Identification of Koumine as a Translocator Protein 18 kDa Positive Allosteric Modulator for the Treatment of Inflammatory and Neuropathic Pain

Abstract

Koumine is an alkaloid that displays notable activity against inflammatory and neuropathic pain, but its therapeutic target and molecular mechanism still need further study. Translocator protein 18 kDa (TSPO) is a vital therapeutic target for pain treatment, and recent research implies that there may be allostery in TSPO. Our previous competitive binding assay hint that koumine may function as a TSPO positive allosteric modulator (PAM). Here, for the first time, we report the pharmacological characterization of koumine as a TSPO PAM. The results imply that koumine might be a high-affinity ligand of TSPO and that it likely acts as a PAM since it could delay the dissociation of ³H-PK11195 from TSPO. Importantly, the allostery was retained in vivo, as koumine augmented Ro5-4864-mediated analgesic and anti-inflammatory effects in several acute and chronic inflammatory and neuropathic pain models. Moreover, the positive allosteric modulatory effect of koumine on TSPO was further demonstrated in cell proliferation assays in T98G human glioblastoma cells. In summary, we have identified and characterized koumine as a TSPO PAM for the treatment of inflammatory and neuropathic pain. Our data lay a solid foundation for the use of the clinical candidate koumine to treat inflammatory and neuropathic pain, further demonstrate the allostery in TSPO, and provide the first proof of principle that TSPO PAM may be a novel avenue for the discovery of analgesics.

Keywords: TSPO; inflammatory pain; koumine; neuropathic pain; positive allosteric modulation.

FIGURE 1

Kinetic profile for Ro5-4864, PK11195, protoporphyrin IX and koumine binding to TSPO proteoliposomes determined using single-cycle kinetics. (A–D) The sensorgram of Ro5-4864 (A), PK11195 (B), protoporphyrin IX (C) and koumine (D) binding to the TSPO proteoliposomes. The Ro5-4864, PK11195 and koumine concentrations were 3.125, 6.25, 12.5, 25 and 50 nM, and the protoporphyrin IX concentrations were 25, 50, 100, 200 and 400 nM. The running buffer contained 10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl and 1% DMSO. Binding data were collected at a flow rate of 30 μl/min. The association and dissociation phases were 180 and 300 s, respectively. The red traces represent the experimental data, and the black traces represent the global fit of the data to a Langmuir 1:1 interaction model. Sensorgrams show blank and reference subtracted data, and a DMSO correction was also applied. A representative result from three independent experiments is presented.

FIGURE 2

Koumine delays the dissociation of ³H-PK11195 from TSPO. (A) Saturation binding curve of specific ³H-PK11195 binding. (B) Binding activity of 0.1 nM ³H-PK11195 in the absence or presence of different concentrations of koumine. (C) The dissociation curves of ³H-PK11195 in the absence or presence of the indicated concentrations of koumine. For (A) and (B), 50 μg of TSPO protein was incubated for 120 min at 4°C with 0.125–32 nM ³H-PK11195 (A) or 0.1 nM PK11195 in the absence or presence of different concentrations of koumine (B). Specific binding is defined as the difference between total binding and nonspecific binding in the presence of 1000-fold excess unlabeled PK11195. In (B), the basal binding activity was the specific binding value (cpm) in the absence of koumine and was defined as 100%. For (C), 50 μg of TSPO protein was incubated for 120 min at 4°C with 10 nM ³H-PK11195 to achieve pre-equilibration, and dissociation was induced by 10 μM PK11195 in the absence or presence of the indicated concentrations of koumine. Specific binding is defined as the difference between total binding and nonspecific binding in the presence of 10 μM PK11195. Abbreviations: KM, koumine. Data are represented as the mean ± SEM of 4–5 independent experiments performed in duplicate (n = 4–5). *p < 0.05, **p < 0.01 vs. vehicle group. Statistical analysis in (B) was performed using one-way ANOVA followed by the LSD post hoc test.

FIGURE 3

Modulation of the in vivo efficacy of Ro5-4864 by koumine in the formalin test in mice. (A,B) Dose response effects of Ro5-4864 (A) or koumine (B) on phase I in the formalin test. (C) Dose response curves for Ro5-4864 in the absence or presence of 0.4 mg/kg koumine. (D,E) Dose response effects of Ro5-4864 (D) or koumine (E) on phase II in the formalin test. (F) Dose response curves for Ro5-4864 in the absence or presence of an inactive dose of koumine. Koumine (0.08–10 mg/kg) or vehicle was injected s. c. 40 min before formalin injection. Ro5-4864 (0.00016–0.5 mg/kg) or vehicle was injected s. c. 30 min before formalin injection. To determine whether koumine potentiated the anti-inflammatory pain effects of Ro5-4864, koumine (0.4 mg/kg, s. c.) or vehicle was injected 10 min before Ro5-4864 (0.00016–0.5 mg/kg, s. c.) injection. Abbreviations; KM: koumine. Data are represented as the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. the corresponding vehicle group. Statistical analysis in (A,B,D,E) were performed using one-way ANOVA followed by the LSD post hoc test and in (C,F) were performed using the independent samples t-test. Each group consisted of 8–10 mice.

FIGURE 4

Modulation of the in vivo efficacy of Ro5-4864 by koumine successive administration in the rat model of CIA. (A,B,D,E) Effects of repeated administrations of Ro5-4864 (A,D) or koumine (B,E) on MWT (A,B) and hind paw volume (D,E). (C,F) Dose response effects on the MWT (C) and hind paw volume (F) of Ro5-4864 in the absence or presence of koumine (1.0 mg/kg). Seven-week-old Lewis rats were immunized with bovine type II collagen in IFA. Immunized rats received Ro5-4864, koumine or vehicle for seven consecutive days beginning from day 29 after the first collagen injection. The MWT was determined on day 35, and the hind paw volume was measured 60 min following the MWT determination for each rat. Koumine or vehicle was injected s. c., and MWT values were determined 60 min after koumine injection. Ro5-4864 or vehicle was injected i. p., and MWT was determined 50 min after Ro5-4864 injection. To determine whether koumine potentiated the effects of Ro5-4864, koumine (1.0 mg/kg, s. c.) or vehicle was injected 10 min before Ro5-4864 (0.0625–1.0 mg/kg, i. p.) injection, and the MWT was determined 50 min after the last injection. Abbreviations; KM: koumine, MWT: mechanical withdrawal threshold. Data are represented as the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. the corresponding vehicle group. Statistical analysis in (A,B,D,E) were performed using one-way ANOVA followed by the LSD post hoc test and in (C,F) were performed using the independent samples t-test. Each group consisted of 6–10 rats.

FIGURE 5

Modulation of the in vivo efficacy of Ro5-4864 by koumine acute administration in the rat model of CIA. (A,B) Effects of acute administration of Ro5-4864 (A) or koumine (B) on the MWT. (C) Dose response effects on the MWT of Ro5-4864 in the absence or presence of koumine (1.0 mg/kg). Seven-week-old Lewis rats were immunized with bovine type II collagen in IFA. Immunized rats received Ro5-4864, koumine or vehicle on day 29 after the first collagen injection. Koumine or vehicle was injected s. c., and the MWT was determined 60 min after koumine injection. Ro5-4864 or vehicle was injected i. p., and the MWT was determined 50 min after Ro5-4864 injection. To determine whether koumine potentiated the effects of Ro5-4864, koumine (1.0 mg/kg, s. c.) or vehicle was injected 10 min before Ro5-4864 (0.0625–1.0 mg/kg, i. p.) injection, and the MWT was determined 50 min after the last injection. Abbreviations; KM: koumine, MWT: mechanical withdrawal threshold. Data are represented as the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. the corresponding vehicle group. Statistical analysis in (A,B) were performed using one-way ANOVA followed by the LSD post hoc test and in (C) was performed using the independent samples t-test. Each group consisted of 7–11 rats.

FIGURE 6

Modulation of the in vivo efficacy of Ro5-4864 by koumine in the rat model of CCI. (A,B) MWT was increased by acute Ro5-4864 (A) or koumine (B) administration in a dose-dependent manner. (C) Dose response effects on the MWT of Ro5-4864 in the absence or presence of koumine (0.28 mg/kg). Koumine (0.28, 1.4 and 7.0 mg/kg) or vehicle was injected s. c. 9 days after CCI surgery, the MWT was determined 60 min after koumine injection. Ro5-4864 (0.0625, 0.125, 0.25, 0.5 and 1.0 mg/kg) or vehicle was injected i. p. 9 days after CCI surgery, the MWT was determined 50 min after Ro5-4864 injection. To determine whether koumine potentiated the analgesic effects of Ro5-4864, koumine (0.28 mg/kg, s. c.) or vehicle was injected 10 min before Ro5-4864 (0.0625–1.0 mg/kg, i. p.) injection on day 9 after CCI surgery, and the MWT was determined 50 min after the last injection. Abbreviations; KM: koumine, MWT: mechanical withdrawal threshold. Data are represented as the mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 vs. the corresponding vehicle group. Statistical analysis in (A,B) were performed using one-way ANOVA followed by the LSD post hoc test and in (C) was performed using the independent samples t-test. Each group consisted of 6–10 rats.

FIGURE 7

Koumine enhances Ro5-4864- and PK11195-mediated antiproliferation effects in T98G human glioblastoma cells. (A,B) Positive modulation of the antiproliferative effect of an EC₂₀ concentration of Ro5-4864 (A) and PK11195 (B) by koumine in T98G human glioblastoma cells. (C,D) Dose response curve of the antiproliferative effect of Ro5-4864 (C) or PK11195 (D) in the absence or presence of 400 μM or 316.3 μM koumine, respectively. Cell proliferation was determined by the BrdU incorporation assay, which was measured at 370 nm (reference wavelength 492 nm). Cells treated with 0.5% DMSO alone were used as controls. Abbreviations: KM, koumine. The values are expressed as the mean ± SEM of 3–5 independent experiments performed in triplicate (n = 3–5), *p < 0.05 vs. the corresponding vehicle group.