Ma, Chen-Ting

The Secretin receptor (SCTR) has been cloned as the first member of class B G protein‐coupled receptors (GPCRs) , giving rise to this highly important group of therapeutic targets, which are crucial for hormonal homeostasis . Despite the wide recognition of their functional importance, the development of potent small molecule ligands for Class B GPCRs remained challenging . To date, no ligands other than secretin peptide and its close analogs are known to interact with SCTRs, despite its therapeutic relevance in multiple diseases, such as heart failure, obesity, diabetes and cancer . In order to enhance our chances for discovering orally bioavailable drug candidates targeting an ‚undruggable’ class B GPCR, we focused our efforts on the identification of positive allosteric modulators (PAMs), which are likely to interact via structurally distinct binding sites and potentiate effects of native hormones and neurotransmitters . We developed a comprehensive tool box with robust high‐throughput screening and lead characterization/optimization assays capable of identifying and characterizing biologically relevant SCTR small molecule modulators. The dominant physiological coupling of SCTRs occurs via Gs proteins. Hence, we utilized this signaling mechanism to establish diverse cAMP accumulation assays suitable for simultaneous detection of PAMs modulating secretin and its potential metabolites. We also established a TR‐FRET based binding assay to further characterize potential PAMs with respect to their kinetic properties. Since functional selectivity has been proven to be therapeutically relevant for this family of membrane proteins, we additionally designed assays to investigate signaling mechanisms beyond Gs protein coupling, such as β‐arrestin recruitment, receptor internalization and calcium flux. High‐throughput screening (HTS) of a 360,000 compound library provided promising sets of hits, which have been further investigated regarding their allosteric activity and mechanism of action. We identified three related scaffolds with significant PAM activity at SCTRs. Selectivity studies revealed that this specific set of compounds demonstrated positive modulation not only on SCTRs but also on GLP‐1Rs (glucagon‐like peptide 1 receptors). Interestingly, dual activation of SCTRs and GLP‐1Rs has been shown to be beneficial for appetite regulation and glucose homeostasis without having proliferative effects on exocrine or total pancreas mass . To conclude, HTS and the development of a diverse panel of binding and functional assays has led to the discovery of small molecules with a unique dual‐acting pharmacological profile and potential to serve as starting points for developing first‐in‐class drug candidates for the treatment of comorbid conditions obesity and diabetes. Support or Funding Information Funding: NHLBI award 5R01HL133501

Abstract Introduction: QSOX1 is a flavin-dependent sulfhydryl oxidase and the only known human enzyme with both disulfide generating and transferring activities. Using tissue microarrays, we previously demonstrated that QSOX1 is over-expressed in multiple tumor types including breast, pancreas, prostate and myeloma. Loss of QSOX1 leads to a significant decrease in tumor cell proliferation and invasion. The over-expression of QSOX1 in tumor cells compared to non-malignant cells combined with its role in tumor cell proliferation and invasion make it an attractive target for potential anti-neoplastic agents. Methods: Using a high throughput screening assay to detect sulfhydryl oxidase activity, we screened a library of compounds for inhibition of the QSOX1 enzyme. We used ROS-GloTM, a luciferase-based assay that detects H2O2, to determine if any compounds in the library inhibited QSOX1-mediated disulfide bond formation using reduced RNAse A as a substrate. QSOX1 generates H2O2 as a by-product of disulfide bond formation. To determine if compounds that showed activity in the enzymatic assay were also active against tumor cells, we treated pancreatic, breast, and myeloma cells with the compounds in a dose-dependent fashion, using non-malignant cells as controls. We assayed growth/viability by MTT and CellTiter-Glo assays. Invasive activity of tumor cells was assessed using matrigel-coated inserts in a modified Boyden chamber assay. Results: High throughput library screening identified 4 compounds that specifically inhibited QSOX1 activity (designated A-D) compared to a glucose oxidase counter-screen. Growth/viability assays showed that compound D suppressed the growth of tumor cells with IC50s between 300nM-750nM, depending on tumor cell line. In non-malignant cells the IC50s for compound D were more than 10-fold higher than in tumor cells. Compounds A and D also inhibited invasion of breast and pancreatic tumor cells through Matrigel. Conclusion: This is the first report of chemical compounds that inhibit QSOX1 enzymatic activity. The discovery of QSOX1 inhibitors with cellular efficacy establishes the chemical tractability of the target. Our data demonstrate that these First-In-Class compounds have potential as anti-neoplastic therapeutics via inhibition of QSOX1. Citation Format: Paul D. Hanavan, Douglas O. Faigel, Chen-Ting Ma, Eduard Sergienko, Nathalie Meurice, Joachim L. Petit, Yvette W. Ruiz, Benjamin A. Katchman, Douglas F. Lake. Small molecule inhibitors of QSOX1 suppress tumor cell growth and invasion. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1790. doi:10.1158/1538-7445.AM2014-1790