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Type 2 diabetes mellitus (T2DM) is a progressive, complex metabolic disorder mainly characterized by insulin resistance and hyperglycemia1. The prevalence of diabetes, of which T2DM accounts for more than 90% of patients, is rising at an alarming rate in certain parts of the world2. Although the exact incidence in China is arguable, the age-adjusted prevalence of diabetes and pre–diabetes was reported to be 9.7% and 15.5%, respectively3, 4. T2DM has thus become a global heath concern due to its complications, such as cardiovascular conditions that result in increased morbidity and mortality. While the currently available anti-diabetic agents are effective in lowering glucose, some of them, including insulin, sulfonylureas and thiazolidinediones, are often limited by weight gain and/or hypoglycemia. However, recent emergence of incretin-based therapies, exemplified by glucagon-like peptide-1 (GLP-1) mimetics and dipeptidyl peptidase-4 (DPP-4) inhibitors, will drastically transform the landscape of diabetes care5. GLP-1 maintains glucose homeostasis through several mechanisms that include, but not limited to, stimulating insulin secretion, suppressing glucagon production, improving β-cell mass and function, inhibiting food intake and slowing gastric emptying6. Therefore, GLP-1 based incretin therapy is designed to target the fundamental defects of T2DM, capable of reducing both glycosylated hemoglobin (HbA1c) and body weight, and has potential benefits on blood pressure, lipids, and other surrogate markers, leading to decreased cardiovascular risk7, 8.

The native GLP-1 has a very short half-life (<2 min) mainly because of the degradation by DPP-4 and neutral endopeptidase (NEP)9. Two approaches have been employed: GLP-1 receptor agonists that mimic the effects of native GLP-1 and DPP-4 inhibitors that increase endogenous GLP-1 levels7. The former is represented by two peptidic mimetics, Exenatide10, 11 and Liraglutide12, approved by the US Food and Drug Administration (FDA) for the treatment of T2DM. Other peptides that are under development include long-acting human GLP-1 analog CJC-113113, GLP-1 analog-Fc fusion protein LY218926514 and pegylated GLP-1 analog LY242875715, recombinant human serum albumin exendin–4 conjugated protein CJC-1134-PC16, Taspoglutide17, as well as AVE001018.

Unlike peptidic mimetics that require injections, DPP-4 inhibitors (such as Sitagliptin, Vildagliptin and Saxagliptin)19, 20, 21 are small in molecular nature, orally active, and transported and stored at ease. Administration of DPP-4 inhibitor has led to improved plasma concentrations of endogenous GLP-1 and marked reduction of HbA1c, but its effect on body weight has been neutral21. Thus, oral formulation of GLP-1 analogs remains a development option22.

It is conceivable that an orally active, non-peptidic GLP-1 receptor agonist could resolve the issues mentioned above. Indeed, this has been the focal point of drug discovery efforts in many multinational pharmaceutical companies. Such efforts have not resulted in any success thus far.

The GLP-1 receptor belongs to the glucagon-secretin B family of the G protein-coupled receptors (GPCRs). A characteristic structural feature of this family is a long and structurally complex extracellular amino-terminal domain (N-domain) containing six conserved cysteine residues that form disulfide bonds important for stabilizing the folded protein. The N-domain is connected to a juxtamembrane domain (J-domain) of the seven membrane-spanning α-helices with intervening loops and a C-terminal tail23. From a biological perspective, class B GPCRs are highly attractive therapeutic targets, but the search for non-peptidic modulators (agonists in particular) has been met with great difficulties. Very few 'druggable' small molecule ligands have been identified for this class of GPCRs. With reference to GLP-1 receptors, only a very small number of non-peptidic agonists have been reported over the years as summarized below in Table 1.

Table 1 The structures of small molecule GLP-1 receptor agonists and their claimed biological activities in vitro and in vivo.
Full size table

Of the compounds listed, diallylmethylamine derivative is more like a DPP-4 inhibitor than a GLP-1 receptor agonist, based on the limited information available in the public domain29. The remaining eight molecules possess fairly diverse structural features. Despite what were described by the inventors for these compounds in terms of orthosteric, allosteric, ago-allosteric, inverse, or partial/full agonists, their functional varieties are manifested in the following manners: (1) inducing both biochemical and cellular responses without in vivo activities24, 27; (2) behaving like an antagonist in cell-based assays (eg, T0632)27; (3) promoting native ligand binding to the receptor in vitro32; (4) demonstrating a full range of GLP-1 properties including in vivo efficacy25, 26; and (5) enhancing GLP-1 activities via binding to the receptor33. Since no distinct commonality could be found in the structures of these five types of compounds, their binding to the GLP-1 receptor must be realized via different sites. Among them, Boc5 is the only one that demonstrated therapeutic benefits in vivo25, 26. While peptidic GLP-1 mimetics displayed some side effects such as nausea and vomiting in the clinic9, both normal and diabetic db/db mice are highly tolerable to Boc525 such that a similar reaction on the conditioned taste aversion required a dose well beyond the therapeutic window26. Long-term toxicity (≥3 month) has yet to be determined. It appears that Boc5 is neither metabolized by nor interacts with the cytochrome P450 in vivo exhibiting a half-life ranging from 12.1 to 35.4 h in mice and rats, respectively when injected intraperitoneally. However, its oral bioavailability is extremely poor due to metabolism by esterase in the gut (unpublished data).

The current binding model of GLP-1 receptor is a two-step mechanism where initially the C-terminal part of the peptide ligand interacts with the N-domain of the receptor, thereby conferring high affinity. In the second step, the N-terminal part of the ligand interacts with the core domain of the receptor (transmembrane helices and connecting loops), leading to activation and signal transduction23. Both the N-domain and J-domain of the receptor are needed for this interaction, and the former is also critical for ligand selectivity between glucagon and GLP-1. Molecular elucidation of such an interaction is nearly impossible due to the inherent complexity of GPCR structures. However, attempts were made using both the crystal structure of human GLP-1 receptor N-terminal extracellular domain34 and computational simulation35. These studies have implicated that activation of GLP-1 receptor by an agonist is related to some intrinsic conformation changes. There may be two major states of the GLP-1 receptor structure: (1) the inactive state, in which the orthosteric agonist-binding site is partially blocked as the consequence of the relative motions between the N-domain and the transmembrane domain; and (2) the active state, in which the orthosteric agonist-binding site is fully accessible35. Binding of an agonist makes a GLP-1 receptor stay in the latter state, as in the case of Boc525, while interaction with an inverse agonist such as T0632 favors the former state23, 27. Compound 2 (quinoxaline derivative), which binds the GLP-1 receptor at an allosteric site, rigidifies its structure with an open binding site to improve the activity of a full agonist35.

Obviously, the exact requirements needed for a small molecule to fully mimic GLP-1 have yet to be understood. Compounds that confer the conventional wisdom36 such as 'Rule of 5' do not display any in vivo bioactivities, while molecules that demonstrate therapeutic benefits in animal models of T2DM and obesity such as Boc5 are considered not druggable because of poor oral bioavailability. Serendipitous discovery of substituted cyclobutanes represented by Boc5 as a new class of GLP-1 receptor agonists led us to believe that a small molecule approach to class B GPCR agonism is no longer a fantasy but a reality37.

Nonetheless, major obstacles still pose great challenges in terms of developing an orally active non-peptidic GLP-1 receptor agonist. To what extent should such a molecule look like from a medicinal chemistry point of view? Can it possess a suitable molecular size that meets the requisite ADME/T profile while maintaining a full range of incretin-like efficacy? Can it be produced economically, so that it can sufficiently compete against its peptidic counterparts? These are the questions that must be addressed in order to sustain the vitality of a research program in this particular area.