Sigma 2 Receptor/Tmem97 Agonists Produce Long Lasting Antineuropathic Pain Effects in Mice

Abstract

Neuropathic pain is an important medical problem with few effective treatments. The sigma 1 receptor (σ1R) is known to be a potential target for neuropathic pain therapeutics, and antagonists for this receptor are effective in preclinical models and are currently in phase II clinical trials. Conversely, relatively little is known about σ2R, which has recently been identified as transmembrane protein 97 (Tmem97). We generated a series of σ1R and σ2R/Tmem97 agonists and antagonists and tested them for efficacy in the mouse spared nerve injury (SNI) model. In agreement with previous reports, we find that σ1R ligands given intrathecally (IT) produce relief of SNI-induced mechanical hypersensitivity. We also find that the putative σ2R/Tmem97 agonists DKR-1005, DKR-1051, and UKH-1114 (K_i ∼ 46 nM) lead to relief of SNI-induced mechanical hypersensitivity, peaking at 48 h after dosing when given IT. This effect is blocked by the putative σ2R/Tmem97 antagonist SAS-0132. Systemic administration of UKH-1114 (10 mg/kg) relieves SNI-induced mechanical hypersensitivity for 48 h with a peak magnitude of effect equivalent to 100 mg/kg gabapentin and without producing any motor impairment. Finally, we find that the TMEM97 gene is expressed in mouse and human dorsal root ganglion (DRG) including populations of neurons that are involved in pain; however, the gene is also likely expressed in non-neuronal cells that may contribute to the observed behavioral effects. Our results show robust antineuropathic pain effects of σ1R and σ2R/Tmem97 ligands, demonstrate that σ2R/Tmem97 is a novel neuropathic pain target, and identify UKH-1114 as a lead molecule for further development.

Keywords: Neuropathic pain; Tmem97; dorsal root ganglion; drug discovery; sigma 1 receptor; sigma 2 receptor.

Figure 1

Norbenzomorphan and methanobenzazocine scaffolds 1 and 2 and the CNS penetrant σ2R/Tmem97 ligands SAS-0132 and DKR-1051.

Figure 2

σ Receptor binding ligands.^aK_i are shown for each ligand at σ1R or σ2R/Tmem97 as the mean of at least two independent experiments ± standard deviation. Siramesine data is from ref . ^aFor all compounds, σ2R/Tmem97 was sourced from rat PC12 cells, with the exception of siramesine, which utilized rat brain homogentate. See Methods Section for more details.

Figure 3

Effect of IT injection of σ1R ligands in the mouse SNI model. Compounds acting on σ1R were tested in the mouse SNI model. Mechanical sensitivity was measured at the indicated time points after IT injection of 10 μg compound. All vehicle groups, n = 6; JWG-1014, n = 5; JSS-1027, n = 5; MFG-1046, n = 4. **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 4

Effect of IT injection of σ2R/Tmem97 ligands in the SNI model. Mechanical sensitivity was measured at the indicated time points after IT injection of 10 μg compound. (A) Effect of siramesine (n = 4) compared to vehicle (n = 6) is shown. (B) Effect of DKR-1005 (n = 6), DKR-1051 (n = 5) and UKH-1114 (n = 11) compared to vehicle (n = 6) is shown. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Figure 5

Effect of UKH-1114 is blocked by the σ2R/Tmem97 antagonist SAS-0132. σ2R/Tmem antagonist SAS-0132 at 10 μg dose given at the same time as UKH-1114, also at 10 μg dose, completely blocked the effect seen with UKH-1114 given alone. n = 6 per group. ****p < 0.0001.

Figure 6

Systemic dosing with UKH-1114 leads to alleviation of neuropathic pain without motor effects. (A) Vehicle (n = 6), gabapentin (100 mg/kg, n = 6), or UKH-1114 (10 mg/kg, n = 6) were given IV and mechanical testing was done at the indicated time points. (B) Rotorod testing was done 48 h after IV injection on an accelerating rotorod reaching a maximum of 40 rotations per minute over 200 s. Latency to fall is shown on the third trial (n = 6 per group). ****p < 0.0001.

Figure 7

Expression analysis for Tmem97 (A), TMEM97 (human), and Tmem97 (mouse) gene expression across orthologous tissues, with greater expression in the mouse and human GI tract and the human DRG. (B) Analysis of mouse single cell data reveals a maximum detection rate of 29% for Tmem97 across all sensory neuron subpopulations as contrasted with 67% or more for known subpopulation marker genes. (C) Cortical expression of Tmem97 as contrasted with the neuronal marker NeuN. Tmem97 expression spans both neuronal and non-neuronal cells, with ~1.5–2.5-fold higher expression in non-neuronal cells.

Figure 8

Synthesis of norbenzomorphan σ2R/Tmem97 ligands. Reagents and conditions (a) 10% Pd/C, H₂, EtOH. (b) K₂CO₃, CH₃CN, 50 °C. (c) Pd(OAc)₂, JohnPhos^®, NaOt-Bu, toluene, 100 °C. (d) TMSI, CH₂Cl₂, then HCl. (e) Na(OAc)₃BH, CH₃COOH, 1,2-dichloroethane.

Figure 9

Synthesis of methanobenzazocine σ2R/Tmem97 ligands. Reagents and conditions (a) TMSI, CH₂Cl₂, then HCl. (b) Na(OAc)₃BH, 1,2-dichloroethane. (c) Pd[P(^tBu)₃]₂, Cs₂CO₃, 1,4-dioxane, 98 °C. (d) 10% Pd/C, H₂, EtOH. (e) K₂CO₃, CH₃CN, 55 °C.