Structural determinants of dual incretin receptor agonism by tirzepatide.

SignificanceTirzepatide is a dual agonist of the glucose-dependent insulinotropic polypeptide receptor (GIPR) and the glucagon-like peptide-1 receptor (GLP-1R), which are incretin receptors that regulate carbohydrate metabolism. This investigational agent has proven superior to selective GLP-1R agonists in clinical trials in subjects with type 2 diabetes mellitus. Intriguingly, although tirzepatide closely resembles native GIP in how it activates the GIPR, it differs markedly from GLP-1 in its activation of the GLP-1R, resulting in less agonist-induced receptor desensitization. We report how cryogenic electron microscopy and molecular dynamics simulations inform the structural basis for the unique pharmacology of tirzepatide. These studies reveal the extent to which fatty acid modification, combined with amino acid sequence, determines the mode of action of a multireceptor agonist.

Tirzepatide is an imbalanced and biased dual GIP and GLP-1 receptor agonist.

Tirzepatide (LY3298176) is a dual GIP and GLP-1 receptor agonist under development for the treatment of type 2 diabetes mellitus (T2DM), obesity, and nonalcoholic steatohepatitis. Early phase trials in T2DM indicate that tirzepatide improves clinical outcomes beyond those achieved by a selective GLP-1 receptor agonist. Therefore, we hypothesized that the integrated potency and signaling properties of tirzepatide provide a unique pharmacological profile tailored for improving broad metabolic control. Here, we establish methodology for calculating occupancy of each receptor for clinically efficacious doses of the drug. This analysis reveals a greater degree of engagement of tirzepatide for the GIP receptor than the GLP-1 receptor, corroborating an imbalanced mechanism of action. Pharmacologically, signaling studies demonstrate that tirzepatide mimics the actions of native GIP at the GIP receptor but shows bias at the GLP-1 receptor to favor cAMP generation over ß-arrestin recruitment, coincident with a weaker ability to drive GLP-1 receptor internalization compared with GLP-1. Experiments in primary islets reveal ß-arrestin1 limits the insulin response to GLP-1, but not GIP or tirzepatide, suggesting that the biased agonism of tirzepatide enhances insulin secretion. Imbalance toward GIP receptor, combined with distinct signaling properties at the GLP-1 receptor, together may account for the promising efficacy of this investigational agent.

Regulation of Endogenous (Male) Rodent GLP-1 Secretion and Human Islet Insulin Secretion by Antagonism of Somatostatin Receptor 5.

Incretin and insulin responses to nutrient loads are suppressed in persons with diabetes, resulting in decreased glycemic control. Agents including sulfonylureas and dipeptidyl peptidase-4 inhibitors (DPP4i) partially reverse these effects and provide therapeutic benefit; however, their modes of action limit efficacy. Because somatostatin (SST) has been shown to suppress insulin and glucagonlike peptide-1 (GLP-1) secretion through the Gi-coupled SST receptor 5 (SSTR5) isoform in vitro, antagonism of SSTR5 may improve glycemic control via intervention in both pathways. Here, we show that a potent and selective SSTR5 antagonist reverses the blunting effects of SST on insulin secretion from isolated human islets, and demonstrate that SSTR5 antagonism affords increased levels of systemic GLP-1 in vivo. Knocking out Sstr5 in mice provided a similar increase in systemic GLP-1 levels, which were not increased further by treatment with the antagonist. Treatment of mice with the SSTR5 antagonist in combination with a DPP4i resulted in increases in systemic GLP-1 levels that were more than additive and resulted in greater glycemic control compared with either agent alone. In isolated human islets, the SSTR5 antagonist completely reversed the inhibitory effect of exogenous SST-14 on insulin secretion. Taken together, these data suggest that SSTR5 antagonism should increase circulating GLP-1 levels and stimulate insulin secretion (directly and via GLP-1) in humans, improving glycemic control in patients with diabetes.

An Allosteric Potentiator of the Dopamine D1 Receptor Increases Locomotor Activity in Human D1 Knock-In Mice without Causing Stereotypy or Tachyphylaxis.

Allosteric potentiators amplify the sensitivity of physiologic control circuits, a mode of action that could provide therapeutic advantages. This hypothesis was tested with the dopamine D1 receptor potentiator DETQ [2-(2,6-dichlorophenyl)-1-((1S,3R)-3-(hydroxymethyl)-5-(2-hydroxypropan-2-yl)-1-methyl-3,4-dihydroisoquinolin-2(1H)-yl)ethan-1-one]. In human embryonic kidney 293 (HEK293) cells expressing the human D1 receptor, DETQ induced a 21-fold leftward shift in the cAMP response to dopamine, with a Kb of 26 nM. The maximum response to DETQ alone was ∼12% of the maximum response to dopamine, suggesting weak allosteric agonist activity. DETQ was ∼30-fold less potent at rat and mouse D1 receptors and was inactive at the human D5 receptor. To enable studies in rodents, an hD1 knock-in mouse was generated. DETQ (3-20 mg/kg orally) caused a robust (∼10-fold) increase in locomotor activity (LMA) in habituated hD1 mice but was inactive in wild-type mice. The LMA response to DETQ was blocked by the D1 antagonist SCH39166 and was dependent on endogenous dopamine. LMA reached a plateau at higher doses (30-240 mg/kg) even though free brain levels of DETQ continued to increase over the entire dose range. In contrast, the D1 agonists SKF 82958, A-77636, and dihydrexidine showed bell-shaped dose-response curves with a profound reduction in LMA at higher doses; video-tracking confirmed that the reduction in LMA caused by SKF 82958 was due to competing stereotyped behaviors. When dosed daily for 4 days, DETQ continued to elicit an increase in LMA, whereas the D1 agonist A-77636 showed complete tachyphylaxis by day 2. These results confirm that allosteric potentiators may have advantages compared with direct-acting agonists.

A Novel Nociceptin Receptor Antagonist LY2940094 Inhibits Excessive Feeding Behavior in Rodents: A Possible Mechanism for the Treatment of Binge Eating Disorder.

Nociceptin/orphanin FQ (N/OFQ), a 17 amino acid peptide, is the endogenous ligand of the ORL1/nociceptin-opioid-peptide (NOP) receptor. N/OFQ appears to regulate a variety of physiologic functions including stimulating feeding behavior. Recently, a new class of thienospiro-piperidine-based NOP antagonists was described. One of these molecules, LY2940094 has been identified as a potent and selective NOP antagonist that exhibited activity in the central nervous system. Herein, we examined the effects of LY2940094 on feeding in a variety of behavioral models. Fasting-induced feeding was inhibited by LY2940094 in mice, an effect that was absent in NOP receptor knockout mice. Moreover, NOP receptor knockout mice exhibited a baseline phenotype of reduced fasting-induced feeding, relative to wild-type littermate controls. In lean rats, LY2940094 inhibited the overconsumption of a palatable high-energy diet, reducing caloric intake to control chow levels. In dietary-induced obese rats, LY2940094 inhibited feeding and body weight regain induced by a 30% daily caloric restriction. Last, in dietary-induced obese mice, LY2940094 decreased 24-hour intake of a high-energy diet made freely available. These are the first data demonstrating that a systemically administered NOP receptor antagonist can reduce feeding behavior and body weight in rodents. Moreover, the hypophagic effect of LY2940094 is NOP receptor dependent and not due to off-target or aversive effects. Thus, LY2940094 may be useful in treating disorders of appetitive behavior such as binge eating disorder, food choice, and overeating, which lead to obesity and its associated medical complications and morbidity.

Pharmacological characterization of the cannabinoid CB1 receptor PET ligand ortholog, [³H]MePPEP.

MePPEP ((3R,5R)-5-(3-methoxy-phenyl)-3-((R)-1-phenyl-ethylamino)-1-(4-trifluoromethyl-phenyl)-pyrrolidin-2-one) is an inverse agonist shown to be an effective PET ligand for labeling cannabinoid CB1 receptors in vivo. [¹¹C]MePPEP and structurally related analogs have been reported to specifically and reversibly label cannabinoid CB1 receptors in rat and non-human primate brains, and [¹¹C]MePPEP has been used in human subjects as a PET tracer. We have generated [³H]MePPEP, an ortholog of [¹¹C]MePPEP, to characterize the molecular pharmacology of the cannabinoid CB1 receptor across preclinical and clinical species. [³H]MePPEP demonstrates saturable, reversible, and single-site high affinity binding to cannabinoid CB1 receptors. In cerebellar membranes purified from brains of rat, non-human primate and human, and cells ectopically expressing recombinant human cannabinoid CB1 receptor, [³H]MePPEP binds cannabinoid CB1 receptors with similar affinity with K(d) values of 0.09 nM, 0.19 nM, 0.14 nM and 0.16 nM, respectively. Both agonist and antagonist cannabinoid ligands compete [³H]MePPEP with predicted rank order potency. No specific binding is present in autoradiographic sections from cannabinoid CB1 receptor knockout mouse brains, demonstrating that [³H]MePPEP selectively binds cannabinoid CB1 receptors in native mouse tissue. Furthermore, [³H]MePPEP binding to anatomical sites in mouse and rat brain is comparable to the anatomical profiles of [¹¹C]MePPEP in non-human primate and human brain in vivo, as well as the binding profiles of other previously described cannabinoid CB1 receptor agonist and antagonist radioligands. Therefore, [³H]MePPEP is a promising tool for translation of preclinical cannabinoid CB1 receptor pharmacology to clinical PET ligand and cannabinoid CB1 receptor inverse agonist therapeutic development.

Structure activity relationship studies of carboxamido-biaryl ethers as opioid receptor antagonists (OpRAs). Part 2.

A series of 6-bicycloaryloxynicotinamides were identified as opioid receptor antagonists at mu, kappa, and delta receptors. Compounds in the 6-(2,3,4,5-tetrahydro-1H-benzo[c]azepin-7-yloxy)nicotinamide scaffold exhibited potent in vitro functional antagonism at all three receptors.

Structure-activity relationship studies of carboxamido-biaryl ethers as opioid receptor antagonists (OpRAs). Part 1.

A structurally unique and new class of opioid receptor antagonists (OpRAs) that bear no structural resemblance with morphine or endogenous opioid peptides has been discovered. A series of carboxamido-biaryl ethers were identified as potent receptor antagonists against mu, kappa and delta opioid receptors. The structure-activity relationship indicated para-substituted aryloxyaryl primary carboxamide bearing an amine tether on the distal phenyl ring was optimal for potent in vitro functional antagonism against three opioid receptor subtypes.

Na+ modulation, inverse agonism, and anorectic potency of 4-phenylpiperidine opioid antagonists.

Differences in the anorectic activity of morphinan (e.g., naltrexone) and 3,4-dimethyl-4-(3-hydroxyphenyl)piperidine (4PP) opioid receptor antagonists have been described. In an attempt to explain these differences, the influence of Na(+) on opioid binding affinity and functional activity of 4PP antagonists was compared to other opioid antagonists. The binding affinities of neutral antagonists were unaffected by the addition of Na(+), whereas that for the peptide, inverse agonist N,N-diallyl-Tyr-Aib-Aib-Phe-Leu-OH (ICI174864) was increased. Similarly, the binding affinities of the 4PP antagonist (3R,4R)-1-((S)-3-hydroxy-3-cyclohexylpropyl)-4-(3-hydroxyphenyl)-3,4-dimethyl-1-piperidine (LY255582) and other 4PP antagonists were increased in the presence of Na(+) with the greatest effects at the delta opioid receptor followed by the mu and kappa opioid receptors, respectively. Similar to ICI174864, 4PP antagonists were found to inhibit basal GTPgamma[(35)S] binding at the delta opioid receptor indicating inverse agonist activity. A correlation was observed between the binding affinities in the presence of Na(+), the inverse agonist potency, and the anorectic potency of 4PP antagonists. These data suggest that 4PP antagonists differ from morphinan antagonists in their inverse agonist activity and suggest a relationship between inverse agonism and anorectic activity.

Na+-dependent high affinity binding of [3H]LY515300, a 3,4-dimethyl-4-(3-hydroxyphenyl)piperidine opioid receptor inverse agonist.

Analogues of 3,4-dimethyl-4-(3-hydroxyphenyl)piperidines are high affinity inverse agonists for micro-, delta- and kappa-opioid receptors. To characterize inverse agonist binding, we synthesized a high specific activity radioligand from this series, [3H]LY515300 (3-[1-((3-cyclohexyl-[3,4-3H(2)])-3(R,S)-hydroxypropyl)-3(R),4(R)-dimethylpiperidin-4-yl]phenol). In membranes expressing cloned human opioid receptors, [3H]LY515300 binding was saturable and exhibited low nonspecific binding. [3H]LY515300 bound with high affinity to the micro- (K(d)=0.07 nM), delta- (K(d)=0.92 nM) and kappa-(K(d)=0.45 nM) opioid receptors. High affinity [3H]LY515300 binding to all opioid receptors was Na(+)-dependent, a characteristic of inverse agonists. Displacement by standard opioid compounds yielded K(i) values consistent with their known opioid receptor affinities. Autoradiographic localization of specific [3H]LY515300 binding in rat and guinea pig brain was high in areas known to express high levels of opioid (particularly micro-opioid receptor) binding sites including the caudate, nucleus accumbens, and nucleus tractus solitarius. Thus, [3H]LY515300 is the first radiolabeled opioid receptor inverse agonist useful for the study of opioid receptors in cell lines and native tissues.

Peptides that regulate food intake: antagonism of opioid receptors reduces body fat in obese rats by decreasing food intake and stimulating lipid utilization.

Agonists to opioid receptors induce a positive energy balance, whereas antagonists at these receptors reduce food intake and body weight in rodent models of obesity. An analog of 3,4-dimethyl-4-(3-hydroxyphenyl)piperidine, LY255582, is a potent non-morphinan antagonist for mu-, kappa-, and delta-receptors (K(i) of 0.4, 2.0, and 5.2 nM, respectively). In the present study, we examined the effects of oral LY255582 treatment on caloric intake, calorie expenditure, and body composition in dietary-induced obese rats. Acute oral treatment of LY255582 produced a dose-dependent decrease in energy intake and respiratory quotient (RQ), which correlated with the occupancy of central opioid receptors. Animals receiving chronic oral treatment with LY255582 for 14 days maintained a negative energy balance that was sustained by increased lipid use. Analysis of body composition revealed a reduction in fat mass accretion, with no change in lean body mass, in animals treated with LY255582. Therefore, chronic treatment with LY255582 reduces adipose tissue mass by reducing energy intake and stimulating lipid use.

GH-releasing peptide-2 increases fat mass in mice lacking NPY: indication for a crucial mediating role of hypothalamic agouti-related protein.

Ghrelin, an endogenous GH secretagogue, is capable of stimulating adiposity in rodents. Because such adiposity was thought to be mediated by hypothalamic NPY neurons, we investigated by which mechanism a synthetic ghrelin receptor agonist, GHRP-2, would generate a positive energy balance in NPY-deficient [Npy(-/-) mice] and wild-type controls. A dose-dependent increase in body weight and food intake was observed during daily sc injections with GHRP-2. Pre- and posttreatment analysis of body composition indicated increased fat mass and bone mass but not lean mass. Respiratory quotient was increased in GHRP-2-treated mice, indicating preservation of fat. Hypothalamic mRNA levels of agouti- related protein (AGRP), an orexigenic melanocortin receptor antagonist, increased after GHRP-2 treatment. Competitive blockade of AGRP action by melanocortin-receptor agonist MT-II prevented GHRP-induced weight gain in Npy(-/-) mice. In conclusion, chronic peripheral treatment with a ghrelin receptor agonist induced a positive energy balance leading to fat gain in the absence of NPY. These effects could be mediated in part by AGRP. To date, there are few therapeutics that can produce a positive energy balance. Ghrelin receptor agonists offer a treatment option for syndromes like anorexia nervosa, cancer cachexia, or AIDS wasting.