Gary E. Schiltz

SCFA Receptors in Pancreatic β Cells: Novel Diabetes Targets?

Nutrient sensing receptors are key metabolic mediators of responses to dietary and endogenously derived nutrients. These receptors are largely G-protein-coupled receptors (GPCRs) and many are gaining significant interest as drug targets with a potential therapeutic role in metabolic diseases. A distinct subclass of nutrient sensing GPCRs, two short chain fatty acid (SCFA) receptors (FFA2 and FFA3) are uniquely responsive to gut microbiota derived nutrients (such as acetate, propionate, and butyrate). Pharmacological, molecular, and genetic studies have investigated their role in organismal glucose metabolism and recently in pancreatic β cell biology. Here, we summarize the present knowledge on the role of these receptors as metabolic sensors in β cell function and physiology, revealing new therapeutic opportunities for type 2 diabetes.

In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy.

Background: GLUT4 is a promising target for multiple myeloma therapy. Results: In silico modeling of GLUT4, followed by virtual screening and validation, led to the identification of GLUT4-selective inhibitors. Conclusion: Despite significant homology between GLUT1 and GLUT4, we have identified GLUT4-selective inhibitors exhibiting cytotoxicity in myeloma. Significance: Potent selective GLUT4 inhibitors are promising cancer therapeutics with both cytostatic and chemosensitizing properties warranting further development. Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics.

Identification of Small-Molecule Inhibitors of Hyperpolarization-Activated Cyclic Nucleotide–Gated Channels

Hyperpolarization-activated cyclic nucleotide–gated (HCN) channels function in the brain to limit neuronal excitability. Limiting the activity of these channels has been proposed as a therapy for major depressive disorder, but the critical role of HCN channels in cardiac pacemaking has limited efforts to develop therapies directed at the channel. Previous studies indicated that the function of HCN is tightly regulated by its auxiliary subunit, tetratricopeptide repeat–containing Rab8b interacting protein (TRIP8b), which is not expressed in the heart. To target the function of the HCN channel in the brain without affecting the channel’s function in the heart, we propose disrupting the interaction between HCN and TRIP8b. We developed a high-throughput fluorescence polarization (FP) assay to identify small molecules capable of disrupting this interaction. We used this FP assay to screen a 20,000-compound library and identified a number of active compounds. The active compounds were validated using an orthogonal AlphaScreen assay to identify one compound (0.005%) as the first confirmed hit for inhibiting the HCN-TRIP8b interaction. Identifying small molecules capable of disrupting the interaction between HCN and TRIP8b should enable the development of new research tools and small-molecule therapies that could benefit patients with depression.

Design and synthesis of potent macrocyclic HIV-1 protease inhibitors involving P1–P2 ligands

A series of potent macrocyclic HIV-1 protease inhibitors have been designed and synthesized. The compounds incorporated 16- to 19-membered macrocyclic rings between a nelfinavir-like P2 ligand and a tyrosine side chain containing a hydroxyethylamine sulfonamide isostere. All cyclic inhibitors are more potent than their corresponding acyclic counterparts. Saturated derivatives showed slight reduction of potency compared to the respective unsaturated derivatives. Compound containing a 16-membered ring as the P1-P2 ligand showed the most potent enzyme inhibitory and antiviral activity.

Discovery of 1,3,4-oxidiazole scaffold compounds as inhibitors of superoxide dismutase expression

The treatment of neurodegenerative diseases is difficult because of multiple etiologies and the interplay of genetics and environment as precipitating factors. In the case of amyotrophic lateral sclerosis (ALS), we have knowledge of a handful of genes that cause disease when mutated. However, drugs to counteract the effect of genetic mutations have not yet been found. One of the causative genes, Cu, Zn-superoxide dismutase (SOD1) is responsible for about 10-15% of the genetically linked autosomal dominant disease. Our rationale was that compounds that reduce expression of the mutant protein would be beneficial to slow onset and/or disease progression. We screened candidate compounds using a cell-based in vitro assay for those that reduce mutant SOD1 (G93A) protein expression. This led to the discovery of 2-[3-iodophenyl)methylsulfanyl]-5pyridin-4-yl-1,3,4-oxadiazole, a known protein kinase inhibitor that decreases G93A-SOD1 expression in vitro and in the brain and spinal cord in vivo. However, this compound has a biphasic dose response curve and a likely toxophore which limit its therapeutic window for chronic disease such as ALS. Therefore, we designed and tested a focused library of analogs for their ability to decrease SOD1 expression in vitro. This exercise resulted in the identification of a lead compound with improved drug-like characteristics and activity. Development of small molecules that reduce the expression of etiologically relevant toxic proteins is a strategy that may also be extended to familial ALS linked to gain of function mutations in other genes.

Discovery and characterization of novel small-molecule CXCR4 receptor agonists and antagonists

The chemokine CXCL12 (SDF-1) and its cognate receptor CXCR4 are involved in a large number of physiological processes including HIV-1 infectivity, inflammation, tumorigenesis, stem cell migration, and autoimmune diseases. While previous efforts have identified a number of CXCR4 antagonists, there have been no small molecule agonists reported. Herein, we describe the identification of a novel series of CXCR4 modulators, including the first small molecules to display agonist behavior against this receptor, using a combination of structure- and ligand-based virtual screening. These agonists produce robust calcium mobilization in human melanoma cell lines which can be blocked by the CXCR4-selective antagonist AMD3100. We also demonstrate the ability of these new agonists to induce receptor internalization, ERK activation, and chemotaxis, all hallmarks of CXCR4 activation. Our results describe a new series of biologically relevant small molecules that will enable further study of the CXCR4 receptor and may contribute to the development of new therapeutics.

Virtual High-Throughput Screening to Identify Novel Activin Antagonists

Activin belongs to the TGFβ superfamily, which is associated with several disease conditions, including cancer-related cachexia, preterm labor with delivery, and osteoporosis. Targeting activin and its related signaling pathways holds promise as a therapeutic approach to these diseases. A small-molecule ligand-binding groove was identified in the interface between the two activin βA subunits and was used for a virtual high-throughput in silico screening of the ZINC database to identify hits. Thirty-nine compounds without significant toxicity were tested in two well-established activin assays: FSHβ transcription and HepG2 cell apoptosis. This screening workflow resulted in two lead compounds: NUCC-474 and NUCC-555. These potential activin antagonists were then shown to inhibit activin A-mediated cell proliferation in ex vivo ovary cultures. In vivo testing showed that our most potent compound (NUCC-555) caused a dose-dependent decrease in FSH levels in ovariectomized mice. The Blitz competition binding assay confirmed target binding of NUCC-555 to the activin A:ActRII that disrupts the activin A:ActRII complexs binding with ALK4-ECD-Fc in a dose-dependent manner. The NUCC-555 also specifically binds to activin A compared with other TGFβ superfamily member myostatin (GDF8). These data demonstrate a new in silico-based strategy for identifying small-molecule activin antagonists. Our approach is the first to identify a first-in-class small-molecule antagonist of activin binding to ALK4, which opens a completely new approach to inhibiting the activity of TGFβ receptor superfamily members. in addition, the lead compound can serve as a starting point for lead optimization toward the goal of a compound that may be effective in activin-mediated diseases.

Targeting Processive Transcription Elongation via SEC Disruption for MYC-Induced Cancer Therapy

The super elongation complex (SEC) is required for robust and productive transcription through release of RNA polymerase II (Pol II) with its P-TEFb module and promoting transcriptional processivity with its ELL2 subunit. Malfunction of SEC contributes to multiple human diseases including cancer. Here, we identify peptidomimetic lead compounds, KL-1 and its structural homolog KL-2, which disrupt the interaction between the SEC scaffolding protein AFF4 and P-TEFb, resulting in impaired release of Pol II from promoter-proximal pause sites and a reduced average rate of processive transcription elongation. SEC is required for induction of heat-shock genes and treating cells with KL-1 and KL-2 attenuates the heat-shock response from Drosophila to human. SEC inhibition downregulates MYC and MYC-dependent transcriptional programs in mammalian cells and delays tumor progression in a mouse xenograft model of MYC-driven cancer, indicating that small-molecule disruptors of SEC could be used for targeted therapy of MYC-induced cancer.

Tissue Tranglutaminase Regulates Interactions between Ovarian Cancer Stem Cells and the Tumor Niche

Cancer progression and recurrence are linked to a rare population of cancer stem cells (CSC). Here, we hypothesized that interactions with the extracellular matrix drive CSC proliferation and tumor-initiating capacity and investigated the functions of scaffold protein tissue transglutaminase (TG2) in ovarian CSC. Complexes formed by TG2, fibronectin (FN), and integrin β1 were enriched in ovarian CSC and detectable in tumors. A function-inhibiting antibody against the TG2 FN-binding domain suppressed complex formation, CSC proliferation as spheroids, tumor-initiating capacity, and stemness-associated Wnt/β-catenin signaling. Disruption of the interaction between TG2 and FN also blocked spheroid formation and the response to Wnt ligands. TG2 and the Wnt receptor Frizzled 7 (Fzd7) form a complex in cancer cells and tumors, leading to Wnt pathway activation. Protein docking and peptide inhibition demonstrate that the interaction between TG2 and Fzd7 overlaps with the FN-binding domain of TG2. These results support a new function of TG2 in ovarian CSC, linked to spheroid proliferation and tumor-initiating capacity and mediated through direct interactions with Fzd7. We propose this complex as a new stem cell target.Significance: These findings reveal a new mechanism by which ovarian CSCs interact with the tumor microenvironment, promoting cell proliferation and tumor initiation. Cancer Res; 78(11); 2990-3001. ©2018 AACR.

Homology modeling of FFA2 identifies novel agonists that potentiate insulin secretion

Critical aspects of maintaining glucose homeostasis in the face of chronic insulin resistance and type 2 diabetes (T2D) are increased insulin secretion and adaptive expansion of beta cell mass. Nutrient and hormone sensing G protein-coupled receptors are important mediators of these properties. A growing body of evidence now suggests that the G protein-coupled receptor, free fatty acid receptor 2 (FFA2), is capable of contributing to the maintenance of glucose homeostasis by acting at the pancreatic beta cell as well as at other metabolically active tissues. We have previously demonstrated that Gαq/11-biased agonism of FFA2 can potentiate glucose stimulated insulin secretion (GSIS) as well as promote beta cell proliferation. However, the currently available Gαq/11-biased agonists for FFA2 exhibit low potency, making them difficult to examine in vivo. This study sought to identify Gαq/11-biased FFA2-selective agonists with potent GSIS-stimulating effects. To do this, we generated an FFA2 homology model that was used to screen a library of 10 million drug-like compounds. Although FFA2 and the related short chain fatty acid receptor FFA3 share 52% sequence similarity, our virtual screen identified over 50 compounds with predicted selectivity and increased potency for FFA2 over FFA3. Subsequent in vitro calcium mobilization assays and GSIS assays resulted in the identification of a compound that can potentiate GSIS via activation of Gαq/11 with 100-fold increased potency compared with previously described Gαq/11-biased FFA2 agonists. These methods and findings provide a foundation for future discovery efforts to identify biased FFA2 agonists as potential T2D therapeutics.