Capsaicin Binding Research Articles

Genetic Influence on Capsaicin Tolerance: Precision Nutrition Implications for Obesity Handling.

It has been suggested that capsaicin (CAP), a major pungent component in chili peppers, can be used as an anti-obesity ingredient due to effects on energy metabolism, but evidence is not consistent. Genetics may account for differences in CAP tolerance and its impact on adiposity status. The aim of this study was to systematically review current evidence concerning the role of genetic polymorphisms influencing CAP tolerance. The present systematic review analyzed and synthesized available evidence concerning associations between genetic polymorphisms and CAP tolerance following the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines. Databases such as PubMed/MEDLINE, Cochrane, Scopus, Google Scholar, SciELO, and LILACS were screened. Out of 228 publications identified, only 6 meet inclusion criteria and were finally included in the final report. Overall, a total of 28 single nucleotide polymorphisms were associated with several CAP tolerance traits including sensitivity to burning/stinging, heat pain, and cough reactions, and detection of bitter taste thresholds. These genetic variants were located within 6 genes involved in key physiological processes such synthesis of tetrahydrobiopterin and nitric oxide production (GCH1), CAP uptake and transduction of thermal stimuli (TRPV1), and bitter taste perception (TAS2R38, TAS2R3, TAS2R4, and TAS2R5). There is evidence about the influence of genetic polymorphisms on CAP tolerance by affecting nociceptive signaling, CAP binding, and bitter tasting. This knowledge may facilitate the design and implementation of innovative CAP-based nutrigenetic strategies for a more precise clinical management of obesity.

The capsaicin binding affinity of wildtype and mutant TRPV1 ion channels

Vanilloids such as capsaicin and resiniferatoxin are highly selective and potent activators for transient receptor potential vanilloid subfamily, member 1, a nociceptor for heat and pain perception. However, the intrinsic vanilloid binding affinity, key for understanding transient receptor potential vanilloid subfamily, member 1 function, remains unknown despite intensive investigations by electrophysiological, structural, and computational methods. In this study, we determined capsaicin binding affinity under physiological conditions by isolating individual binding steps to each subunit with concatemers. We estimated the capsaicin association constant of a wildtype subunit to be in the order of 106M-1 and that of the Y511A mutant subunit to be a hundred times lower, in the order of 104M-1. The Y511A mutation, located at the entrance of the vanilloid binding pocket, reduces binding affinity without a noticeable effect on activation gating. We further affirmed that there is little cooperativity between vanilloid binding steps. Models based on independent binding and equally cooperative subunit gating can accurately describe capsaicin activation.

Capsaicin binds the N-terminus of Hsp90, induces lysosomal degradation of Hsp70, and enhances the anti-tumor effects of 17-AAG (Tanespimycin)

Heat shock protein 90 (Hsp90) and its co-chaperones promote cancer, and targeting Hsp90 holds promise for cancer treatment. Most of the efforts to harness this potential have focused on targeting the Hsp90 N-terminus ATP binding site. Although newer-generation inhibitors have shown improved efficacy in aggressive cancers, induction of the cellular heat shock response (HSR) by these inhibitors is thought to limit their clinical efficacy. Therefore, Hsp90 inhibitors with novel mechanisms of action and that do not trigger the HSR would be advantageous. Here, we investigated the mechanism by which capsaicin inhibits Hsp90. Through mutagenesis, chemical modifications, and proteomic studies, we show that capsaicin binds to the N-terminus of Hsp90 and inhibits its ATPase activity. Consequently, capsaicin and its analogs inhibit Hsp90 ATPase-dependent progesterone receptor reconstitution in vitro. Capsaicin did not induce the HSR, instead, it promoted the degradation of Hsp70 through the lysosome-autophagy pathway. Remarkably, capsaicin did not induce degradation of the constitutively expressed cognate Hsc70, indicating selectivity for Hsp70. Combined treatments of capsaicin and the Hsp90 inhibitor 17-AAG improved the anti-tumor efficacy of 17-AAG in cell culture and tridimensional tumor spheroid growth assays using breast and prostate cancer models. Consistent with this, in silico docking studies revealed that capsaicin binding to the ATP binding site of Hsp90 was distinct from classical N-terminus Hsp90 inhibitors, indicating a novel mechanism of action. Collectively, these findings support the use of capsaicin as a chemical scaffold to develop novel Hsp90 N-terminus inhibitors as well as its ability to be a potential cancer co-therapeutic.

Characterization of the interaction between capsaicin and porcine myofibrillar proteins

Flavor binding or release behavior acts as an important role in the sensory characteristics of meat products. The interaction between capsaicin and porcine myofibrillar proteins (MPs) was investigated using spectroscopic methods and molecular docking simulations. The capsaicin quenched the fluorescence of MPs via static quenching mechanisms, indicating the interaction between MPs and capsaicin. The binding constant (1.20 × 105, 3.98 × 105 and 16.98 × 105 L/mol for 298, 303 and 310 K, respectively) and the number of binding sites (1.57, 1.74 and 2.01 for 298, 303 and 310 K, respectively) for capsaicin binding to MPs increased with the increase of temperature. Fluorescence and Raman spectroscopic analyses suggested that capsaicin exhibited no obvious effect on the conformation of MPs. The thermodynamic analysis revealed that interactions between capsaicin and MPs were spontaneous processes. The positive ΔH (169.05 kJ/mol) and ΔS (0.66 kJ/(mol·K)) indicated that the binding of capsaicin to MPs was predominantly driven by hydrophobic force, which was further corroborated by molecular docking simulations. The study provides insights into the mechanism of the interaction between capsaicin and MPs, which can guide the modulation of the spicy flavor during the meat processing to improve the sensory characteristics of meat products.

Cost-Effective Pipeline for a Rational Design and Selection of Capsaicin Analogues Targeting TRPV1 Channels.

Transient receptor potential (TRP) channels constitute a large group of membrane receptors associated with sensory pathways in vertebrates. One of the most studied is TRPV1, a polymodal receptor tuned for detecting heat and pungent compounds. Specific inhibition of the nociceptive transduction at the peripheral nerve represents a convenient approach to pain relief. While acting as a chemoreceptor, TRPV1 shows high sensitivity and selectivity for capsaicin. In contrast to the drugs available on the market that target the inflammatory system, TRPV1 antagonists act as negative modulators of nociceptive transduction. Therefore, the development of compounds modulating TRPV1 activity has expanded dramatically over time. Experimental data suggest that most agonist and antagonist drugs interact at or near capsaicin's binding site. In particular, the properties of capsaicin's head play an essential role in modulating potency and affinity. Here, we explored a cost-efficient pipeline to predict the effects of introducing chemical modifications into capsaicin's head region. An extensive set of molecules was selected by first considering the geometrical properties of capsaicin's binding site and then molecular docking. Finally, the novel ligands were ranked by combining molecular and pharmacokinetic predictions.

Energetics and structural determinations of TRPV1 activation by vanilloids.

TRPV1 ion channel is a prototypical nociceptor. Vanilloids with similar structures, such as capsaicin, resiniferatoxin (RTX), and capsazepine, exhibit highly variable gating effects, from being a potent agonist to being an antagonist. To understand the structural and mechanistic differences in gating between these vanilloids, we first isolated intermediates states with varying number of bound RTX, using concatemers of wildtype rat TRPV1 subunits (RTX binds irreversibly) and Y511A mutant subunits (RTX binds reversibly). Equilibrium measurements from these concatemers revealed that binding of RTX to each subunit contributes 1.8 kcal/mol towards stabilizing the open state. Replacing RTX with capsaicin (whose binding to wildtype or mutant subunit is reversible) revealed that the Y511A mutation reduced capsaicin binding affinity by two orders of magnitude; capsaicin exerts a similar stabilization energy as RTX. Finally, we compared the energetic differences between RTX and two derivatives (5’-iRTX and 6’-iRTX), in which a one-atom difference makes the analogous molecules exhibit completely different gating effects on TRPV1: RTX is a full agonist, 6’-iRTX is a partial agonist, and 5’iRTX is an antagonist. We found that compared with RTX, 6’-iRTX only makes a small contribution (0.3 kcal/mol) to stabilize the open state, while 5’-iRTX contributes 1.2 kcal/mol to stabilize the close state. Combining energetic information with computational modeling and structural details of ligand-channel interactions allows a better understanding of TRPV1 activation and nociception.

Molecular characterization of capsaicin binding interactions with ovalbumin and casein

Capsaicin (CAP) rich diets may help with a variety of human pathophysiological conditions; however, CAP administration is difficult due to its high pungency and limited water solubility. This study comprehensively explored the mechanism of CAP binding with ovalbumin (OVA) and casein (CAS) by multi-spectroscopic, thermodynamics, and molecular docking simulation at pH 7.4, as well as the prospect of employing these food proteins as CAP carriers. The findings demonstrated that CAP could interact with OVA/CAS and increase their fluorescence intensity, which was followed by specific protein conformational changes. According to the ITC data, the interaction between CAP and OVA/CAS proceeded spontaneously, with hydrogen bonding and hydrophobic interactions governing the binding process. The binding constant Ka for the CAP-OVA and CAP-CAS complexes was determined by ITC to be 1.07 ± 0.21 ⅹ 105 M−1 and 2.22 ± 0.14 ⅹ 105 M−1, respectively. Molecular docking analysis further indicated the existence of a high affinity CAP binding site on OVA and CAS, supporting the experimental findings. Moreover, the data demonstrated that CAP binding interactions with these proteins led to the formation of complexes with about 97% CAP encapsulation efficiency, contributing to a synergistic enhancement in their antioxidant activity. Therefore, this study implies that OVA and CAS have great potential for being utilized as edible delivery vehicles for lipophilic bioactive molecules such as CAP.

A Potential Route of Capsaicin to Its Binding Site in the TRPV1 Ion Channel.

Transient receptor potential (TRP) ion channels are important pharmacological targets because of their role in the perception of pain, and so, understanding their chemical regulation is essential for the development of analgesic drugs. Among the currently known TRP channel chemical agonists, capsaicin, the active compound of chili pepper, is probably the most exhaustively studied. The availability of the three-dimensional structure of the vanilloid receptor 1 (TRPV1) has fueled computational studies revealing the molecular details of capsaicin binding modes. Although this is a significant step, a comprehensible binding mechanism or pathway is invaluable for targeting TRP channels in modern pharmacology. In the present work, free-energy and enhanced sampling techniques have been used to explore a possible membrane-mediated pathway for capsaicin to enter the TRPV1 binding pocket where capsaicin accesses the protein starting at the extracellular milieu through the outer leaflet and into its binding site in the protein. The main states visited along this route have been characterized and include (i) a bound state in agreement with the binding mode “head-down, tail-up” and (ii) an alternative state corresponding to a “head-up, tail-down” binding mode. In agreement with previous reports, binding is mediated by both hydrogen bonds and van der Waals interactions, and residue Y511 is crucial for stabilizing the bound state and during the binding process. Together, these results provide a foundation to further understand TRPV channels, and they could be used to guide therapeutic design of selective inhibitors potentially leading to novel avenues for pharmacological applications targeting the TRPV1 channel.

Vanilloid agonist-mediated activation of TRPV1 channels requires coordinated movement of the S1–S4 bundle rather than a quiescent state

Transient receptor potential vanilloid1 (TRPV1) channel plays an important role in a wide range of physiological and pathological processes, and a comprehensive understanding of TRPV1 gating will create opportunities for therapeutic intervention. Recent incredible advances in cryo-electron microscopy (cryo-EM) have yielded high-resolution structures of all TRPV subtypes (TRPV1–6) and all of them share highly conserved six transmembrane (TM) domains (S1–S6). As revealed by the open structures of TRPV1 in the presence of a bound vanilloid agonist (capsaicin or resiniferatoxin), TM helicesS1 to S4 form a bundle that remains quiescent during channel activation, highlighting differences in the gating mechanism of TRPV1 and voltage-gated ion channels. Here, however, we argue that the structural dynamics rather than quiescence of S1–S4 domains is necessary for capsaicin-mediated activation of TRPV1. Using fluorescent unnatural amino acid (flUAA) incorporation and voltage-clamp fluorometry (VCF) analysis, we directly observed allostery of the S1–S4 bundle upon capsaicin binding. Covalent occupation of VCF-identified sites, single-channel recording, cell apoptosis analysis, and exploration of the role of PSFL828, a novel non-vanilloid agonist we identified, have collectively confirmed the essential role of this coordinated S1–S4 motility in capsaicin-mediated activation of TRPV1. This study concludes that, in contrast to cryo-EM structural studies, vanilloid agonists are also required for S1–S4 movement during TRPV1 activation. Redefining the gating process of vanilloid agonists and the discovery of new non-vanilloid agonists will allow the evaluation of new strategies aimed at the development of TRPV1 modulators.

Synthesis, structural and spectroscopic characterization, in silico study, and antinociceptive effect in adult zebrafish of 2-(4-isobutylphenyl) -N'-phenylpropanohydrazide

• Structural characterization of a novel ibuprofen derivative. • Vibrational analysis was carried out. • Electronic absorption spectrum was obtained. • Molecular docking and ADMET studies were carried out. • The novel compound was not toxic, and affected locomotor activity of the Zebrafish. In this work, an ibuprofen derivative 2-(4-isobutylphenyl) -N'-phenylpropanohydrazide (ACPHZN) was synthesized and characterized by NMR, ATR-FTIR, FT-Raman, and UV-Vis spectroscopy, while their structural and spectroscopic properties were investigated using DFT calculations. In vivo study using animal model in an adult Zebrafish ( Danio rerio ), and molecular docking was performed. In addition, molecular descriptors of the properties of absorption, distribution, metabolism and excretion, and toxicity (ADMET) were obtained. The data calculated for ibuprofen derivative are in accordance with the experimental values. From the assays in adult zebrafish, it was found that the ibuprofen derivative was non-toxic and exhibited analgesic properties through the TRPA1, TRPV1 and TRPM8 channels. Molecular docking revealed six interactions of ACPHZN with residues of the capsaicin binding site, and a more favorable affinity energy (-9.0 kcal / mol). ADMET studies suggest that ACPHZN has a pharmacological principle as an oral drug based on a longer half-life in the human body.

In-vitro binding analysis and inhibitory effect of capsaicin on lipase

Capsaicin is the main active ingredient in chilis. This study selected capsazepine as a comparative model and revealed the binding mechanism and inhibitory effect of capsaicin on lipase in vitro. Electrochemical results showed capsaicin has a moderate affinity to lipase with a 7.413 × 104 L/mol binding constant, which is higher than capsazepine (3.388 × 104 L/mol). Structural analysis displayed that capsaicin/capsazepine caused a slight disturbance (less than 1%) to the secondary structure of lipase. The capsaicin binding epitope on the lipase was characterized by nuclear magnetic resonance. H47, H48 and H49 were the protons mainly involved in the interaction. In the comparison model, the H27 and H26 were involved in the capsazepine-lipase system. Hydrogen proton binding epitope analysis combined with molecular docking showed the main elements involved in the binding details. Enzyme kinetics results showed that capsaicin could inhibit the activity of lipase, resulting in a decrease in its ability to hydrolyze the substrate varying hydrolysis rate from 0% to 33.33% in different capsaicin concentrations. The biological model in vitro established in this study will support the development of weight-loss-related functional foods.

Hydroxylation, Epoxidation, and Dehydrogenation of Capsaicin by a Microbial Promiscuous Cytochrome P450 105D7.

Cytochrome P450 enzymes (P450s) are versatile biocatalysts, which insert a molecular oxygen into inactivated C-H bonds under mild conditions. CYP105D7 from Streptomyces avermitilis has been reported as a bacterial substrate-promiscuous P450 which catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, compactin and steroids. In this study, CYP105D7 catalyzes hydroxylation, epoxidation and dehydrogenation of capsaicin, a pharmaceutical agent, revealing its functional diversity. The kinetic parameters of the CYP105D7 oxidation of capsaicin were determined as Km =311.60±87.30 μM and kcat =2.01±0.33 min-1 . In addition, we conducted molecular docking, mutagenesis and substrate binding analysis, indicating that Arg81 plays crucial role in the capsaicin binding and catalysis. To our best knowledge, this study presents the first report to illustrate that capsaicin can be catalyzed by prokaryotic P450s.

Bovine alpha-lactalbumin assemblies with capsaicin: Formation, interactions, loading and physiochemical characterization

Numerous human conditions can benefit from diets rich in proteins and bioactives, such as capsaicin (CAP), yet their effective delivery is a sensorial, scientific and technological challenge. This study hypothesized that CAP can form various complexes with native bovine alpha-lactalbumin (holo-ALA) and decalcified-ALA (apo-ALA). Calorimetric and spectroscopic techniques reveals ALA-CAP molecular complexation is spontaneous, exothermic and accompanied by various conformational changes. ITC shows the interaction stoichiometry (n) and binding constant (Kb) for holo-ALA to be 0.87 ± 0.03, 1.54 ± 0.23 × 105 M−1 and for apo-ALA to be 0.64 ± 0.09, 9.41 ± 2.16 × 104 M−1. Molecular docking further elucidates that hydrogen bonds govern CAP binding to holo-ALA while hydrophobic interactions dominate binding to apo-ALA in a structural cleft. Finally, this work shows these interactions along with controlled aggregation can be utilized to form CAP-loaded colloids with encapsulation efficiency of 47.1 ± 1.0%. Thus, this study shows great promise in the prospective use of ALA as an edible delivery vehicle for CAP.

Enhancement of Lipolysis in 3T3-L1 Adipocytes by Nitroarene Capsaicinoid Analogs

Transient receptor potential vanilloid 1 (TRPV1) activation by capsaicin binding increased intracellular calcium influx and stimulated adipocyte-to-adipocyte communication, leading to lipolysis. Generally, enhancement of π-stacking capabilities improves certain binding interactions. Notably, nitroarenes exhibit strong binding interactions with aromatic amino acid side chains in proteins. New capsaicinoid analogs were designed by substitution of the OCH3 group with a nitrogen dioxide (NO2) group on the vanillyl ring to investigate how π-stacking interactions in capsaicinoid analogs contribute to lipolysis. Capsaicinoid analogs, nitro capsaicin (5), and nitro dihydrocapsaicin (6) were prepared in moderate yields via coupling of a nitroaromatic amine salt and fatty acids. Oil Red O staining and triglyceride assays with 10 µM loading of capsaicin (CAP), dihydrocapsaicin (DHC), 5, and 6 were performed to investigate their effect on lipolysis in 3T3-L1 adipocytes. Both assay results indicated that 5 and 6 decreased lipid accumulation by 13.6% and 14.7%, respectively, and significantly reduced triglyceride content by 26.9% and 28.4%, respectively, in comparison with the control experiment. Furthermore, the decrease in triglyceride content observed in response to nitroarene capsaicinoid analogs was approximately 2-folds higher than that of CAP and DHC. These results arose from the NO2 group augmented π-π stacking with Tyr511 and the attractive charge interaction with Glu570 affecting binding interactions with TRPV1 receptors.

Multiple single nucleotide polymorphisms of the transient receptor potential vanilloid 1 (TRPV1) genes associate with cough sensitivity to capsaicin in healthy subjects

BackgroundCough is a common symptom in several respiratory diseases and may occur in healthy subjects as a defense mechanism against noxious inhalants. Cough response is mediated by transient receptor potential vanilloid-1 (TRPV1) expressed by C-fibers in the airways. Capsaicin (CPS) activates TRPV1 and is regularly used as a tool to study cough response. Although single nucleotide polymorphisms (SNPs) of TRPV1 are implicated in CPS binding, their role in cough response is not fully elucidated. AimsIn this study we investigated the relationship between capsaicin cough challenge sensitivity and multiple TRPV1 polymorphisms. MethodsThe dose-response of cough induced by CPS inhalation was determined in 20 unselected healthy volunteers and the concentration of CPS causing two coughs (C2) was calculated. The SNPs I585V(rs8065080), T505A(rs17633288), T469I(rs224534), I315 M(rs222747), P91S(rs222749), and K2N(rs9894618) were characterized in blood DNA from each subject. The association between combinations of TRPV1 SNPs and CPS sensitivity of each subject was assessed by linear regression. ResultsAll subjects were wild type for T505A and K2N, while they exhibited two to six SNPs with high capsaicin responsiveness. The major contribution to CPS sensitivity in vivo (C2) was due to four combined SNPs: 315 M, 585I, 469I and 91S (p = 0.015). We found, however, that the presence of a minimum of two polymorphisms, such as 91S combined with 315 M (p = 0.032) or 91S with 585I (p = 0.025), was sufficient to detect an effect on C2. ConclusionCapsaicin cough challenge sensitivity in healthy subjects is dependent on multiple TRPV1 polymorphisms.

Direct Anandamide Activation of TRPV1 Produces Divergent Calcium and Current Responses

In the brainstem nucleus of the solitary tract (NTS), primary vagal afferent neurons express the transient receptor potential vanilloid subfamily member 1 (TRPV1) at their central terminals where it contributes to quantal forms of glutamate release. The endogenous membrane lipid anandamide (AEA) is a putative TRPV1 agonist in the brain, yet the extent to which AEA activation of TRPV1 has a neurophysiological consequence is not well established. We investigated the ability of AEA to activate TRPV1 in vagal afferent neurons in comparison to capsaicin (CAP). Using ratiometric calcium imaging and whole-cell patch clamp recordings we confirmed that AEA excitatory activity requires TRPV1, binds competitively at the CAP binding site, and has low relative affinity. While AEA-induced increases in peak cytosolic calcium were similar to CAP, AEA-induced membrane currents were significantly smaller. Removal of bath calcium increased the AEA current with no change in peak CAP currents revealing a calcium sensitive difference in specific ligand activation of TRPV1. Both CAP- and AEA-activated TRPV1 currents maintained identical reversal potentials, arguing against a major difference in ion selectivity to resolve the AEA differences in signaling. In contrast with CAP, AEA did not alter spontaneous glutamate release at NTS synapses. We conclude: (1) AEA activation of TRPV1 is markedly different from CAP and produces different magnitudes of calcium influx from whole-cell current; and (2) exogenous AEA does not alter spontaneous glutamate release onto NTS neurons. As such, AEA may convey modulatory changes to calcium-dependent processes, but does not directly facilitate glutamate release.

Kinetic Analysis of Vanilloid-Induced Activation in TRPV1 Channel

The capsaicin receptor, TRPV1 ion channel, is a polymodal nociceptor and attractive drug target. Recent advances in cryo-electron microscopy revealed the vanilloid-binding pocket in high resolution, which allowed atomic interactions mediating capsaicin binding to be proposed. In the current model, capsaicin adopts a “tail-up, head-down” pose; key interactions include two hydrogen bonds formed between the amide of capsaicin and Thr551 and between the hydroxyl on the vanillyl group and Glu571 (of mouse TRPV1). Guided by this model, the vanilloid-insensitive TRPV2 channel has been successfully transformed to be vanilloid-sensitive with minimal alterations. In this study, we provide further evidence from kinetic and equilibrium analyses in support of the capsaicin binding model. Eliminating the key hydrogen bonds by point mutations right-shifted the concentration-response relationship, which was achieved by accelerating the current OFF rate and decelerating the ON rate. These observations are consistent with the expectation that mutations would weaken capsaicin binding. Surprisingly, though resiniferatoxin (RTX) has been proposed to bind in a similar fashion as capsaicin, kinetic and equilibrium analyses revealed that removing these crucial hydrogen bonds did not alter RTX activation kinetics as they did capsaicin, suggesting that RTX may behave differently.

Structural Characterization of Ligand-Specific Interactions in TRPV1 Channel: Gating Mechanism by Capsaicin and Capsazepine

TRPV1 channels are polymodal receptors whose open probability is under command of several stimuli such as temperature, pH and ligands. Several studies have involved TRPV1 channels in pain sensation related to inflammatory responses and tissue damage, leading the pharmaceutical industry to the search of novel compounds able to modulate their function in order to develop novel pain relief therapies. Capsazepine is the best known TRPV1 antagonist and the residues accounting for their binding to TRPV1 channels have been identified by mutagenesis and specie-specific effects. Nevertheless, the molecular mechanism underlying capsazepine-mediated channel inhibition has not been described at molecular level to date. We performed molecular dynamics simulations of the TRPV1 channel in the open state in the absence and the presence of Capsazepine and Capsaicin in order to identify the interactions accounting of TRPV1 channel. Even when Capsazepine shares in part their binding site with the well-known TRPV1 channel agonist Capsaicin, their ligand orientation resulted to be different. We also observed a dynamic hydrogen bond network at the neighbors of the vanilloid binding pocket that differs with those observed in both the absence of ligand and in the presence of capsaicin. Our results suggest that the subtle differences at the interaction network at the capsaicin binding pocket leads to completely opposite effects at the channel activation gate.

Structural mechanism underlying capsaicin binding and activation of the TRPV1 ion channel.

Capsaicin bestows spiciness by activating TRPV1 channel with exquisite potency and selectivity. Capsaicin-bound channel structure was previously resolved by cryo-EM at 4.2-to-4.5 Å resolution, however important details required for mechanistic understandings are unavailable: capsaicin was registered as a small electron density, reflecting neither its chemical structure nor specific ligand-channel interactions. We obtained the missing atomic-level details by iterative computation, which were confirmed by systematic site-specific functional tests. We observed that the bound capsaicin takes “tail-up, head-down” configurations. The vanillyl and amide groups form specific interactions to anchor its bound position, while the aliphatic tail may sample a range of conformations, making it invisible in cryo-EM images. Capsaicin stabilizes the open state by “pull-and-contact” interactions between the vanillyl group and the S4-S5 linker. Our study provided a structural mechanism for the agonistic function of capsaicin and its analogs, and demonstrated an effective approach to obtain atomic level information from cryo-EM structures.

Capsaicin Interaction with TRPV1 Channels in a Lipid Bilayer: Molecular Dynamics Simulation

Transient receptor potential vanilloid subtype 1 (TRPV1) is a heat-sensitive ion channel also involved in pain sensation, and is the receptor for capsaicin, the active ingredient of hot chili peppers. The recent structures of TRPV1 revealed putative ligand density within the S1 to S4 voltage-sensor-like domain of the protein. However, questions remain regarding the dynamic role of the lipid bilayer in ligand binding to TRPV1. Molecular dynamics simulations were used to explore behavior of capsaicin in a 1-palmitoyl-2-oleoyl phosphatidylcholine bilayer and with the target S1–S4 transmembrane helices of TRPV1. Equilibrium simulations reveal a preferred interfacial localization for capsaicin. We also observed a capsaicin molecule flipping from the extracellular to the intracellular leaflet, and subsequently able to access the intracellular TRPV1 binding site. Calculation of the potential of mean force (i.e., free energy profile) of capsaicin along the bilayer normal confirms that it prefers an interfacial localization. The free energy profile indicates that there is a nontrivial but surmountable barrier to the flipping of capsaicin between opposing leaflets of the bilayer. Molecular dynamics of the S1–S4 transmembrane helices of the TRPV1 in a lipid bilayer confirm that Y511, known to be crucial to capsaicin binding, has a distribution along the bilayer normal similar to that of the aromatic group of capsaicin. Simulations were conducted of the TRPV1 S1–S4 transmembrane helices in the presence of capsaicin placed in the aqueous phase, in the lipid, or docked to the protein. No stable interaction between ligand and protein was seen for simulations initiated with capsaicin in the bilayer. However, interactions were seen between TRPV1 and capsaicin starting from the cytosolic aqueous phase, and capsaicin remained stable in the majority of simulations from the docked pose. We discuss the significance of capsaicin flipping from the extracellular to the intracellular leaflet and mechanisms of binding site access by capsaicin.