Zhengping Ma

Eleven Amino Acid Glucagon-like Peptide-1 Receptor Agonists with Antidiabetic Activity

Glucagon-like peptide 1 (GLP-1) is a 30 or 31 amino acid peptide hormone that contributes to the physiological regulation of glucose homeostasis and food intake. Herein, we report the discovery of a novel class of 11 amino acid GLP-1 receptor agonists. These peptides consist of a structurally optimized 9-mer, which is closely related to the N-terminal 9 amino acids of GLP-1, linked to a substituted C-terminal biphenylalanine (BIP) dipeptide. SAR studies resulted in 11-mer GLP-1R agonists with similar in vitro potency to the native 30-mer. Peptides 21 and 22 acutely reduced plasma glucose excursions and increased plasma insulin concentrations in a mouse model of diabetes. These peptides also showed sustained exposures over several hours in mouse and dog models. The described 11-mer GLP-1 receptor agonists represent a new tool in further understanding GLP-1 receptor pharmacology that may lead to novel antidiabetic agents.

Discovery and Structure−Activity Relationships of Trisubstituted Pyrimidines/Pyridines as Novel Calcium-Sensing Receptor Antagonists

The trisubstituted pyrimidine 1 was identified through high-throughput screening as a novel calcium-sensing receptor (CaSR) antagonist. Small molecule CaSR antagonists and/or negative allosteric modulators have the potential to act as an anabolic agent for the treatment of osteoporosis. The investigation of structure-activity relationships around 1 resulted in the identification of 18c and 18d, which showed efficacy at promoting PTH release in vivo and exhibited improved potency and solubility over the original lead 1.

Characterization of Agonist-Induced Motilin Receptor Trafficking and Its Implications for Tachyphylaxis

The motilin receptor (MR) is a member of the seven-transmembrane receptor family and is expressed throughout the gastrointestinal tract of humans and other species. Motilin, the natural MR peptide ligand, has profound stimulatory effects on gastrointestinal contractility, indicating a therapeutic potential for MR modulators. However, long-term clinical use of certain MR agonists is limited by tachyphylaxis, a reduced responsiveness to repeated compound exposure. This study was meant to characterize the ligand-induced endocytosis of MR and to test whether receptor trafficking contributes to tachyphylaxis. A cell-based assay was developed by fusing a green fluorescent protein (GFP) moiety to the motilin receptor, and high-content biology instrumentation was used to quantify time and dose dependence of MR-GFP endocytosis. Maximal internalization of MR-GFP was induced after 45 min of constant exposure to 80 nM motilin. This process was disrupted by nocodazole, suggesting an essential role for microtubules. Internalized MR-GFP vesicles disappeared within 15 to 45 min of motilin withdrawal but did not overlap with the lysosomal compartment, indicating that MR-GFP escaped degradation and was recycled back to the plasma membrane. It is noteworthy that the kinetics of MR-GFP redistribution varied substantially when stimulated with motilin, erythromycin, 6,9-hemiacetal 8,9-anhydro-4″-deoxy-3′-N-desmethyl-3′-N-ethylerythromycin B (ABT-229), or N-[(1S)-1-[[[(1S)-1-(aminocarbonyl)-3-phenylpropyl]amino]carbonyl]-3-phenylpropyl]-2′-(1,3-benzodioxol-5-ylmethyl)tetrahydro-1′,3′-dioxo-spiro[piperidine-4,5′(6′H)-[1H][1,2,4]triazolo[1,2-a]pyridazine]-8′-carboxamide (BMS-591348) at equipotent doses for Ca2+-mobilization. Retardation of the intracellular MR-GFP sorting cycle seemed to correlate with the tachyphylaxis-inducing properties of each compound, but not its EC50. These results indicate that MR internalization, desensitization, and resensitization are ligand-dependent and that appropriate screening strategies may enable the development of small molecule agonists with ideal combinations of these distinct properties.

A novel direct homogeneous assay for ATP citrate lyase

ATP citrate lyase (ACL) is a cytosolic enzyme that catalyzes the synthesis of acetyl-CoA and oxaloacetate using citrate, CoA, and ATP as substrates and Mg2+ as a necessary cofactor. The ACL-dependent synthesis of acetyl-CoA is thought to be an essential step for the de novo synthesis of fatty acids and cholesterol. For this reason, inhibition of ACL has been pursued as a strategy to treat dyslipidemia and obesity. Traditionally, ACL enzyme activity is measured indirectly by coupling to enzymes such as malate dehydrogenase or chloramphenicol acetyl transferase. In this report, however, we describe a novel procedure to directly measure ACL enzyme activity. We first identified a convenient method to specifically detect [14C]acetyl-CoA without detecting [14C]citrate by MicroScint-O. Using this detection system, we devised a simple, direct, and homogeneous ACL assay in 384-well plate format that is suitable for high-throughput screening. The current assay consists of 1) incubation of ACL enzyme with [14C]citrate and other substrates/cofactors CoA, ATP, and Mg2+, 2) EDTA quench, 3) addition of MicroScint-O, the agent that specifically detects product [14C]acetyl-CoA, and 4) detection of signal by TopCount. This unique ACL assay may provide more efficient identification of new ACL inhibitors and allow detailed mechanistic characterization of ACL/inhibitor interactions.

Pyridine amides as potent and selective inhibitors of 11β-hydroxysteroid dehydrogenase type 1

Several series of pyridine amides were identified as selective and potent 11beta-HSD1 inhibitors. The most potent inhibitors feature 2,6- or 3,5-disubstitution on the pyridine core. Various linkers (CH(2)SO(2), CH(2)S, CH(2)O, S, O, N, bond) between the distal aryl and central pyridyl groups are tolerated, and lipophilic amide groups are generally favored. On the distal aryl group, a number of substitutions are well tolerated. A crystal structure was obtained for a complex between 11beta-HSD1 and the most potent inhibitor in this series.

Potent biphenyl- and 3-phenyl pyridine-based inhibitors of acetyl-CoA carboxylase.

We report the synthesis and enzymatic evaluation of potent inhibitors of acetyl-CoA carboxylases (ACCs) containing biphenyl or 3-phenyl pyridine cores. These compounds inhibit both ACC1 and ACC2, or are moderately selective for either enzyme, depending on side chain substitution. Typical activities of the most potent compounds in this class are in the low double-digit to single-digit nanomolar range in in vitro assays using human ACC1 and ACC2 enzymes.

Cell-based assay of MGAT2-driven diacylglycerol synthesis for profiling inhibitors: use of a stable isotope-labeled substrate and high-resolution LC/MS.

To demonstrate monoacylglycerol acyltransferase 2 (MGAT2)-mediated enzyme activity in a cellular context, cells of the murine secretin tumor cell-1 line of enteroendocrine origin were used to construct human MGAT2-expressing recombinant cell lines. Low throughput and utilization of radiolabeled substrate in a traditional TLC technique were circumvented by development of a high-resolution LC/MS platform. Monitoring incorporation of stable isotope-labeled D31-palmitate into diacylglycerol (DAG) allowed selective tracing of the cellular DAG synthesis activity. This assay format dramatically reduced background interference and increased the sensitivity and the signal window compared with the TLC method. Using this assay, several MGAT2 inhibitors from different chemotypes were characterized. The described cell-based assay adds a new methodology for the development and evaluation of MGAT2 inhibitors for the treatment of obesity and type 2 diabetes.