Discovery of a Human Neuromedin U Receptor 1-Selective Hexapeptide Agonist with Enhanced Serum Stability.

Neuromedin U (NMU) activates two NMU receptors (NMUR1 and NMUR2) and is a useful antiobesity drug lead. We report discovery of a hexapeptide agonist, 2-thienylacetyl-Trp1-Phe(4-F)2-Arg3-Pro4-Arg5-Asn6-NH2 (4). However, the NMUR1 selectivity and serum stability of this agonist were unsatisfactory. Through a structure-activity relationship study focused on residue 2 of agonist 4, serum stability, and pharmacokinetic properties, we report here the discovery of a novel NMUR1 selective hexapeptide agonist 7b that suppresses body weight gain in mice.

Withania somnifera root extract prolongs analgesia and suppresses hyperalgesia in mice treated with morphine.

Previous studies demonstrated that Withania somnifera Dunal (WS), a safe medicinal plant, prevents the development of tolerance to the analgesic effect of morphine. In the present study, we investigated whether WS extract (WSE) (100 mg/kg, i.p.) may also modulate the analgesic effect induced by acute morphine administration (2.5, 5, 10 mg/kg, s.c.) in the tail-flick and in the hot plate tests, and if it may prevent the development of 2.5 mg/kg morphine-induced rebound hyperalgesia in the low intensity tail-flick test. Further, to characterize the receptor(s) involved in these effects, we studied, by receptor-binding assay, the affinity of WSE for opioid (µ, δ, k), cannabinoid (CB1, CB2), glutamatergic (NMDA), GABAergic (GABAA, GABAB), serotoninergic (5HT2A) and adrenergic (α2) receptors. The results demonstrated that (i) WSE alone failed to alter basal nociceptive threshold in both tests, (ii) WSE pre-treatment significantly protracted the antinociceptive effect induced by 5 and 10 mg/kg of morphine only in tail-flick test, (iii) WSE pre-treatment prevented morphine-induced hyperalgesia in the low intensity tail-flick test, and (iv) WSE exhibited a high affinity for the GABAA and moderate affinity for GABAB, NMDA and δ opioid receptors. WSE prolongs morphine-induced analgesia and suppresses the development of morphine-induced rebound hyperalgesia probably through involvement of GABAA, GABAB, NMDA and δ opioid receptors. This study suggests the therapeutic potential of WSE as a valuable adjuvant agent in opioid-sparing therapies.

Development of a neuromedin U-human serum albumin conjugate as a long-acting candidate for the treatment of obesity and diabetes. Comparison with the PEGylated peptide.

Neuromedin U (NMU) is an endogenous peptide implicated in the regulation of feeding, energy homeostasis, and glycemic control, which is being considered for the therapy of obesity and diabetes. A key liability of NMU as a therapeutic is its very short half-life in vivo. We show here that conjugation of NMU to human serum albumin (HSA) yields a compound with long circulatory half-life, which maintains full potency at both the peripheral and central NMU receptors. Initial attempts to conjugate NMU via the prevalent strategy of reacting a maleimide derivative of the peptide with the free thiol of Cys34 of HSA met with limited success, because the resulting conjugate was unstable in vivo. Use of a haloacetyl derivative of the peptide led instead to the formation of a metabolically stable conjugate. HSA-NMU displayed long-lasting, potent anorectic, and glucose-normalizing activity. When compared side by side with a previously described PEG conjugate, HSA-NMU proved superior on a molar basis. Collectively, our results reinforce the notion that NMU-based therapeutics are promising candidates for the treatment of obesity and diabetes.

Identification of non-peptidic neuromedin U receptor modulators by a robust homogeneous screening assay.

AIM: To develop a homogeneous binding assay for high-throughput screening (HTS) of hit compounds at human neuromedin U receptor (hNMU-R) 1 and to identify non-peptidic small molecule hNMU-R modulators through functional assessments and structure-activity relationship (SAR) analyses. METHODS: Membrane preparations of Chinese hamster ovary cells (CHO-K1) stably expressing hNMU-R1, [125I]hNMU-25, and wheat germ agglutinin-coupled microbeads were used to develop an HTS assay based on scintillation proximity assay (SPA) technology. This method was applied to a large-scale screening campaign against a diverse library of 36,000 synthetic compounds or natural products and subsequent confirmation studies. CHO-K1 cells stably expressing full-length hNMU-R1 or hNMU-R2 and a calcium-sensitive dye were employed to functionally measure intracellular calcium mobilization upon ligand stimulation. Preliminary SAR was determined based on limited structural modifications. RESULTS: The Ki value (0.7 nmol/L) of hNMU-25 (the natural ligand) at hNMU-R1 measured by the SPA method was consistent with that reported in the literature, and the Z'factor for this HTS assay was 0.81. A total of 100 hits, showing more than 30% competitive inhibition on [125I]hNMU-25 binding to hNMU-R1, were identified initially, 3 of which were confirmed thereafter to have reasonable hNMU-R1-binding affinities and similar chemical structures. Based on their common molecular skeleton, 203 analogs were synthesized and tested. Among the 16 analogs that retained variable hNMU-R1- binding abilities, 2 elicited calcium influx in both hNMU-R1 and hNMU-R2-expressing cells, but none displayed antagonist activity. CONCLUSION: The homogeneous hNMU-R1 binding assay is an efficient and robust tool for screening potential hNMU-R modulators. Two non-selective hNMU-R agonists discovered are of low molecular weight nature with novel chemical structures. The preliminary SAR investigation suggests that both the triphenyl and guanidinol groups are crucial to the bioactivities observed.

Functional characterization of cell lines for high-throughput screening of human neuromedin U receptor subtype 2 specific agonists using a luciferase reporter gene assay.

We developed a functional cell-based high-throughput screening (HTS) assay to identify modulators of the human neuromedin subtype 2 receptor. This assay utilized the signal transduction pathway of hNMU2R, which is positively coupled to adenylyl cyclase and downstream calcium signal pathways. We describe in detail a robust, sensitive, and functional assay for the hNMU2R G-protein-coupled receptor expressed in human embryonic kidney (HEK)-293 cells, whose activity was reflected by a luciferase reporter gene transcriptionally regulated by a 3-repeat serum response element (SRE)-3 repeat multiple response element (MRE)-3 repeat cyclic AMP (cAMP) response element (CRE)-VIP mini promoter. The HEK 293 clonal cell line, stably co-transfected with the 3xSRE/3xMRE/3xCRE/VIP mini promoter-driven luciferase and pCDNA3.1-NMU2R plasmid, was selected by active geneticin sulfate and their ability to express luciferase with a forskolin challenge following hNMU plus forskolin, known to activate intracellular signal transduction. Then the cell density, incubation time, dimethyl sulfoxide (DMSO) concentration used to screen the hNMU receptor subtype 2 specific agonist were optimized, and whether intrinsic luminescent substance of extracts isolated from traditional Chinese herbs disturbs luminescence of luciferase expressed in HEK293 cells was considered. The optimal incubation time was found to be between 8 and 9h, the cell density and DMSO concentrations were optimized from 3x10(4) to 6x10(4), and less than 2%, respectively. Our data show that hNMU2R luci-HEK293 cells and their assay exhibit a low background and ideal model for high-throughput screening. These results demonstrate that this reporter gene assay is useful for pharmacological analysis, and is amenable to HTS for human NMU2R agonists.

Substance identification by quantitative characterization of oscillatory activity in murine spinal cord networks on microelectrode arrays.

This paper presents a novel and comprehensive method to identify substances on the basis of electrical activity and is a substantial improvement for drug screening. The spontaneous activity of primary neuronal networks is influenced by neurotransmitters, ligands, and other substances in a similar fashion as known from in vivo pharmacology. However, quantitative methods for the identification of substances through their characteristic effects on network activity states have not yet been reported. We approached this problem by creating a database including native activity and five drug-induced oscillatory activity states from extracellular multisite recordings from microelectrode arrays. The response profiles consisted of 30 activity features derived from the temporal distribution of action potentials, integrated burst properties, calculated coefficients of variation, and features of Gabor fits to autocorrelograms. The different oscillatory states were induced by blocking neurotransmitter receptors for: (i) GABA(A); (ii) glycine; (iii) GABA(A) and glycine; (iv) all major synaptic types except AMPA, and (v) all major synapses except NMDA. To test the identification capability of the six substance-specific response profiles, five blind experiments were performed. The response features from the unknown substances were compared to the database using proximity measures using the normalized Euclidian distance to each activity state. This process created six identification coefficients where the smallest correctly identified the unknown substances. Such activity profiles are expected to become substance-specific 'finger prints' that classify unique responses to known and unknown substances. It is anticipated that this kind of approach will help to quantify pharmacological responses of networks used as biosensors.