performance toward each spiciness related vanilloid compound repeatably: 1 aM capsaicin, 10 aM dihydrocapsaicin, 1 fM piperine, 10nM allicin, and 1 pM AITC. The binding mechanism is also examined by simulation. Furthermore, the elimination of the burning sensation on the tongue after eating spicy foods is not investigated. Based on the synthesis of micelles composed of casein protein (which is contained in skim milk) that remove pungent compounds bound to TRPV1 nanodisc, the deactivation of TRPV1 is investigated, and the electrode is reusable that mimics electronic tongue.

Highly Efficient Real-Time TRPV1 Screening Methodology for Effective Drug Candidates.

Transient receptor potential vanilloid 1 (TRPV1) agonists that bind to the vanilloid pocket are being actively studied in the pharmaceutical industry to develop novel treatments for chronic pain and cancer. To discover synthetic vanilloids without the side effect of capsaicin, a time-consuming process of drug candidate selection is essential to a myriad of chemical compounds. Herein, we propose a novel approach to field-effect transistors for the fast and facile screening of lead vanilloid compounds for the development of TRPV1-targeting medications. The graphene field-effect transistor was fabricated with human TRPV1 receptor protein as the bioprobe, and various analyses (SEM, Raman, and FT-IR) were utilized to verify successful manufacture. Simulations of TRPV1 with capsaicin, olvanil, and arvanil were conducted using AutoDock Vina/PyMOL to confirm the binding affinity. The interaction of the ligands with TRPV1 was detected via the fabricated platform, and the collected responses corresponded to the simulation analysis.

A Novel Curcumin Isomer, cis‐transCurcumin, Activates Adenosine Receptor Subtypes A1 And A3: Potential for Pain Therapy

A wide variety of plants produce compounds that contain the 4‐hydroxy‐3‐methoxybenzyl or “vanillyl” group. These vanilloid compounds include such well‐known food molecules as vanillin, capsaicin, gingerol, and curcumin. Vanilloid compounds have been widely studied for their medicinal properties, and one property that most of them have in common is the ability to induce analgesia. Studies have indicated multiple potential cellular mechanisms for this analgesic activity, including TRPV1 receptors, opioid receptors, and adenosine receptors (ARs). There are only a few studies on the interaction of vanilloid compounds with ARs, and to our knowledge no previous study has measured the binding affinity of any vanilloid compound with either of the two AR subtypes that are linked to the inhibitory Gαsubunit (subtypes A1and A3). Our study objective was to assess the binding affinity and receptor activation of a novel curcumin isomer, cis‐transcurcumin (CTCUR), at AR subtypes A1and A3. Since agonism of these subtypes is associated with analgesia, we hypothesized that CTCUR would be an agonist at AR subtypes A1 and A3. Two lines of Chinese hamster ovary cells were used, each transfected to overexpress one of the AR subtypes. Cell survival assays were performed to assess toxicity. Competitive binding assays and confocal microscopy were performed to measure binding affinity. Computer docking was also performed, to assess location of binding. Assays of intracellular cAMP levels were performed to measure receptor activation. Survival results indicate that CTCUR is not toxic to CHO cells at any of the concentrations tested (the highest being 100 μM). Competition assay results indicate that CTCUR binds to subtype A1 with a Ki of 306 nM and to subtype A3 with a Ki of 400 nM. Microscopy results confirm sub‐micromolar affinity for A1, but not for A3. Docking results confirm sub‐micromolar affinity for both subtypes, and furthermore indicate that CTCUR binds to the “toggle switch” domain of α helix 6 that is important for receptor activation. Intracellular cAMP results indicate that CTCUR acts as an agonist of ARs. Thus, we fail to reject our hypothesis. These results are of interest because they provide the first in vitro confirmation of the hypothesis that vanilloid compounds can activate ARs, in accordance with the results of previous studies in vivo. Vanilloid compounds that activate ARs are therefore worthy of further study, as a potential class of analgesic therapeutics.

Heat-dependent opening of TRPV1 in the presence of capsaicin

Noxious heat (> 42°C) and capsaicin are two well-known stimuli for nociception. Our ability to sense noxious heat and spiciness is owed to the transient receptor potential vanilloid member 1 (TRPV1), a calcium-permeable ion channel expressed in primary sensory nerve terminals. TRPV1 also serves as a sensor for other vanilloid compounds and is a member of the TRP channel superfamily. Here we present six high-resolution cryo-EM structures of apo and capsaicin-bound full-length TRPV1 reconstituted into lipid nanodiscs over a range of temperatures.

Profile of David Julius and Ardem Patapoutian: 2021 Nobel Laureates in Physiology or Medicine

Three apparently unrelated findings led to the discovery of the temperature receptors: a better understanding of pain-sensing, the molecular nature of the channels in charge of touch and proprioception, and the 2021 Nobel Prize in Physiology or Medicine bestowed to David Julius and Ardem Patapoutian. In 1953, state-of-the-art research in temperature sensing concluded that temperature afferent fibers came in two flavors, “cold” and “warm,” characterized by discharging by a lowering in temperature and an increase in temperature, respectively (1). Hensel and Zotterman had previously described the sensitizing effect of menthol upon cold receptors (2). It is also an old observation that acute application of capsaicin, the active component of chili peppers, causes intense pain, and that its repeated administration renders animals insensitive to the action of this vanilloid compound (3). The fact that capsaicin induces a depolarization of ventral root neurons from the isolated rat spinal cord suggested to Theriault et al. (4) that capsaicin’s acute and chronic effects on nociception could be explained by a direct action on primary sensory neurons. On the other hand, McCarter et al. (5) demonstrated the existence of mechanosensitive currents in rat dorsal root ganglion (DRG) cells, clearly pointing to the existence of mechanosensitive ion channels as being responsible for the sense of touch. David Julius. Image credit: University of California, San Francisco. Since temperature is an “intensive” thermodynamic parameter (i.e., one that does not depend on the size of the system), it was difficult to think of specialized temperature receptors. However, the seminal 1997 Nature paper from Julius’ laboratory demonstrated that living beings have evolved to transduce all stimuli coming from the external world using ion channels, including temperature (6). Caterina et al. (6) used an expression cloning strategy to search for the capsaicin receptor and found a single clone in which capsaicin induced a significant … [↵][1]1To whom correspondence may be addressed. Email: ramon.latorre{at}uv.cl. [1]: #xref-corresp-1-1

Capsaicin Attenuates Lipopolysaccharide-Induced Inflammation and Barrier Dysfunction in Intestinal Porcine Epithelial Cell Line-J2.

Capsaicin is a spicy, highly pungent, colorless, vanilloid compound found in chili peppers with anti-inflammatory, antioxidant, anti-cancer, and analgesic properties. However, the protective effects of capsaicin on the pig intestine during inflammation are yet to be explored. This study investigated the effects of capsaicin on the gut inflammatory response, intestinal epithelial integrity, and gene expression level of nutrient transporters in a model of lipopolysaccharide (LPS)-induced inflammation in non-differentiated intestinal porcine epithelial cell line-J2 (IPEC-J2). The results showed that the pre-treatment of cells with capsaicin (100 μM) significantly decreased the gene expression and secretion of proinflammatory cytokines induced by LPS through Toll-like receptor 4 (TLR4)/NF-κB signaling pathway. In addition, pre-treatment of cells with capsaicin also increased both gene and protein abundance of tight junction proteins. Furthermore, pre-treatment cells with capsaicin significantly increased trans-epithelial electrical resistance (TEER) and decreased permeability of fluorescein isothiocyanate-dextran (FD4) from the apical side to the basolateral side compared with the control (P < 0.05). Additionally, pre-treatment of cells with capsaicin upregulated the mRNA abundance of nutrients transporters such as Na+/glucose cotransporter 1 (SGLT1). These results suggested that capsaicin could attenuate LPS-induced inflammation response through TLR4/NF-κB pathway and improve barrier integrity and glucose absorption.

Novel piperazine urea derivatives as highly potent transient receptor potential vanilloid 1 (TRPV1) antagonists

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel with high permeability to Ca2+, which can be activated by low pH, noxious heat and vanilloid compounds such as capsaicin. TRPV1 has been proved to be very important in the process of pain production and is considered to be a highly effective analgesic target. In this work, three series of new piperazine urea TRPV1 antagonists were designed, synthesized and evaluated based on classical TRPV1 antagonists BCTC and GRT12360. Among them, N-(4,6-dimethylpyridin-2-yl)-4-(2-(pyrrolidin-1-yl)benzyl)piperazine-1-carboxamide (5ac) was finally identified, which had excellent TRPV1 antagonistic activity (IC50 (CAP) = 9.80 nM), good bioavailability and did not cause side effects of hyperthermia. In the study of molecular docking, the compound 5ac fitted well with the amino acid residues on rTRPV1 through hydrophobic interaction. Collectively, compound 5ac is an efficient TRPV1 antagonist and can be used as a candidate for the development of analgesic drugs.

TRPV1-Targeted Drugs in Development for Human Pain Conditions.

The transient receptor potential vanilloid-1 (TRPV1) is a non-specific cation channel known for its sensitivity to pungent vanilloid compound (i.e. capsaicin) and noxious stimuli, including heat, low pH or inflammatory mediators. TRPV1 is found in the somatosensory system, particularly primary afferent neurons that respond to damaging or potentially damaging stimuli (nociceptors). Stimulation of TRPV1 evokes a burning sensation, reflecting a central role of the channel in pain. Pharmacological and genetic studies have validated TRPV1 as a therapeutic target in several preclinical models of chronic pain, including cancer, neuropathic, postoperative and musculoskeletal pain. While antagonists of TRPV1 were found to be a valuable addition to the pain therapeutic toolbox, their clinical use has been limited by detrimental side effects, such as hyperthermia. In contrast, capsaicin induces a prolonged defunctionalisation of nociceptors and thus opened the door to the development of a new class of therapeutics with long-lasting pain-relieving effects. Here we review the list of TRPV1 agonists undergoing clinical trials for chronic pain management, and discuss new indications, formulations or combination therapies being explored for capsaicin. While the analgesic pharmacopeia for chronic pain patients is ancient and poorly effective, modern TRPV1-targeted drugs could rapidly become available as the next generation of analgesics for a broad spectrum of pain conditions.

Evidence that the TRPV1 S1-S4 membrane domain contributes to thermosensing

Sensing and responding to temperature is crucial in biology. The TRPV1 ion channel is a well-studied heat-sensing receptor that is also activated by vanilloid compounds, including capsaicin. Despite significant interest, the molecular underpinnings of thermosensing have remained elusive. The TRPV1 S1-S4 membrane domain couples chemical ligand binding to the pore domain during channel gating. Here we show that the S1-S4 domain also significantly contributes to thermosensing and couples to heat-activated gating. Evaluation of the isolated human TRPV1 S1-S4 domain by solution NMR, far-UV CD, and intrinsic fluorescence shows that this domain undergoes a non-denaturing temperature-dependent transition with a high thermosensitivity. Further NMR characterization of the temperature-dependent conformational changes suggests the contribution of the S1-S4 domain to thermosensing shares features with known coupling mechanisms between this domain with ligand and pH activation. Taken together, this study shows that the TRPV1 S1-S4 domain contributes to TRPV1 temperature-dependent activation.

Capsaicin and Its Analogues Impede Nocifensive Response of Caenorhabditis elegans to Noxious Heat.

Capsaicin is the most abundant pungent molecule identified in red chili peppers, and it is widely used for food flavoring, in pepper spray for self-defense devices and recently in ointments for the relief of neuropathic pain. Capsaicin and several other related vanilloid compounds are secondary plant metabolites. Capsaicin is a selective agonist of the transient receptor potential channel, vanilloid subfamily member 1 (TRPV1). After exposition to vanilloid solution, Caenorhabditis elegans wild type (N2) and mutants were placed on petri dishes divided in quadrants for heat stimulation. Thermal avoidance index was used to phenotype each tested C. elegans experimental groups. The data revealed for the first-time that capsaicin can impede nocifensive response of C. elegans to noxious heat (32-35 °C) following a sustained exposition. The effect was reversed 6 h post capsaicin exposition. Additionally, we identified the capsaicin target, the C. elegans transient receptor potential channel OCR-2 and not OSM-9. Further experiments also undoubtedly revealed anti-nociceptive effect for capsaicin analogues, including olvanil, gingerol, shogaol and curcumin.

Contributions of the Transmembrane Domain to Heat Activation of Human TRPV1

Temperature sensation and regulation are crucial physiological processes in biology. Transient receptor potential vanilloid-1 (TRPV1) is a highly studied cation channel that is largely responsible for thermosensation, as well as nociception. Along with being activated by high temperatures, the receptor is also activated by capsaicin, the pungent vanilloid compound in chili peppers. Chemical ligand binding has been well-characterized functionally and structurally, but despite significant interest, the molecular mechanisms behind thermosensation have remained unclear. Studies outside of our lab have indicated that the human TRPV1 pore domain (hV1-PD) is a factor in the thermosensitivity of human TRPV1, and additional studies conducted within our lab of the human TRPV1 sensing domain (hV1-SD) have shown that this domain contributes significantly to the heat activation mechanism of human TRPV1. These data illuminate a need for further investigation of human TRPV1 to elucidate the molecular underpinnings of thermosensation. Furthermore, these studies point to the human TRPV1 transmembrane domain (hV1-TMD) as being a critical region within the channel responsible for heat activation. This research utilizes a variety biophysical techniques including negative-stain electron microscopy (EM) and solution nuclear magnetic resonance (NMR) spectroscopy to probe the hV1-TMD for its contributions to heat activation of the human TRPV1 channel.

6-Paradol and its glucoside improve memory disorder in mice.

In this study, the effects of 6-paradol (6P) and 6-paradol-β-glucoside (6PG) on neuritogenesis were investigated using PC12 cells. Treatment with 200 μM 6P or 6PG and nerve growth factor (NGF) (5 ng mL-1) increased the number of elongated dendritic cells 8.7 and 5.4 times, respectively, compared to that with NGF (5 ng mL-1) treatment alone. 6P and 6PG did not stimulate the phosphorylation of extracellular regulated protein kinases (ERK)1/2 and cAMP response element-binding protein (CREB) in the tropomyosin receptor kinase A (TrkA) pathway as their activities were suppressed by the pathway inhibitor, k252a. 6P enhanced Ca2+ influx into the cells, whereas 6PG had no effect on Ca2+ influx, although it stimulated PC12 cell differentiation. High-performance liquid chromatography (HPLC) analysis of 6PG in PC12 culture medium suggested that 6PG was deglycosylated to generate 6P, which exhibited the effect. Furthermore, the bioactivities of 6P and 6PG were investigated in mice, and the results revealed that they ameliorated short-term memory loss in animals during behavioral testing.

Capsaicin-Induced Impairment of Functional Network Dynamics in Mouse Hippocampus via a TrpV1 Receptor-Independent Pathway: Putative Involvement of Na+/K+-ATPase

The vanilloid compound capsaicin (Cp) is best known to bind to and activate the transient receptor potential vanilloid receptor-1 (TrpV1). A growing number of studies use capsaicin as a tool to study the role of TrpV1 in the central nervous system (CNS). Although most of capsaicin’s CNS effects have been reported to be mediated by TrpV1 activation, evidence exists that capsaicin can also trigger functional changes in hippocampal activity independently of TrpV1. Recently, we have reported that capsaicin induces impairment in hippocampal gamma oscillations via a TrpV1-independent pathway. Here, we dissect the underlying mechanisms of capsaicin-induced alterations to functional network dynamics. We found that capsaicin induces a reduction in action potential (AP) firing rate and a subsequent loss of synchronicity in pyramidal cell (PC) spiking activity in hippocampus. Moreover, capsaicin induces alterations in PC spike-timing since increased first-spike latency was observed after capsaicin treatment. First-spike latency can be regulated by the voltage-dependent potassium current D (ID) or Na+/K+-ATPase. Selective inhibition of ID via low 4-AP concentration and Na+/K+-ATPase using its blocker ouabain, we found that capsaicin effects on AP spike timing were completely inhibited by ouabain but not with 4-AP. In conclusion, our study shows that capsaicin in a TrpV1-independent manner and possibly involving Na+/K+-ATPase activity can impair cognition-relevant functional network dynamics such as gamma oscillations and provides important data regarding the use of capsaicin as a tool to study TrpV1 function in the CNS.

Structural mechanisms underlying activation of TRPV1 channels by pungent compounds in gingers.

Like chili peppers, gingers produce pungent stimuli by a group of vanilloid compounds that activate the nociceptive transient receptor potential vanilloid 1 (TRPV1) ion channel. How these compounds interact with TRPV1 remains unclear. We used computational structural modelling, functional tests (electrophysiology and calcium imaging), and mutagenesis to investigate the structural mechanisms underlying ligand-channel interactions. The potency of three principal pungent compounds from ginger -shogaol, gingerol, and zingerone-depends on the same two residues in the TRPV1 channel that form a hydrogen bond with the chili pepper pungent compound, capsaicin. Computational modelling revealed binding poses of these ginger compounds similar to those of capsaicin, including a "head-down tail-up" orientation, two specific hydrogen bonds, and important contributions of van der Waals interactions by the aliphatic tail. Our study also identified a novel horizontal binding pose of zingerone that allows it to directly interact with the channel pore when bound inside the ligand-binding pocket. These observations offer a molecular level explanation for how unique structures in the ginger compounds affect their channel activation potency. Mechanistic insights into the interactions of ginger compounds and the TRPV1 cation channel should help guide drug discovery efforts to modulate nociception.

Structure-activity relationship for vanilloid compounds from extract of Zingiber officinale var rubrum rhizomes: effect on extracellular melanogenesis inhibitory activity

The components in the methanol extract of Zingiber officinale var rubrum (red ginger) were isolated by a series of column chromatography and identified by Nuclear Magnetic Resonance (NMR) and Matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOFMS). Thirteen components, including eleven vanilloid compounds, were isolated from the extract. All the isolated components reduced extracellular melanin contents. Structure-activity relationship studies suggested that elongation of the carbon chain of shogaol and gingerol increased the extracellular melanogenesis inhibitory activity, and that the carbonyl and hydroxyl groups on the carbon chain in gingerol played an important role in this effect. In order to reveal the importance of the vanillyl group for the melanogenesis inhibitory activity, 6-gingerol (1) and 6-shogaol (5), which were the main compounds in the extract, were glucosylated and their effects were evaluated. The glucosylated 6-shogaol resulted in improved melanogenesis inhibitory activity, while the glucosylated 6-gingerol had no such effect. These results indicated different mechanisms for the melanogenesis inhibitory activity of the two vanilloid compounds.

Conserved Allosteric Pathways for Activation of TRPV3 Revealed through Engineering Vanilloid-Sensitivity

The Transient Receptor Potential Vanilloid 1 (TRPV) channel is activated by an array of stimuli, including heat and vanilloid compounds. The TRPV1 homologues TRPV2 and TRPV3 are also activated by heat, but sensitivity to vanilloids and many other agonists is not conserved among TRPV subfamily members. It was recently discovered that four mutations in TRPV2 are sufficient to render the channel sensitive to the TRPV1-specific vanilloid agonist resiniferatoxin (RTx). Here, we show that mutation of six residues in TRPV3 corresponding to the vanilloid site in TRPV1 is sufficient to engineer RTx binding. However, robust activation of TRPV3 by RTx requires facilitation of channel opening by introducing mutations in the pore, temperatures > 30°C, or sensitization with another agonist. Our results demonstrate that the energetics of channel activation can determine the apparent sensitivity to a stimulus and suggest that allosteric pathways for activation are conserved in the TRPV family.

Conserved allosteric pathways for activation of TRPV3 revealed through engineering vanilloid-sensitivity.

The Transient Receptor Potential Vanilloid 1 (TRPV) channel is activated by an array of stimuli, including heat and vanilloid compounds. The TRPV1 homologues TRPV2 and TRPV3 are also activated by heat, but sensitivity to vanilloids and many other agonists is not conserved among TRPV subfamily members. It was recently discovered that four mutations in TRPV2 are sufficient to render the channel sensitive to the TRPV1-specific vanilloid agonist resiniferatoxin (RTx). Here, we show that mutation of six residues in TRPV3 corresponding to the vanilloid site in TRPV1 is sufficient to engineer RTx binding. However, robust activation of TRPV3 by RTx requires facilitation of channel opening by introducing mutations in the pore, temperatures > 30°C, or sensitization with another agonist. Our results demonstrate that the energetics of channel activation can determine the apparent sensitivity to a stimulus and suggest that allosteric pathways for activation are conserved in the TRPV family.