Discovery of small molecule antagonists of chemokine receptor CXCR6 that arrest tumor growth in SK-HEP-1 mouse xenografts as a model of hepatocellular carcinoma.

The chemokine system plays an important role in mediating a proinflammatory microenvironment for tumor growth in hepatocellular carcinoma (HCC). The CXCR6 receptor and its natural ligand CXCL16 are expressed at high levels in HCC cell lines and tumor tissues and receptor expression correlates with increased neutrophils in these tissues contributing to poor prognosis in patients. Availability of pharmacologcal tools targeting the CXCR6/CXCL16 axis are needed to elucidate the mechanism whereby neutrophils are affected in the tumor environment. We report the discovery of a series of small molecules with an exo-[3.3.1]azabicyclononane core. Our lead compound 81 is a potent (EC50 = 40 nM) and selective orally bioavailable small molecule antagonist of human CXCR6 receptor signaling that significantly decreases tumor growth in a 30-day mouse xenograft model of HCC.

Measurement of Pyridine Nucleotides in Biological Samples Using LC-MS/MS.

Pyridine nucleotides which include NAD+, NADH, NADP, and NADPH play vital roles in many different biological processes. These metabolites can be accurately quantified in a wide variety of biological samples using LC-MS/MS. The quality and precision of these measurements was enhanced using heavy isotope-labeled internal standards and carefully crafted protocols for sample processing.

Allosteric Inhibition of Ubiquitin-like Modifications by a Class of Inhibitor of SUMO-Activating Enzyme.

Ubiquitin-like (Ubl) post-translational modifications are potential targets for therapeutics. However, the only known mechanism for inhibiting a Ubl-activating enzyme is through targeting its ATP-binding site. Here we identify an allosteric inhibitory site in the small ubiquitin-like modifier (SUMO)-activating enzyme (E1). This site was unexpected because both it and analogous sites are deeply buried in all previously solved structures of E1s of ubiquitin-like modifiers (Ubl). The inhibitor not only suppresses SUMO E1 activity, but also enhances its degradation in vivo, presumably due to a conformational change induced by the compound. In addition, the lead compound increased the expression of miR-34b and reduced c-Myc levels in lymphoma and colorectal cancer cell lines and a colorectal cancer xenograft mouse model. Identification of this first-in-class inhibitor of SUMO E1 is a major advance in modulating Ubl modifications for therapeutic aims.

Molecular mechanism of a covalent allosteric inhibitor of SUMO E1 activating enzyme.

E1 enzymes activate ubiquitin (Ub) and ubiquitin-like modifiers (Ubls) in the first step of Ub/Ubl conjugation cascades and represent potential targets for therapeutic intervention in cancer and other life-threatening diseases. Here, we report the crystal structure of the E1 enzyme for the Ubl SUMO in complex with a recently discovered and highly specific covalent allosteric inhibitor (COH000). The structure reveals that COH000 targets a cryptic pocket distinct from the active site that is completely buried in all previous SUMO E1 structures and that COH000 binding to SUMO E1 is accompanied by a network of structural changes that altogether lock the enzyme in a previously unobserved inactive conformation. These structural changes include disassembly of the active site and a 180° rotation of the catalytic cysteine-containing SCCH domain, relative to conformational snapshots of SUMO E1 poised to catalyze adenylation. Altogether, our study provides a molecular basis for the inhibitory mechanism of COH000 and its SUMO E1 specificity, and also establishes a framework for potential development of molecules targeting E1 enzymes for other Ubls at a cryptic allosteric site.

Repurposing antimalarial aminoquinolines and related compounds for treatment of retinal neovascularization.

Neovascularization is the pathological driver of blinding eye diseases such as retinopathy of prematurity, proliferative diabetic retinopathy, and wet age-related macular degeneration. The loss of vision resulting from these diseases significantly impacts the productivity and quality of life of patients, and represents a substantial burden on the health care system. Current standard of care includes biologics that target vascular endothelial growth factor (VEGF), a key mediator of neovascularization. While anti-VGEF therapies have been successful, up to 30% of patients are non-responsive. Therefore, there is a need for new therapeutic targets, and small molecule inhibitors of angiogenesis to complement existing treatments. Apelin and its receptor have recently been shown to play a key role in both developmental and pathological angiogenesis in the eye. Through a cell-based high-throughput screen, we identified 4-aminoquinoline antimalarial drugs as potent selective antagonists of APJ. The prototypical 4-aminoquinoline, amodiaquine was found to be a selective, non-competitive APJ antagonist that inhibited apelin signaling in a concentration-dependent manner. Additionally, amodiaquine suppressed both apelin-and VGEF-induced endothelial tube formation. Intravitreal amodaiquine significantly reduced choroidal neovascularization (CNV) lesion volume in the laser-induced CNV mouse model, and showed no signs of ocular toxicity at the highest doses tested. This work firmly establishes APJ as a novel, chemically tractable therapeutic target for the treatment of ocular neovascularization, and that amodiaquine is a potential candidate for repurposing and further toxicological, and pharmacokinetic evaluation in the clinic.

Discovery of Novel Small-Molecule Inducers of Heme Oxygenase-1 That Protect Human iPSC-Derived Cardiomyocytes from Oxidative Stress.

Oxidative injury to cardiomyocytes plays a critical role in cardiac pathogenesis following myocardial infarction. Transplantation of stem cell-derived cardiomyocytes has recently progressed as a novel treatment to repair damaged cardiac tissue but its efficacy has been limited by poor survival of transplanted cells owing to oxidative stress in the post-transplantation environment. Identification of small molecules that activate cardioprotective pathways to prevent oxidative damage and increase survival of stem cells post-transplantation is therefore of great interest for improving the efficacy of stem cell therapies. This report describes a chemical biology phenotypic screening approach to identify and validate small molecules that protect human-induced pluripotent stem cell cardiomyocytes (hiPSC-CMs) from oxidative stress. A luminescence-based high-throughput assay for cell viability was used to screen a diverse collection of 48,640 small molecules for protection of hiPSC-CMs from peroxide-induced cell death. Cardioprotective activity of "hit" compounds was confirmed using impedance-based detection of cardiomyocyte monolayer integrity and contractile function. Structure-activity relationship studies led to the identification of a potent class of compounds with 4-(pyridine-2-yl)thiazole scaffold. Examination of gene expression in hiPSC-CMs revealed that the hit compound, designated cardioprotectant 312 (CP-312), induces robust upregulation of heme oxygenase-1, a marker of the antioxidant response network that has been strongly correlated with protection of cardiomyocytes from oxidative stress. CP-312 therefore represents a novel chemical scaffold identified by phenotypic high-throughput screening using hiPSC-CMs that activates the antioxidant defense response and may lead to improved pharmacological cardioprotective therapies.

Ultra-High-Throughput Screening of Natural Product Extracts to Identify Proapoptotic Inhibitors of Bcl-2 Family Proteins.

Antiapoptotic Bcl-2 family proteins are validated cancer targets composed of six related proteins. From a drug discovery perspective, these are challenging targets that exert their cellular functions through protein-protein interactions (PPIs). Although several isoform-selective inhibitors have been developed using structure-based design or high-throughput screening (HTS) of synthetic chemical libraries, no large-scale screen of natural product collections has been reported. A competitive displacement fluorescence polarization (FP) screen of nearly 150,000 natural product extracts was conducted against all six antiapoptotic Bcl-2 family proteins using fluorochrome-conjugated peptide ligands that mimic functionally relevant PPIs. The screens were conducted in 1536-well format and displayed satisfactory overall HTS statistics, with Z'-factor values ranging from 0.72 to 0.83 and a hit confirmation rate between 16% and 64%. Confirmed active extracts were orthogonally tested in a luminescent assay for caspase-3/7 activation in tumor cells. Active extracts were resupplied, and effort toward the isolation of pure active components was initiated through iterative bioassay-guided fractionation. Several previously described altertoxins were isolated from a microbial source, and the pure compounds demonstrate activity in both Bcl-2 FP and caspase cellular assays. The studies demonstrate the feasibility of ultra-high-throughput screening using natural product sources and highlight some of the challenges associated with this approach.

Synthesis and physicochemical characterization of novel phenotypic probes targeting the nuclear factor-kappa B signaling pathway.

Activation of nuclear factor-kappa B (NF-κB) and related upstream signal transduction pathways have long been associated with the pathogenesis of a variety of inflammatory diseases and has recently been implicated in the onset of cancer. This report provides a synthetic and compound-based property summary of five pathway-related small-molecule chemical probes identified and optimized within the National Institutes of Health-Molecular Libraries Probe Center Network (NIH-MLPCN) initiative. The chemical probes discussed herein represent first-in-class, non-kinase-based modulators of the NF-κB signaling pathway, which were identified and optimized through either cellular phenotypic or specific protein-target-based screening strategies. Accordingly, the resulting new chemical probes may allow for better fundamental understanding of this highly complex biochemical signaling network and could advance future therapeutic translation toward the clinical setting.

Identification of Inhibitors of NOD1-Induced Nuclear Factor-κB Activation.

NOD1 (nucleotide-binding oligomerization domain 1) protein is a member of the NLR (NACHT and leucine rich repeat domain containing proteins) protein family, which plays a key role in innate immunity as a sensor of specific microbial components derived from bacterial peptidoglycans and induction of inflammatory responses. Mutations in NOD proteins have been associated with various inflammatory diseases that affect NF-κB (nuclear factor κB) activity, a major signaling pathway involved in apoptosis, inflammation, and immune response. A luciferase-based reporter gene assay was utilized in a high-throughput screening program conducted under the NIH-sponsored Molecular Libraries Probe Production Center Network program to identify the active scaffolds. Herein, we report the chemical synthesis, structure-activity relationship studies, downstream counterscreens, secondary assay data, and pharmacological profiling of the 2-aminobenzimidazole lead (compound 1c, ML130) as a potent and selective inhibitor of NOD1-induced NF-κB activation.

Isolation, synthesis, and biological activity of aphrocallistin, an adenine-substituted bromotyramine metabolite from the Hexactinellida sponge Aphrocallistes beatrix.

A new adenine-substituted bromotyrosine-derived metabolite designated as aphrocallistin (1) has been isolated from the deep-water Hexactinellida sponge Aphrocallistes beatrix. Its structure was elucidated on the basis of spectral data and confirmed through a convergent, modular total synthetic route that is amenable toward future analogue preparation. Aphrocallistin inhibits the growth of a panel of human tumor cell lines with IC(50) values ranging from 7.5 to >100 microM and has been shown to induce G1 cell cycle arrest in the PANC-1 pancreatic carcinoma cell line. Aphrocallistin has been fully characterized in the NCI cancer cell line panel and has undergone in vitro ADME pharmacological profiling.