Bombesin receptor subtype 3 (BRS-3) is a G protein coupled receptor whose natural ligand is unknown. We developed potent, selective agonist (Bag-1, Bag-2) and antagonist (Bantag-1) ligands to explore BRS-3 function. BRS-3-binding sites were identified in the hypothalamus, caudal brainstem, and several midbrain nuclei that harbor monoaminergic cell bodies. Antagonist administration increased food intake and body weight, whereas agonists increased metabolic rate and reduced food intake and body weight. Prolonged high levels of receptor occupancy increased weight loss, suggesting a lack of tachyphylaxis. BRS-3 agonist effectiveness was absent in Brs3(-/Y) (BRS-3 null) mice but was maintained in Npy(-/-)Agrp(-/-), Mc4r(-/-), Cnr1(-/-), and Lepr(db/db) mice. In addition, Brs3(-/Y) mice lost weight upon treatment with either a MC4R agonist or a CB1R inverse agonist. These results demonstrate that BRS-3 has a role in energy homeostasis that complements several well-known pathways and that BRS-3 agonists represent a potential approach to the treatment of obesity.

Macrophages are widely recognized as cells that play a central role in the regulation of immune and inflammatory activities, as well as tissue remodeling. The execution of these activities is mediated by complex and multifactorial processes involving macrophage products (1). In response to antigens such as LPS, 1 macrophages secrete proinflammatory cytokines and oxidants such as TNF␣, IL-6, IL-1␤, IL-12, and nitric oxide (1). TNF␣ and IL-6 are important macrophage secretory products that contribute to pathophysiological changes associated with several acute and chronic inflammatory conditions, including septic shock, autoimmune diseases, wasting, rheumatoid arthritis, inflammatory bowel disease, and respiratory distress syndrome (2-4). A number of regulatory molecules termed macrophage-deactivating factors have been the focus of considerable research (5-9). These molecules are believed to prevent the excessive production of proinflammatory mediators, including TNF␣ and IL-6. Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two multifunctional neuropeptides whose primary immunomodulatory function is anti-inflammatory in nature. VIP and PACAP inhibit several macrophage functions, including phagocytosis, respiratory burst, and chemotaxis (reviewed in Ref. 10), as well as LPS-induced IL-6 production (11). Furthermore, we have recently demonstrated that VIP and PACAP protect mice from endotoxic shock presumably through the inhibition of TNF␣ and IL-6 production.2 VIP was also reported to suppress TNF␣ production in human peripheral blood cells (12,13).Both VIP and PACAP interact with a family of three VIP/ PACAP receptors, VIP 1 R, and VIP 2 R, which exhibit similar affinities for the two neuropeptides and activate primarily the adenylate cyclase system, and PACAP-R, which exhibits a 2-3 orders of magnitude higher affinity for PACAP than for VIP and activates both the adenylate cyclase and phospholipase C systems (reviewed in Ref. 14). Peritoneal macrophages have been described to possess VIP 1 R and PACAP-R (15-17).LPS is a major stimulus for the production of proinflammatory cytokines, including TNF␣, from macrophages (1). TNF␣ synthesis is controlled at several levels. Whereas post-transcriptional, translational, and post-translational mechanisms play important roles, TNF␣ transcription appears to be the primary regulatory site. Although the TNF␣ promoter contains a complex array of transactivating binding sites, the kB and CRE elements appear essential for maximal TNF␣

Tumor necrosis factor-alpha (TNF-alpha) production accompanies CNS insults of all kinds. Because the neuropeptide vasoactive intestinal peptide (VIP) and the structurally related peptide pituitary adenylyl cyclase-activating polypeptide (PACAP) have potent anti-inflammatory effects in the periphery, we investigated whether these effects extend to the CNS. TNF-alpha mRNA was induced within 2 hr after rat spinal cord transection, and its upregulation was suppressed by a synthetic VIP receptor agonist. Cultured rat microglia were used to examine the mechanisms underlying this inhibition because microglia are the likely source of TNF-alpha in injured CNS. In culture, increases in TNF-alpha mRNA resulting from lipopolysaccharide (LPS) stimulation were reduced significantly by 10(-7) m VIP and completely eliminated by PACAP at the same concentration. TNF-alpha protein levels were reduced 90% by VIP or PACAP at 10(-7) m. An antagonist of VPAC(1) receptors blocked the action of VIP and PACAP, and a PAC(1) antagonist blocked the action of PACAP. A direct demonstration of VIP binding on microglia and the existence of mRNAs for VPAC(1) and PAC(1) (but not VPAC(2)) receptors argue for a receptor-mediated effect. The action of VIP is cAMP-mediated because (1) activation of cAMP by forskolin mimics the action; (2) PKA inhibition by H89 reverses the neuropeptide-induced inhibition; and (3) the lipophilic neuropeptide mimic, stearyl-norleucine(17) VIP (SNV), which does not use a cAMP-mediated pathway, fails to duplicate the inhibition. We conclude that VIP and PACAP inhibit the production of TNF-alpha from activated microglia by a cAMP-dependent pathway.

Bombesin receptor subtype-3 (BRS-3) is an orphan G proteincoupled receptor implicated in the regulation of energy homeostasis. Here, we report the biologic effects of a highly optimized BRS-3 agonist, ( -5046). Single oral doses of MK-5046 inhibited 2-h and overnight food intake and increased fasting metabolic rate in wild-type but not Brs3 knockout mice. Upon dosing for 14 days, MK-5046 at 25 mg ⅐ kg Ϫ1 ⅐ day Ϫ1reduced body weight of diet-induced obese mouse by 9% compared with vehicle-dosed controls. In mice, 50% brain receptor occupancy was achieved at a plasma concentration of 0.34 Ϯ 0.23 M. With chronic dosing, effects on metabolic rate, rather than food intake, seem to be the predominant mechanism for weight reduction by MK-5046. The compound also effectively reduced body weight in rats and caused modest increases in body temperature, heart rate, and blood pressure. These latter effects on temperature, heart rate, and blood pressure were transient in nature and desensitized with continued dosing. MK-5046 is the first BRS-3 agonist with properties suitable for use in larger mammals. In dogs, MK-5046 treatment produced statistically significant and persistent weight loss, which was initially accompanied by increases in body temperature and heart rate that abated with continued dosing. Our results demonstrate antiobesity efficacy for MK-5046 in rodents and dogs and further support BRS-3 agonism as a new approach to the treatment of obesity.

We report the development and characterization of compound 22 (MK-5046), a potent, selective small molecule agonist of BRS-3 (bombesin receptor subtype-3). In pharmacological testing using diet-induced obese mice, compound 22 caused mechanism-based, dose-dependent reductions in food intake and body weight.