Sergienko, Eduard

G protein-coupled receptors (GPCRs) are among the most common drug targets, with numerous agonists and antagonists approved for clinical use. More recently, it has been appreciated that drugs can also modulate the action of natural agonists of these receptors, thus providing unique clinical advantages. Here, we describe the identification and characterization of a small molecule positive allosteric modulator (PAM) of secretin action at the class B G protein-coupled secretin receptor. This phenyl(trifluoromethyl)-pyrimidine can occupy the secretin receptor without stimulating its internalization, yet priming it to enhance both the potency and efficacy of the action of natural secretin. This is also shown to exhibit its effects on cells expressing low numbers of these receptors, without enhancing the effects of other structurally related hormones acting at other class B GPCRs. The mechanism responsible for this PAM effect is the slowing of the off-rate of receptor-bound secretin. This compound can serve as a lead to the development of other drugs that enhance the action of natural endogenous secretin and can be utilized to explore the potential therapeutic utility of such compounds.

Abstract Mitochondrial creatine kinase (MtCK) is a key enzyme in energy buffering and homeostasis in cells. It catalyzes transfer of phosphoryl group from ATP to creatine. Overexpression of MtCK occurs in many cancer cells to meet elevated energy demands, which is associated with poor prognosis. This suggests that MtCK may be a promising target for cancer therapeutics. We sought to discover first-in-class selective inhibitors of MtCK with diverse mechanisms of action using high-throughput screening, biochemical characterization and cryo-EM studies. Through these studies, we identified diverse types of compounds that modulate activity of MtCK in vitro , including fast-equilibrium and time-dependent orthosteric and allosteric inhibitors. Select hits were subjected to in vitro enzymatic and binding assays to assess MtCK inhibition and binding. A subset of inhibitors was advanced into structural studies using cryo-EM resulting in the molecular structure of MtCK with and without bound substrates in complex with an allosteric uncompetitive inhibitor that we discovered. These studies identified compound’s unique binding pocket on MtCK and established the molecular steps manifesting in the apparent uncompetitive mode of inhibition. We demonstrate that the compound stabilizes an active site loop in a closed conformation restricting access of the creatine substrate to and the release of phosphocreatine product from its binding pocket. These findings establish chemical tools suitable for validation of MtCK as a promising breast cancer target with high therapeutic potential and build a foundation for future structure-guided optimization of the hit compounds of MtCK we identified and de novo rational design of novel MtCK inhibitors. Abstract Figure

As part of an ongoing effort to develop a drug targeting the type 1 cholecystokinin receptor (CCK1R) to help prevent and/or treat obesity, we recently performed a high throughput screening effort of small molecules seeking candidates that enhanced the action of the natural agonist, CCK, thus acting as positive allosteric modulators without exhibiting intrinsic agonist action. Such probes would be expected to act in a temporally finite way to enhance CCK action to induce satiety during and after a meal and potentially even modulate activity at the CCK1R in a high cholesterol environment present in some obese patients. The current work focuses on the best scaffold, representing tetracyclic molecules identified through high throughput screening we previously reported. Extensive characterization of the two top “hits” from the previous effort demonstrated them to fulfill the desired pharmacologic profile. We undertook analog-by-catalog expansion of this scaffold using 65 commercially available analogs. In this effort, we were able to eliminate an off-target effect observed for this scaffold while retaining its activity as a positive allosteric modulator of CCK1R in both normal and high cholesterol membrane environments. These insights should be useful in the rational medicinal chemical enhancement of this scaffold and in the future development of candidates to advance to pre-clinical proof-of-concept and to clinical trials.

Class B G protein‐coupled receptors (GPCRs) are a highly important group of therapeutic targets with strong physiologic implications in cardiovascular, metabolic, or gastrointestinal systems. Endogenously activated by moderate‐length peptide hormones, class B GPCRs entertain a complex binding mechanism to induce stimulatory signaling processes. The secretin receptor (SCTR) is a prototypical member of this receptor family, having clinical relevance in multiple diseases including heart failure, digestive disorders, diabetes and obesity. [1‐4] However, its endogenous ligand secretin and peptide‐based analogs are the only ligands known to interact with SCTRs, likely due to challenges in class B GPCR drug discovery [5]. To find orally available therapeutic leads targeting this “undruggable” receptor, we focused on identifying positive allosteric modulators (PAMs), which act via structurally distinct binding pockets and potentiate hormonal signaling of native agonists. Thus, we developed a testing funnel tailored to discover SCTR small molecule modulators. After screening a 12,000‐compound library with a unique primary assay that used a mixture of stimulators, we identified three structurally related scaffolds (A‐C) enhancing peptidic SCTR activation [5]. Applying a comprehensive toolbox with binding and functional assays, we studied structure‐activity relationships of a well‐considered set of commercially available analogs incorporating these three scaffolds. Our investigations afforded an optimized analog B2, which showed substantial positive cooperativity to low‐potent peptide agonists and significantly decelerated secretin dissociation. Due to the innate electrophilicity of scaffold set A, we recognized a structural similarity to PAMs acting on glucagon‐like peptide‐1 receptor (GLP‐1R), a prominent member of class B GPCR family. We further demonstrated that a subset of SCTR PAMs was able to augment not only SCTR signaling but also GLP‐1R functions. A recent study described that dual activation of SCTRs and GLP‐1Rs was benefiting glucose homeostasis and appetite regulation while avoiding proliferative effects on pancreas mass [6]. Determining cooperativity between PAMs and selected orthosteric peptides, we established distinct selectivity and probe dependency profiles. To decipher reversible and covalent mechanisms‐of‐action as well as potential binding sites of novel PAMs, we conducted glutathione reactivity, cAMP‐washout and receptor mutational studies. [7] To conclude, we discovered a unique class of SCTR modulators providing useful pharmacologic tools to study SCTR‐ or GLP‐1R‐specific activation pathways and explore novel allosteric binding sites with potential to instigate the development of first‐in‐class therapeutics for treating comorbid conditions obesity and diabetes. [7] References [1] Annu Rev Pharmacol Toxicol, 2020 , 60 , 89‐107; [2] Trends in Biochemical Sciences, 2017 , 12 , 946‐960; [3] Experimental Physiology, 2013 , 5 , 973‐987; [4] Current Neuropharmacology, 2007 , 3 , 168‐179; [5] SLAS Discovery, 2021 , 26 , 1‐16; [6] Journal of Peptide Science, 2017 , 12 , 845‐854; [7] submitted to Biochemical Pharmacology 2021 .

Abstract Purpose: Quiescin Sulfhydryl Oxidase 1 (QSOX1) is an enzyme over-expressed by tumor cells. Our group and others have shown that QSOX1 is involved in invasion and metastasis. We previously reported that a small molecule, SBI-183, inhibited the enzymatic activity of QSOX1 and suppressed tumor cell invasion in vitro and metastasis of MDA-MB-231 breast tumor cells in vivo. In our current work we tested SPX-009, an analog of SBI-183 with increased potency, to suppress invasion and metastasis in vitro and in vivo. Methods: Commercially available analogs for SBI-183 were screened in a cell-free fluorescent screening assay for the ability to inhibit QSOX1. Analog compounds that inhibited QSOX1 in a concentration-dependent manner were then tested for the ability to inhibit growth and invasion of triple negative breast cancer (TNBC) cells (MDA-MB-231), sarcomatoid kidney cancer cells (RCJ-41T2) and pancreatic adenocarcinoma cells (MIAPaCa2). MTT assays were used to measure the effect of the compounds on tumor growth. 2D invasion assays and 3D tumor spheroid assays were employed to measure the effect of the analogs on invasion. One potent analog (SPX-009) was evaluated for the ability to suppress tumor growth and metastasis in mice bearing MDA-MB-231-luc breast cancer orthotopic xenografts. Results: Three of 53 analogs inhibited the enzymatic activity of QSOX1 at levels similar to the parent compound. However, one of those compounds, SPX-009, suppressed invasion of breast, kidney and pancreas tumor cells in 2D and 3D invasion assays at >10-fold lower concentrations than the parent compound (SBI-183). In a non-metastatic xenograft of kidney cancer SBI-183 suppressed primary kidney tumor growth by 51%, but in a metastatic xenograft breast cancer model (MDA-MB-231-luc TNBC), SBI-183 reduced lung metastasis by 76% compared to control. Human-mouse xenograft studies are ongoing with the more potent analog (SPX-009) and will also be presented. Conclusions: Chemical inhibitors of QSOX1 have a minor effect on tumor cell viability, but a major effect on tumor cell invasion in vitro and metastasis in vivo. This finding suggests that combining a QSOX1 inhibitor with a cytotoxic agent might provide a means to inhibit metastasis of cells that are resistant to cytotoxic agents. Citation Format: Amber Fifield, Thai H. Ho, Douglas O. Faigel, Sergei Svarovsky, Eduard Sergienko, Thomas C. Chung, Douglas F. Lake. Chemical inhibitors of quiescin sulfhydryl oxidase (QSOX1) suppress tumor invasion and metastasis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5323.

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 Quiescin sulfhydryl oxidase 1 (QSOX1) is an enzyme overexpressed by many different tumor types. QSOX1 catalyzes the formation of disulfide bonds in proteins. Because short hairpin knockdowns (KD) of QSOX1 have been shown to suppress tumor growth and invasion in vitro and in vivo, we hypothesized that chemical compounds inhibiting QSOX1 enzymatic activity would also suppress tumor growth, invasion, and metastasis. High throughput screening using a QSOX1-based enzymatic assay revealed multiple potential QSOX1 inhibitors. One of the inhibitors, known as “SBI-183,” suppresses tumor cell growth in a Matrigel-based spheroid assay and inhibits invasion in a modified Boyden chamber, but does not affect viability of nonmalignant cells. Oral administration of SBI-183 inhibits tumor growth in 2 independent human xenograft mouse models of renal cell carcinoma. We conclude that SBI-183 warrants further exploration as a useful tool for understanding QSOX1 biology and as a potential novel anticancer agent in tumors that overexpress QSOX1.

Hepatic cystogenesis in polycystic liver disease is associated with increased levels of cyclic adenosine monophosphate (cAMP) in cholangiocytes lining liver cysts. Takeda G protein receptor 5 (TGR5), a G protein–coupled bile acid receptor, is linked to cAMP and expressed in cholangiocytes. Therefore, we hypothesized that TGR5 might contribute to disease progression. We examined expression of TGR5 and Gα proteins in cultured cholangiocytes and in livers of animal models and humans with polycystic liver disease. In vitro, we assessed cholangiocyte proliferation, cAMP levels, and cyst growth in response to (1) TGR5 agonists (taurolithocholic acid, oleanolic acid [OA], and two synthetic compounds), (2) a novel TGR5 antagonist (m‐tolyl 5‐chloro‐2‐[ethylsulfonyl] pyrimidine‐4‐carboxylate [SBI‐115]), and (3) a combination of SBI‐115 and pasireotide, a somatostatin receptor analogue. In vivo, we examined hepatic cystogenesis in OA‐treated polycystic kidney rats and after genetic elimination of TGR5 in double mutant TGR5−/− ;Pkhd1del2/del2 mice. Compared to control, expression of TGR5 and Gαs (but not Gαi and Gαq) proteins was increased 2‐fold to 3‐fold in cystic cholangiocytes in vitro and in vivo. In vitro, TGR5 stimulation enhanced cAMP production, cell proliferation, and cyst growth by ∼40%; these effects were abolished after TGR5 reduction by short hairpin RNA. OA increased cystogenesis in polycystic kidney rats by 35%; in contrast, hepatic cystic areas were decreased by 45% in TGR5‐deficient TGR5−/−;Pkhd1del2/del2 mice. TGR5 expression and its colocalization with Gαs were increased ∼2‐fold upon OA treatment. Levels of cAMP, cell proliferation, and cyst growth in vitro were decreased by ∼30% in cystic cholangiocytes after treatment with SBI‐115 alone and by ∼50% when SBI‐115 was combined with pasireotide. Conclusion: TGR5 contributes to hepatic cystogenesis by increasing cAMP and enhancing cholangiocyte proliferation; our data suggest that a TGR5 antagonist alone or concurrently with somatostatin receptor agonists represents a potential therapeutic approach in polycystic liver disease. (Hepatology 2017;66:1197‐1218).

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