Novel selective, potent naphthyl TRPM8 antagonists identified through a combined ligand- and structure-based virtual screening approach

Abstract

Transient receptor potential melastatin 8 (TRPM8), a nonselective cation channel, is the predominant mammalian cold temperature thermosensor and it is activated by cold temperatures and cooling compounds, such as menthol and icilin. Because of its role in cold allodynia, cold hyperalgesia and painful syndromes TRPM8 antagonists are currently being pursued as potential therapeutic agents for the treatment of pain hypersensitivity. Recently TRPM8 has been found in subsets of bladder sensory nerve fibres, providing an opportunity to understand and treat chronic hypersensitivity. However, most of the known TRPM8 inhibitors lack selectivity, and only three selective compounds have reached clinical trials to date. Here, we applied two virtual screening strategies to find new, clinics suitable, TRPM8 inhibitors. This strategy enabled us to identify naphthyl derivatives as a novel class of potent and selective TRPM8 inhibitors. Further characterization of the pharmacologic properties of the most potent compound identified, compound 1, confirmed that it is a selective, competitive antagonist inhibitor of TRPM8. Compound 1 also proved itself active in a overreactive bladder model in vivo. Thus, the novel naphthyl derivative compound identified here could be optimized for clinical treatment of pain hypersensitivity in bladder disorders but also in different other pathologies.

Figure 1

Derived pharmacophore through SMARTS strings. (A) Schematization of the derived pharmacophore model. (B) SMARTS strings encoding for the pharmacophore.

Figure 2

TRPM8-Compound 1 complex, detail of the binding site. (A) Top view, perpendicular to the membrane plane, extra- to intracellular perspective. The naphthyl moiety of the ligand, here highlighted in cyan and represented in CPK, well fits into the sub-pocket framed by Ile746, Tyr745, Leu750 and Leu806 (in grey and CPK). (B) Side view, perpendicular to the transmembrane helices. Compound 1, represented in cyan and ball-end-stick, lands parallel to TM helices 2 and 3 surface, here in red cartoon. Dotted blue line represents the Hydrogen bond between the amine group of Compound 1 and the carboxyl function of Asp802, interaction further strengthened by opposite charge attraction. The furane ring locates between Leu750 and Phe794 (in grey and CPK).

Figure 3

Docking results obtained by LiGen on the benchmarking dataset. Distribution of the active compounds within the entire ranking as subdivided into 100 bins, resulting from the Docking campaign targeted in an 8 Å radius sphere around Asp802. Active compounds and enrichment factor (EF) are reported for each top N %.

Figure 4

Naphthyl derivatives. Structure and TRPM8 inhibition values for the naphthyl derivatives identified by the HTS campaign (the %inhibition values are obtained at 10 mM).

Figure 5

Effects of individual TRPM8 point mutations on calcium responses induced by cooling agent 10 and icilin. Shown data represent mean ± standard error of the mean (SEM) of quadruplicate determinations of a representative experiment (n = 4). Statistical analysis was performed using unpaired Student’s t test with GraphPad Prism (95% confidence interval). (A) Dose response curves of icilin on mutant TRPM8 receptors. The substitutions Y745A, D802A and N799A abrogated agonist activity. The I746A substitution significantly (p < 0.05) shifted pIC₅₀ of Icilin from 6.6 to 6.9. (B) Dose response curves of Cooling Agent 10 on mutant TRPM8 receptors. The substitution I746A strongly reduced agonist activity in term of efficacy while D802A significantly (p < 0.0001) shifted the pIC₅₀ of the agonist from 5.1 to 4.5. Other mutations did not have effects on Cooling agent 10 activity.

Figure 6

Effects of individual TRPM8 point mutations on Compound 1-mediated inhibition of calcium responses. Mutant and wt TRPM8-transfected cells were exposed to increasing concentrations of Compound 1 and then stimulated with icilin (A) or Cooling agent 10 (B) at the specific EC80 concentration. Shown data represent mean ± SEM of quadruplicate determinations of a representative experiment (n = 2). Statistical analysis was performed using unpaired Student’s t test with GraphPad Prism (95% confidence interval). (A) Dose response curves of Compound 1 activity on Icilin activation. The substitution I746A significantly (p = 0.003) reduced compound 1 inhibition shifting pIC50 from 7.2 to 7.7. (B) Dose response curves of Compound 1 inhibition of Cooling Agent 10 activation of TRPM8 receptor. The substitution D802A shifted its pIC50 from 7.2 to 5.5 significantly decreasing (p < 0.0001) Compound 1 potency, while N799A significantly increase Compound 1 potency (p < 0.0001) shifting pIC50 from 7.2 to 8.0.

Figure 7

Validation of TRPM8 antagonist compound 1 by orthogonal assays and compound selectivity. (A) Seven points dose-response curves of compound 1 inhibition of cooling Agent 10 and Icilin-induced TRPM8 activation using an intracellular calcium mobilization assay. Compound 1 pIC50s against cooling agent 10 and icilin are similar (pIC50 7.38 and 7.23 respectively). Shown data represent the mean ± SEM (error bars) of triplicate determinations of a representative experiment (n = 2). (B) Dose-response curve of compound 1 tested by using cold temperatures as stimulus. Compound 1 pIC50 against cold is similar to that calculated for chemical agonists (pIC50 6.73). Shown data represent the mean ± SEM (error bars) of triplicate determinations of a representative experiment (n = 2). (C) Test of compound 1 activity at manual patch clamp. Outward currents were elicited upon addition of cooling agent 10 at + 40 mV, in the presence and in the absence of compound 1. Compound 1 addition completely abrogated electrical impulse transmission. (D) Compound 1 selectivity towards TRPM8 related receptors TRPA1, TRPV1, TRPV4. Compound 1 is completely inactive against all TRPM8 related receptors tested (p < 0.001). Shown data represent the mean ± SEM (error bars) of triplicate determinations of a representative experiment (n = 2).

Figure 8

In vivo effects of compound 1 in isovolumetric bladder rat model. The results were given as mean values ± standard error of the mean (sem) of the measurement in 8 different rats. (A) Effects of intravenous administration of vehicle (on the left) and compound 1 at 10 mg/kg (on the right) on micturition frequency (MF). No significant changes were observed upon vehicle administration (on the left), while compound 1 (on the right) significantly reduced MF at 30 min (p < 0.01). (B) Effects of intravenous administration of vehicle and compound 1 at 10 mg/kg on inhibition time. Compound 1 significantly enhanced inhibition time (p < 0.05). (C) Effects of intravescical administration of vehicle and compound 1 at 2.268 mg. Compound 1 significantly increased (p < 0.01) the threshold volume inducing RBC.