MCH-R1 Antagonist GPS18169, a Pseudopeptide, Is a Peripheral Anti-Obesity Agent in Mice

Abstract

Melanin-concentrating hormone (MCH) is a 19 amino acid long peptide found in the brain of animals, including fishes, batrachians, and mammals. MCH is implicated in appetite and/or energy homeostasis. Antagonists at its receptor (MCH-R1) could be major tools (or ultimately drugs) to understand the mechanism of MCH action and to fight the obesity syndrome that is a worldwide societal health problem. Ever since the deorphanisation of the MCH receptor, we cloned, expressed, and characterized the receptor MCH-R1 and started a vast medicinal chemistry program aiming at the discovery of such usable compounds. In the present final work, we describe GPS18169, a pseudopeptide antagonist at the MCH-R1 receptor with an affinity in the nanomolar range and a Ki for its antagonistic effect in the 20 picomolar range. Its metabolic stability is rather ameliorated compared to its initial parent compound, the antagonist S38151. We tested it in an in vivo experiment using high diet mice. GPS18169 was found to be active in limiting the accumulation of adipose tissues and, correlatively, we observed a normalization of the insulin level in the treated animals, while no change in food or water consumption was observed.

Keywords: MCH; MCH-R1 antagonist; diet-induced obesity; pseudopeptide; treatment.

Figure 1

Two structures of peptides representative of the families bearing either a lactam bridge (GPS12731) or a triazole bridge (GPS15290). The lactam bridge was built between a glutamic acid (or aspartic acid for other pseudopeptides) and ornithine (or a lysine) side chains. The triazole bridge was synthesized between an azido-homoalanine and a homopropargylglycine. The red circles point at the bridge structure.

Figure 2

Comparative view of the relative intensity of GPS15290 metabolites in vitro in hepatocytes. Green: mouse metabolites, blue: rat metabolites. The starting pseudopeptide is GPS15290: pGua-[N₃-hAla-MLR-βhVal-RP-hPra]-W-OH (mass = 1352.7); M1 is (hPra -W)-(N₃-hAla-pGua) (mass = 604.2); M2 is R-βhVal-RP (mass = 540.3); M3 is (hPra-W)-(N₃-hAla-pGua)-M (mass = 735.3); M4 is βhVal-RP-(hPra-W)-(N₃-hAla-pGua) (mass = 970.5); M5 is P-hPra-(N₃-hAla -pGua)-MLR-βhVal-R (mass = 1184.6); M6 is pGua-[(N₃-hAla + O^-)-(M+O)LR-βhVal-RP-hPra]-W-OH, oxidations on Met and on N₃-hAla (mass = 1384.7); M7 is R-βhVal-RP-(hPra-W)-((N₃-hAla + O^-)-pGua), oxidation on N₃-hAla (mass = 1142.6); M8 is pGua-[N₃-hAla-MLR-βhVal-RP-hPra] (mass is 1166.6); M9 is pGua-[N₃-hAla-(M+O)LR-βhVal-RP-hPra]-W-OH oxidation on Met (mass = 1368.7); M10 is R-βhVal-RP-(hPra-W)-(N₃-hAla-pGua) (mass = 1126.6); M11, with a mass of 1179.5 remained unknown; M12 is (hPra-W)-(N₃-hAla-pGua)-ML (mass = 848.4); M13 is LR-βhVal-RP-hPra-(N₃-hAla-pGua) (mass = M1184.6); M14 is pGua-[N₃-hAla-(M+O)LR-βhVal-RP-hPra]-W-OH, oxidation on Met and opening by hydrolysis (mass is 1386.7); M15 is pGua-[(N₃-hAla + O^-)-MLR-βhVal-RP-hPra]-W-OH oxidation on Met and opening by hydrolysis (mass = 1386.7); M16 is MLR-βhVal-RP-(hPra-W)-(N₃-hAla-pGua) (mass is 1370.7); M17 is pGua-[(N₃-hAla + O^-)-MLR-βhVal-RP-hPra]-(W + O)-OH, oxidations on Met and on Trp (mass is 1384.7); M18 is pGua-[N₃-hAla-MLR-βhVal-RP-hPra]-W-OH, opening by hydrolysis (mass is 1370.7); M19 is pGua-[(N₃-hAla + O^-)-MLR-βhVal-RP-hPra]-W-OH, oxidation on N₃-hAla (mass = 1368.7); M20 is pGua-[N₃-hAla-MLR-βhVal-RP-hPra]-W-OH, opening by hydrolysis (mass is 1370.7). These attributions were carried out based on ms/ms data.

Figure 3

Examples of isotherms obtained with the two antagonistic pseudopeptides, GPS13290 and GPS18169. The assays were binding displacement experiments (panels A, B) of labelled MCH at membranes overexpressing the human MCH-R1 or MCH-induced cytosolic Ca²⁺ ion mobilization inhibition in living cells (panels C, D). The ligand was [¹²⁵I]-[Phe¹³,Tyr¹⁹]-MCH. Independent experiments were performed at least twice using different batches of membranes from stably transfected CHO cells, and each point was obtained in triplicate. Concentration isotherms were obtained using eight concentrations of each product from 10–11 to 10–6 M. The data represent the mean ± SD of the triplicate measure. Experiments were run at least twice, independently.

Figure 4

Water (A) and food (B) intake in high-fat diet-induced obesity model in C57BL/6 mice. Male C57BL/6 mice were fed a high-fat diet (HFD, 60% of calories) or a standard chow diet (STD) from 4 weeks of age for 8 weeks before treatment. Once obesity was installed, GPS18169, at 5 and 10 mg/kg and vehicle (WFI) was administered by intraperitoneal injection (IP) once daily for 12 weeks after 8 weeks of HFD feeding. Food (B) and water (A) intake were recorded twice weekly. ⁺ p < 0.05, vs. Vehicle (High-fat Diet); two-way ANOVA followed by Bonferroni test. Green squares: vehicle and normal diet; red squares: vehicle and high fat diet; orange triangles: GPS18169 (10 mg/kg) and high fat diet; greenish-yellow triangles: GPS18169 (5mg/kg) and high fat diet. Complete individual data are presented in Table S4 (Water) and Table S5 (Food).

Figure 5

Body weight changes in high-fat diet-induced obesity model in C57Bl/6 mice. Male C57BL/6 mice were fed a high-fat diet (HFD) (60% of calories) or a standard chow diet (STD) from 4 weeks of age for 8 weeks before treatment. Test article, GPS18169, at 5 and 10 mg/kg and vehicle (WFI) were administered by intraperitoneal injection (IP) once daily for 12 weeks after 8 weeks of HFD feeding. The body weight was measured thrice a week. Complete individual data are presented in Table S6 (body weight changes).