Novel TRPV1 Channel Agonists With Faster and More Potent Analgesic Properties Than Capsaicin

Abstract

The transient receptor potential vanilloid 1 (TRPV1) ion channel is a member of the family of Transient Receptor Potential (TRP) channels that acts as a molecular detector of noxious signals in primary sensory neurons. Activated by capsaicin, heat, voltage and protons, it is also well known for its desensitization, which led to the medical use of topically applied TRPV1 agonist capsaicin for its long-lasting analgesic effects. Here we report three novel small molecules, which were identified using a Structure-Based Virtual Screening for TRPV1 from the ZINC database. The three compounds were tested using electrophysiological assays, which confirmed their capsaicin-like agonist activity. von Frey filaments were used to measure the analgesic effects of the compounds applied topically on tactile allodynia induced by intra-plantar carrageenan. All compounds had anti-nociceptive activity, but two of them showed faster and longer lasting analgesic effects than capsaicin. The present results suggest that TRPV1 agonists different from capsaicin could be used to develop topical analgesics with faster onset and more potent effects.

Keywords: TRPV1; allodynia; analgesic; capsaicin; carrageenan; drug discovery; transient receptor potential vanilloid 1 channels; von frey.

Figure 1

Workflow of TRPV1 Virtual Screening. (A) A SBVS over a ZINC Database with 112,935 compounds was performed using the TRPV1 channel structure as target. Results were filtered by excluding all molecules with binding energy below −7 kcal/mol, which reduced the candidate molecules to 1,000. A restriction by LogP values compatible with the ADME criteria was subsequently applied, followed by clustering and bibliography-based selection, which rendered five potential candidate molecules. (B) Structural comparison between capsaicin, the novel TRPV1 channel agonists found in this study and Resiniferatoxin. The molecules were decomposed into three regions, called A, B, and C. A-region comprises the Vanilloid ring, B-Region comprises a linker containing the electron donor–acceptor pair and C-region represents the hydrophobic region of each molecule.

Figure 2

In vitro evaluation of putative TRPV1 agonists. (A) Representative experiments of TEVC recordings in Xenopus Oocytes expressing TRPV1 channels during a +30 mV pulse at increasing concentrations of compounds 1 (A), 2 (B), and 3 (C). (D) Dose–response curve of putative TRPV1 agonists. Steady-state currents elicited by a voltage pulse at +30 mV in the presence of compound 1 (filled circles), compound 2 (empty circles), compound 3 (inverted triangles), and capsaicin (red circles) normalized by the currents elicited in the presence of capsaicin (I_x/I_capsaicin) as a function of capsaicin or test compound concentration (X). The data was fitted using Hill Equation: $\frac{Ix}{lcapsaicin}=Imax*{\left[X\right]}^n/({EC}_{50}^n+{\left[X\right]}^n)$. The following parameters were estimated for compounds 1, 2, 3, and capsaicin: EC₅₀ of 53 ± 6 nM, 53 ± 4.3 nM, 92 ± 10 nM, and 440 ± 66 nM respectively; Hill coefficients (n) of 0.73, 0.9, 1.06, and 0.45 respectively and maximal effect was 0.8, 0.73, 0.89, and 1, respectively.

Figure 3

Effects of compounds on tactile allodynia produced by carrageenan-induced inflammatory pain in rats. The effects on tactile thresholds (g) using von Frey filaments before (0) and at different times (1–9, 24 h) after the subcutaneous injection (arrow) of saline (open circles) or carrageenan followed by application of vehicle (cream alone; filled squares), or together with increasing concentrations equivalent to the EC₅₀ (gray triangle), 10xEC₅₀ (red cross), 100xEC₅₀ (purple rhomboid) and 1,000xEC₅₀ (green inverted triangle) of compound 1 (A), compound 2 (B), compound 3 (C), and capsaicin (D). Statistics are shown in Table 4.

Figure 4

Ratio of tactile thresholds between the different compounds and vehicle or capsaicin. The results of tactile thresholds for compound 1 (A), compound 2 (B), compound 3 (C), and capsaicin (D) were divided by the thresholds obtained for the vehicle group. The results of tactile thresholds for compound 1 (E), compound 2 (F), compound 3 (G) were divided by the thresholds obtained for the capsaicin group. Note that compounds 1 and 2 show effects at earlier time points compared to capsaicin. Concentrations: EC₅₀ (gray circle, 10xEC₅₀ (red cross), 100XEC₅₀ (purple rhomboid), 1000xEC₅₀ (green inverted triangle).

Figure 5

Paw withdrawal thresholds (PWT) and maximal possible effect (%MPE) to extrapolate the median effective dose (ED50). The PWT was estimated for compound 1 (A), compound 2 (B), compound 3 (C), and capsaicin (D). Concentrations: EC₅₀ (gray circle, 10xEC₅₀ (red cross), 100XEC₅₀ (purple rhomboid), 1,000xEC₅₀ (green inverted triangle). The ED₅₀ was extrapolated at 50% from the linear fit of the %MPE values at the log10 of each concentration for compound 1 (E), compound 2 (F), compound 3 (G), and capsaicin (H).