Small molecule dual-inhibitors of TRPV4 and TRPA1 for attenuation of inflammation and pain

Abstract

TRPV4 ion channels represent osmo-mechano-TRP channels with pleiotropic function and wide-spread expression. One of the critical functions of TRPV4 in this spectrum is its involvement in pain and inflammation. However, few small-molecule inhibitors of TRPV4 are available. Here we developed TRPV4-inhibitory molecules based on modifications of a known TRPV4-selective tool-compound, GSK205. We not only increased TRPV4-inhibitory potency, but surprisingly also generated two compounds that potently co-inhibit TRPA1, known to function as chemical sensor of noxious and irritant signaling. We demonstrate TRPV4 inhibition by these compounds in primary cells with known TRPV4 expression - articular chondrocytes and astrocytes. Importantly, our novel compounds attenuate pain behavior in a trigeminal irritant pain model that is known to rely on TRPV4 and TRPA1. Furthermore, our novel dual-channel blocker inhibited inflammation and pain-associated behavior in a model of acute pancreatitis - known to also rely on TRPV4 and TRPA1. Our results illustrate proof of a novel concept inherent in our prototype compounds of a drug that targets two functionally-related TRP channels, and thus can be used to combat isoforms of pain and inflammation in-vivo that involve more than one TRP channel. This approach could provide a novel paradigm for treating other relevant health conditions.

Figure 1. Modifications of tool compound GSK205 for improved targeting of TRPV4.

The synthesized compounds differed in the highlighted part of the molecule, changed residue indicated with arrow. Compound 16-19 compound was synthesized to incorporate two modifications from two compounds, 16-8 and 16-18, found most potent in anti-TRPV4 screening assays (see Fig. 2).

Figure 2. Assessment of 16-… compounds in N2a cells with directed expression of TRPV4.

(A) Ca⁺⁺ imaging screening of all compounds in N2A cells with directed expression of TRPV4 (rat). The cells were stimulated with TRPV4-selective activator compound, GSK101 (5 nM) in the presence of 5 μM of the respective inhibitor. The number on each bar corresponds to average peak ∆Ca⁺⁺ concentrations in ≈100 cells. Inset: micrographs of pseudo-colored cells before and after activation with 5nM GSK101, in addition note the corresponding time course of the averaged Ca⁺⁺ signal (fura-2 Ca⁺⁺ imaging). Except for compound 16-43C, the difference to vehicle control reach the level of statistical significance p < 0.01 (one-way ANOVA). (B) Dose-response of the most potent, “winner” compounds in TRPV4-expressing N2a cells. The IC₅₀ were; 0.45 ± 0.05 μM (16-8), 0.59 ± 0.12 μM (16-18), 0.81 ± 0.1 μM (16-19), 4.19 ± 0.71 μM (GSK205). Plot generated from averaged peak ∆Ca⁺⁺ concentration of ≥75 cells per data-point.

Figure 3. TRPV4 channel inhibition by compounds 16-8 and 16-19 – patch-clamp e-phys.

(A) Current-voltage relationship of TRPV4-mediated currents after activation with 5 nM GSK101. Recordings were performed in TRPV4-GFP+ N2a cells. The representative traces represent an average of ≈12 sweeps. In all experiments, cells were pre-incubated with the respective compound (5 μM) for 5 minutes. (B) Average current densities at −100mV/+100 mV were significantly diminished by inhibitors (*P < 0.05; one-way ANOVA; n ≥ 5 cells/group).

Figure 4. Compound 16-8 inhibits TRPV4 in I˚ cells more potently than GSK205.

(A) I˚ articular chondrocytes (pig); dose-response comparison between the most potent compound, 16-8, and GSK205 in response to stimulation with 5 nM GSK101. Inset: Chondrocytes responding to activation with GSK101, fura-2 Ca⁺⁺ imaging; right-hand image taken at 5 sec after GSK101 application. 16-8 was significantly more potent than GSK205 (mean ± SEM, n = 6 independent expts, n ≥ 25 cells/expt; *p < 0.05, t-test). Ordinate shows average peak ∆Ca⁺⁺ concentrations. (B) I˚ astrocytes (rat); dose-response comparison between 16-8 and GSK205 in response to 5 nM GSK101. Inset: Astrocytes responding to activation with GSK101; right-hand image taken at 5 sec after GSK101 application (mean ± SEM, n = 5 independent expts, n ≥ 200 cells/expt; *p < 0.05, t-test). Ordinate shows average peak ∆Ca⁺⁺ concentrations.

Figure 5. Compounds 16-8 and 16-19 also potently inhibit TRPA1, not TRPV1-3.

(A) Specificity vs TRPV1-3. Both 16-8 and 16-19 (5 μM each) compounds did not inhibit TRPV1, −2 or −3 channels (all mouse isoforms), directed over-expression in N2a cells and subsequent Ca⁺⁺ imaging. Mean±SEM is shown, ≥100 cells per condition. (B) Dose-dependent inhibition of TRPA1 (mouse, directed expression in N2a cells) by GSK 205, 16-8 and 16-19, activation with 100 μM mustard oil, resulting in IC₅₀ of 5.56 ± 0.4 μM (GSK205), 0.41 ± 0.37 μM (16-19), 0.43 ± 0.3 μM (16-8). Plot generated from averaged peak ∆Ca⁺⁺ concentration of ≥75 cells per data-point.

Figure 6. Cellular toxicity studies of compounds 16-8 and 16-19.

N2a cells were subjected to increasing concentrations of compounds 16-8 and 16-19, resulting cell viability was analyzed for the next 48 h. (A) Time course of cell viability in the presence of various concentrations of 16-8. Note clear reduction at 40 and 80 μM. (B) As in (A), for compound 16-19, with similar outcome. Representative result of 2 independent experiments.

Figure 7. 16-8 and 16-19 effectively attenuate formalin-evoked trigeminal irritant pain.

(A) Time-course of nocifensive behavior in WT mice following whisker-pad injection of 4% formalin. The mice were pre-injected (i.p., 10 mg/kg; 15 min before formalin) with GSK205, 16-8 or 16-19. Note effective reduction of nocifensive behavior in the late “neural” phase by compounds 16-8, 16-19, not by GSK205. (B) Cumulative response binned into 3 phases: acute phase (0–5 min), interphase (5–15 min), and late “neural” phase (15–45 min). Note significant reduction of nocifensive behavior in the late phase by 16-8, 16-19, not GSK205 (*P < 0.01 vs vehicle and GSK205, one-way ANOVA). (C) As in (A), but also including Trpv4^−/− mice. Compounds were applied i.p. 15 min before formalin challenge, at 10 mg/kg except established TRPA1 blocker, A967079 (25 mg/kg). Previously-established attenuated nocifensive behavior in early and late phase in Trpv4^−/− mice was recapped, which was reduced further by TRPA1 blocker, A967079. (D) As in (B), plus inclusion of Trpv4^−/− mice. Robust effects of TRPA1-blocker, A967079, were mimicked equi-potently by 16-8 and 16-19 for early phase, and by 16-8 for late phase, partially by 16-19 for late phase. (A,C) show averaged behavioral metrics per time-point, bars in (B,D) represent mean ± SEM; for (D) ^*P < 0.05; ^#P < 0.005, one-way ANOVA; for all panels n = 5–8 mice/group.

Figure 8. Compound 16-8 attenuates acute pancreatitis and improves pain behavior.

(A) Caerulein-evoked acute pancreatitis causes pancreatic edema, which is eliminated by compound 16-8 (10 mg/kg, applied at 30 min before first exposure to caerulein). (B) Caerulein-evoked acute pancreatitis strongly elevates cellular toxicity marker amylase in serum. Amylase is reduced, but not significantly, in 16-8 treated animals. (C) caerulein-evoked acute pancreatitis causes elevated myelo-peroxidase (MPO) activity in serum, a marker for infiltration of inflammatory cells into the pancreas. MPO activity is significantly reduced in 16-8 treated mice. (D) caerulein-evoked acute pancreatitis can be readily demonstrated histologically, exemplified in the micrograph panels shown. Note increased pancreas inflammation in the middle-panel vs non-inflamed pancreas in vehicle-control challenged mice, and its attenuation by treatment with compound 16-8. (E) Bar diagram shows quantitation of inflammatory histologic parameters as shown in (D). Note significant increase of inflammation-index in caerulein acute pancreatitis mice, and its significant reduction upon treatment with compound 16-8. (F) Caerulein-evoked acute pancreatitis causes pain behavior, significantly reduced by compound 16-8. Note greatly reduced activity over the 6 h test period in caerulein-induced acute pancreatitis. This nocifensive behavior is greatly improved in response to systemic application of compound 16-8. Results are expressed as mean ± SEM; n = 6 mice/group; *P < 0.05 (one-way ANOVA).