Ana B. Bueno

Differential Activation and Modulation of the Glucagon-like Peptide-1 Receptor by Small Molecule Ligands

The glucagon-like peptide-1 receptor (GLP-1R) is a major therapeutic target for the treatment of type 2 diabetes due to its role in glucose homeostasis. Despite the availability of peptide-based GLP-1R drugs for treatment of this disease, there is great interest in developing small molecules that can be administered orally. The GLP-1R system is complex, with multiple endogenous and clinically used peptide ligands that exhibit different signaling biases at this receptor. This study revealed that small molecule ligands acting at this receptor are differentially biased to peptide ligands and also from each other with respect to the signaling pathways that they activate. Furthermore, allosteric small molecule ligands were also able to induce bias in signaling mediated by orthosteric ligands. This was dependent on both the orthosteric and allosteric ligand as no two allosteric-orthosteric ligand pairs could induce the same signaling profile. We highlight the need to profile compounds across multiple signaling pathways and in combination with multiple orthosteric ligands in systems such as the GLP-1R where more than one endogenous ligand exists. In the context of pleiotropical coupling of receptors and the interplay of multiple pathways leading to physiologic responses, profiling of small molecules in this manner may lead to a better understanding of the physiologic consequences of biased signaling at this receptor. This could enable the design and development of improved therapeutics that have the ability to fine-tune receptor signaling, leading to beneficial therapeutic outcomes while reducing side effect profiles.

Small molecule drug discovery at the glucagon-like peptide-1 receptor.

The therapeutic success of peptide glucagon-like peptide-1 (GLP-1) receptor agonists for the treatment of type 2 diabetes mellitus has inspired discovery efforts aimed at developing orally available small molecule GLP-1 receptor agonists. Although the GLP-1 receptor is a member of the structurally complex class B1 family of GPCRs, in recent years, a diverse array of orthosteric and allosteric nonpeptide ligands has been reported. These compounds include antagonists, agonists, and positive allosteric modulators with intrinsic efficacy. In this paper, a comprehensive review of currently disclosed small molecule GLP-1 receptor ligands is presented. In addition, examples of “ligand bias” and “probe dependency” for the GLP-1 receptor are discussed; these emerging concepts may influence further optimization of known molecules or persuade designs of expanded screening strategies to identify novel chemical starting points for GLP-1 receptor drug discovery.

Positive Allosteric Modulation of the Glucagon-like Peptide-1 Receptor by Diverse Electrophiles

Therapeutic intervention to activate the glucagon-like peptide-1 receptor (GLP-1R) enhances glucose-dependent insulin secretion and improves energy balance in patients with type 2 diabetes mellitus. Studies investigating mechanisms whereby peptide ligands activate GLP-1R have utilized mutagenesis, receptor chimeras, photo-affinity labeling, hydrogen-deuterium exchange, and crystallography of the ligand-binding ectodomain to establish receptor homology models. However, this has not enabled the design or discovery of drug-like non-peptide GLP-1R activators. Recently, studies investigating 4-(3-benzyloxyphenyl)-2-ethylsulfinyl-6-(trifluoromethyl)pyrimidine (BETP), a GLP-1R-positive allosteric modulator, determined that Cys-347 in the GLP-1R is required for positive allosteric modulator activity via covalent modification. To advance small molecule activation of the GLP-1R, we characterized the insulinotropic mechanism of BETP. In guanosine 5′-3-O-(thio)triphosphate binding and INS1 832-3 insulinoma cell cAMP assays, BETP enhanced GLP-1(9–36)-NH2-stimulated cAMP signaling. Using isolated pancreatic islets, BETP potentiated insulin secretion in a glucose-dependent manner that requires both the peptide ligand and GLP-1R. In studies of the covalent mechanism, PAGE fluorography showed labeling of GLP-1R in immunoprecipitation experiments from GLP-1R-expressing cells incubated with [3H]BETP. Furthermore, we investigated whether other reported GLP-1R activators and compounds identified from screening campaigns modulate GLP-1R by covalent modification. Similar to BETP, several molecules were found to enhance GLP-1R signaling in a Cys-347-dependent manner. These chemotypes are electrophiles that react with GSH, and LC/MS determined the cysteine adducts formed upon conjugation. Together, our results suggest covalent modification may be used to stabilize the GLP-1R in an active conformation. Moreover, the findings provide pharmacological guidance for the discovery and characterization of small molecule GLP-1R ligands as possible therapeutics.

Biochemical and kinetic characterization of BACE1: investigation into the putative species-specificity for β- and β′-cleavage sites by human and murine BACE1

β‐amyloid peptides (Aβ) are produced by a sequential cleavage of amyloid precursor protein (APP) by β‐ and γ‐secretases. The lack of Aβ production in beta‐APP cleaving enzyme (BACE1)–/– mice suggests that BACE1 is the principal β‐secretase in mammalian neurons. Transfection of human APP and BACE1 into neurons derived from wild‐type and BACE1–/– mice supports cleavage of APP at the canonical β‐secretase site. However, these studies also revealed an alternative BACE1 cleavage site in APP, designated as β′, resulting in Aβ peptides starting at Glu11. The apparent inability of human BACE1 to make this β′‐cleavage in murine APP, and vice versa, led to the hypothesis that this alternative cleavage was species‐specific. In contrast, the results from human BACE1 transgenic mice demonstrated that the human BACE1 is able to cleave the endogenous murine APP at the β′‐cleavage site. To address this discrepancy, we designed fluorescent resonance energy transfer peptide substrates containing the β‐ and β′‐cleavage sites within human and murine APP to compare: (i) the enzymatic efficiency; (ii) binding kinetics of a BACE1 active site inhibitor LY2039911; and (iii) the pharmacological profiles for human and murine recombinant BACE1. Both BACE1 orthologs were able to cleave APP at the β‐ and β′‐sites, although with different efficiencies. Moreover, the inhibitory potency of LY2039911 toward recombinant human and native BACE1 from mouse or guinea pig was indistinguishable. In summary, we have demonstrated, for the first time, that recombinant BACE1 can recognize and cleave APP peptide substrates at the postulated β′‐cleavage site. It does not appear to be a significant species specificity to this cleavage.

Discovery of 6-(4-{[5-Cyclopropyl-3-(2,6-dichlorophenyl)isoxazol-4-yl]methoxy}piperidin-1-yl)-1-methyl-1H-indole-3-carboxylic Acid: A Novel FXR Agonist for the Treatment of Dyslipidemia

The farnesoid X receptor (FXR) is a member of the metabolic subfamily of nuclear receptors. Several FXR agonists have been reported in the literature to have profound effects on plasma lipids in animal models. To discover novel and effective therapies for dyslipidemia and atherosclerosis, we have developed a series of potent FXR agonists that robustly lower plasma LDL and vLDL in LDLr-/- mice. To this end the novel piperidinylisoxazole system LY2562175 was discovered. This molecule is a potent and selective FXR agonist in vitro and has robust lipid modulating properties, lowering LDL and triglycerides while raising HDL in preclinical species. The preclinical ADME properties of LY2562175 were consistent with enabling once daily dosing in humans, and it was ultimately advanced to the clinic for evaluation in humans. The synthesis and biological profile of this molecule is discussed.

Mechanisms to Elevate Endogenous Glucagon-Like Peptide-1 (GLP-1) Beyond Injectable GLP-1 Analogues and Metabolic Surgery

Therapeutic engineering of glucagon-like peptide 1 (GLP-1) has enabled development of new medicines to treat type 2 diabetes. These injectable analogs achieve robust glycemic control by increasing concentrations of “GLP-1 equivalents” (∼50 pmol/L). Similar levels of endogenous GLP-1 occur after gastric bypass surgery, and mechanistic studies indicate glucose lowering by these procedures is driven by GLP-1. Therefore, because of the remarkable signaling and secretory capacity of the GLP-1 system, we sought to discover mechanisms that increase GLP-1 pharmacologically. To study active GLP-1, glucose-dependent insulinotropic polypeptide receptor (Gipr)–deficient mice receiving background dipeptidyl peptidase 4 (DPP4) inhibitor treatment were characterized as a model for evaluating oral agents that increase circulating GLP-1. A somatostatin receptor 5 antagonist, which blunts inhibition of GLP-1 release, and agonists for TGR5 and GPR40, which stimulate GLP-1 secretion, were investigated alone and in combination with the DPP4 inhibitor sitagliptin; these only modestly increased GLP-1 (∼5–30 pmol/L). However, combining molecules to simultaneously intervene at multiple regulatory nodes synergistically elevated active GLP-1 to unprecedented concentrations (∼300–400 pmol/L), drastically reducing glucose in Gipr null and Leprdb/db mice in a GLP-1 receptor–dependent manner. Our studies demonstrate that complementary pathways can be engaged to robustly increase GLP-1 without invasive surgical or injection regimens.

Optimization of Hydroxyethylamine Transition State Isosteres as Aspartic Protease Inhibitors by Exploiting Conformational Preferences.

NMR conformational analysis of a hydroxyethylamine peptide isostere developed as an aspartic protease inhibitor shows that it is a flexible architecture. Cyclization to form pyrrolidines, piperidines, or morpholines results in a preorganization of the whole system in solution. The resulting conformation is similar to the conformation of the inhibitor in the active site of BACE-1. This entropic gain results in increased affinity for the enzyme when compared with the acyclic system. For morpholines 27 and 29, the combination of steric and electronic factors is exploited to orient substituents toward S1, S1, and S2 pockets both in the solution and in the bound states. These highly preorganized molecules proved to be the most potent compounds of the series. Additionally, the morpholines, unlike the pyrrolidine and piperidine analogues, have been found to be brain penetrant BACE-1 inhibitors.

Erratum: Differential activation and modulation of the glucagon-like peptide-1 receptor by small molecular ligands (Molecular Pharmacology (2013) 83 (822-834))

In the above article [Wootten, D, Savage EE, Willard FS, Bueno AB, Sloop KW, Christopoulos A, and Sexton PM (2013) Mol Pharmacol 83: [822–834][1]], the structure drawing of TT15 in Fig. 1 is wrong. The structure should be as represented below. The name of the compound in the abbreviations on pagesIn the above article [Wootten, D, SavageEE,Willard FS, BuenoAB, SloopKW,Christopoulos A, and Sexton PM (2013) Mol Pharmacol 83:822–834], the structure drawing of TT15 in Fig. 1 is wrong. The structure should be as represented below. The name of the compound in the abbreviations on pages 822 and 823 and in Tables 1, 2, and 4 is also incorrect; it should be TT15, (2S)-2-[[(8S)-7-benzoyl-3-[4-[(3,4-dichlorophenyl)methoxy]phenyl]-2oxo-1,6,8,9-tetrahydropyrido[4,3-g][1,4]benzoxazine-8-carbonyl]amino]-3-[4-(4-cyanophenyl)phenyl]propanoic acid (IUPAC name), as indicated in the supplemental material. Intermediate 5 of the synthesis of TT15 (see supplemental material) corresponds to “intermediate A” in Mjalli, A. M. M. US Patent 7, 727, 983 B2 “Oxadiazoanthracene compounds for the treatment of diabetes,” Granted patent, Editor, TransTech Pharma.