Publications by authors named "Juan Marugan"

147 Publications

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

Sci Rep 2021 Jan 22;11(1):2121. Epub 2021 Jan 22.

Department of Microbiology and Immunology, Columbia University Irving Medical Center, New York, NY, 10032, USA.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.
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http://dx.doi.org/10.1038/s41598-021-81486-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7822874PMC
January 2021

Fluoxazolevir inhibits hepatitis C virus infection in humanized chimeric mice by blocking viral membrane fusion.

Nat Microbiol 2020 12 31;5(12):1532-1541. Epub 2020 Aug 31.

Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, USA.

Fluoxazolevir is an aryloxazole-based entry inhibitor of hepatitis C virus (HCV). We show that fluoxazolevir inhibits fusion of HCV with hepatic cells by binding HCV envelope protein 1 to prevent fusion. Nine of ten fluoxazolevir resistance-associated substitutions are in envelope protein 1, and four are in a putative fusion peptide. Pharmacokinetic studies in mice, rats and dogs revealed that fluoxazolevir localizes to the liver. A 4-week intraperitoneal regimen of fluoxazolevir in humanized chimeric mice infected with HCV genotypes 1b, 2a or 3 resulted in a 2-log reduction in viraemia, without evidence of drug resistance. In comparison, daclatasvir, an approved HCV drug, suppressed more than 3 log of viraemia but is associated with the emergence of resistance-associated substitutions in mice. Combination therapy using fluoxazolevir and daclatasvir cleared HCV genotypes 1b and 3 in mice. Fluoxazolevir combined with glecaprevir and pibrentasvir was also effective in clearing multidrug-resistant HCV replication in mice. Fluoxazolevir may be promising as the next generation of combination drug cocktails for HCV treatment.
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http://dx.doi.org/10.1038/s41564-020-0781-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7677215PMC
December 2020

Safety assessment of metarrestin in dogs: A clinical candidate targeting a subnuclear structure unique to metastatic cancer cells.

Regul Toxicol Pharmacol 2020 Oct 30;116:104716. Epub 2020 Jun 30.

National Center for Advancing Translational Sciences, NIH, Rockville, Maryland, USA. Electronic address:

Pancreatic cancer is a leading cause of cancer-related deaths in the U.S. Ninety percent of patients with stage IV pancreatic cancer die within one year of diagnosis due to complications of metastasis. A metastatic potential of cancer cells has been shown to be closely associated with formation of perinucleolar compartment (PNC). Metarrestin, a first-in-class PNC inhibitor, was evaluated for its toxicity, toxicokinetics, and safety pharmacology in beagle dogs following every other day oral (capsule) administration for 28 days to support its introduction into clinical trials. The study consisted of four dose groups: vehicle; 0.25, 0.75 and 1.50 mg/kg/dose. Metarrestin reached its maximum concentration in blood at 3 h (overall median T) across all doses with a mean t over 168 h of 55.5 h. Dose dependent increase in systemic exposure (C and AUC) with no sex difference was observed on days 1 and 27. Metarrestin accumulated from Day 1 to Day 27 at all dose levels and in both sexes by an overall factor of about 2.34. No mortality occurred during the dosing period; however, treatment-related clinical signs of toxicity consisting of hypoactivity, shaking/shivering, thinness, irritability, salivation, abnormal gait, tremors, ataxia and intermittent seizure-like activity were seen in both sexes at mid and high dose groups. Treatment-related effects on body weight and food consumption were seen at the mid and high dose levels. Safety pharmacology study showed no treatment-related effects on blood pressure, heart rate, corrected QT, PR, RR, or QRS intervals, or respiratory function parameters (respiratory rate, tidal volume, minute volume). There were no histopathological changes observed, with the exception of transient thymic atrophy which was considered to be non-adverse. Based primarily on clinical signs of toxicity, the No Observed Adverse Effect Level (NOAEL) in dogs was considered to be 0.25 mg/kg metarrestin after every other day dosing for 28 days with a mean of male and female C = 82.5 ng/mL and AUC = 2521 h*ng/mL, on Day 27.
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http://dx.doi.org/10.1016/j.yrtph.2020.104716DOI Listing
October 2020

Identification of a G-Protein-Independent Activator of GIRK Channels.

Cell Rep 2020 06;31(11):107770

Nash Family Department of Neuroscience, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA. Electronic address:

G-protein-gated inwardly rectifying K (GIRK) channels are essential effectors of inhibitory neurotransmission in the brain. GIRK channels have been implicated in diseases with abnormal neuronal excitability, including epilepsy and addiction. GIRK channels are tetramers composed of either the same subunit (e.g., homotetramers) or different subunits (e.g., heterotetramers). Compounds that specifically target subsets of GIRK channels in vivo are lacking. Previous studies have shown that alcohol directly activates GIRK channels through a hydrophobic pocket located in the cytoplasmic domain of the channel. Here, we report the identification and functional characterization of a GIRK1-selective activator, termed GiGA1, that targets the alcohol pocket. GiGA1 activates GIRK1/GIRK2 both in vitro and in vivo and, in turn, mitigates the effects of a convulsant in an acute epilepsy mouse model. These results shed light on the structure-based development of subunit-specific GIRK modulators that could provide potential treatments for brain disorders.
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http://dx.doi.org/10.1016/j.celrep.2020.107770DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7401321PMC
June 2020

Chlorcyclizine Inhibits Viral Fusion of Hepatitis C Virus Entry by Directly Targeting HCV Envelope Glycoprotein 1.

Cell Chem Biol 2020 Jul 7;27(7):780-792.e5. Epub 2020 May 7.

Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892, USA. Electronic address:

Chlorcyclizine (CCZ) is a potent hepatitis C virus (HCV) entry inhibitor, but its molecular mechanism is unknown. Here, we show that CCZ directly targets the fusion peptide of HCV E1 and interferes with the fusion process. Generation of CCZ resistance-associated substitutions of HCV in vitro revealed six missense mutations in the HCV E1 protein, five being in the putative fusion peptide. A viral fusion assay demonstrated that CCZ blocked HCV entry at the membrane fusion step and that the mutant viruses acquired resistance to CCZ's action in blocking membrane fusion. UV cross-linking of photoactivatable CCZ-diazirine-biotin in both HCV-infected cells and recombinant HCV E1/E2 protein demonstrated direct binding to HCV E1 glycoprotein. Mass spectrometry analysis revealed that CCZ cross-linked to an E1 sequence adjacent to the putative fusion peptide. Docking simulations demonstrate a putative binding model, wherein CCZ binds to a hydrophobic pocket of HCV E1 and forms extensive interactions with the fusion peptide.
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http://dx.doi.org/10.1016/j.chembiol.2020.04.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7368827PMC
July 2020

Identification of a Small-Molecule Inhibitor That Disrupts the SIX1/EYA2 Complex, EMT, and Metastasis.

Cancer Res 2020 06 27;80(12):2689-2702. Epub 2020 Apr 27.

Department of Pharmacology, University of Colorado Anschutz Medical Campus, Aurora, Colorado.

Metastasis is the major cause of mortality for patients with cancer, and dysregulation of developmental signaling pathways can significantly contribute to the metastatic process. The Sine oculis homeobox homolog 1 (SIX1)/eyes absent (EYA) transcriptional complex plays a critical role in the development of multiple organs and is typically downregulated after development is complete. In breast cancer, aberrant expression of SIX1 has been demonstrated to stimulate metastasis through activation of TGFβ signaling and subsequent induction of epithelial-mesenchymal transition (EMT). In addition, SIX1 can induce metastasis via non-cell autonomous means, including activation of GLI-signaling in neighboring tumor cells and activation of VEGFC-induced lymphangiogenesis. Thus, targeting SIX1 would be expected to inhibit metastasis while conferring limited side effects. However, transcription factors are notoriously difficult to target, and thus novel approaches to inhibit their action must be taken. Here we identified a novel small molecule compound, NCGC00378430 (abbreviated as 8430), that reduces the SIX1/EYA2 interaction. 8430 partially reversed transcriptional and metabolic profiles mediated by SIX1 overexpression and reversed SIX1-induced TGFβ signaling and EMT. 8430 was well tolerated when delivered to mice and significantly suppressed breast cancer-associated metastasis without significantly altering primary tumor growth. Thus, we have demonstrated for the first time that pharmacologic inhibition of the SIX1/EYA2 complex and associated phenotypes is sufficient to suppress breast cancer metastasis. SIGNIFICANCE: These findings identify and characterize a novel inhibitor of the SIX1/EYA2 complex that reverses EMT phenotypes suppressing breast cancer metastasis.
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http://dx.doi.org/10.1158/0008-5472.CAN-20-0435DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7510951PMC
June 2020

Identification of Small Molecule Enhancers of Immunotherapy for Melanoma.

Sci Rep 2020 03 30;10(1):5688. Epub 2020 Mar 30.

Division of Preclinical Innovation, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, Maryland, USA.

Small molecule based targeted therapies for the treatment of metastatic melanoma hold promise but responses are often not durable, and tumors frequently relapse. Response to adoptive cell transfer (ACT)-based immunotherapy in melanoma patients are durable but patients develop resistance primarily due to loss of antigen expression. The combination of small molecules that sustain T cell effector function with ACT could lead to long lasting responses. Here, we have developed a novel co-culture cell-based high throughput assay system to identify compounds that could potentially synergize or enhance ACT-based immunotherapy of melanoma. A BRAF mutant melanoma cell line, SB-3123 which is resistant to Pmel-1-directed ACT due to low gp100 expression levels was used to develop a homogenous time resolve fluorescence (HTRF), screening assay. This high throughput screening assay quantitates IFNγ released upon recognition of the SB-3123 melanoma cells by Pmel-1 CD8 T-cells. A focused collection of approximately 500 small molecules targeting a broad range of cellular mechanisms was screened, and four active compounds that increased melanoma antigen expression leading to enhanced IFNγ production were identified and their in vitro activity was validated. These four compounds may provide a basis for enhanced immune recognition and design of novel therapeutic approaches for patients with BRAF mutant melanoma resistant to ACT due to antigen downregulation.
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http://dx.doi.org/10.1038/s41598-020-62369-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7105471PMC
March 2020

Validation and Characterization of Five Distinct Novel Inhibitors of Human Cytomegalovirus.

J Med Chem 2020 04 27;63(8):3896-3907. Epub 2020 Mar 27.

Department of Pediatrics, Division of Infectious Disease, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, United States.

The critical consequences of human cytomegalovirus (HCMV) infection in the transplant population and in congenitally infected infants, the limited treatment options for HCMV, and the rise of resistant mutants toward existing therapies has fueled the search for new anti-HCMV agents. A pp28-luciferase recombinant HCMV was used as a reporter system for high-throughput screening of HCMV inhibitors. Approximately 400 000 compounds from existing libraries were screened. Subsequent validation assays using resynthesized compounds, several virus strains, and detailed virology assays resulted in the identification of five structurally unique and selective HCMV inhibitors, active at sub to low micromolar concentrations. Further characterization revealed that each compound inhibited a specific stage of HCMV replication. One compound was also active against herpes simplex virus (HSV1 and HSV2), and another compound was active against Epstein-Barr virus (EBV). Drug combination studies revealed that all five compounds were additive with ganciclovir or letermovir. Future studies will focus on optimization of these new anti-HCMV compounds along with mechanistic studies.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01501DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7386824PMC
April 2020

Metabolism and pharmacokinetics characterization of metarrestin in multiple species.

Cancer Chemother Pharmacol 2020 04 17;85(4):805-816. Epub 2020 Mar 17.

Division of Preclinical Innovation (DPI), National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, 9800 Medical Center Drive, Rockville, MD, 20850, USA.

Purpose: Metarrestin is a first-in-class pyrrolo-pyrimidine-derived small molecule targeting a marker of genome organization associated with metastasis and is currently in preclinical development as an anti-cancer agent. Here, we report the in vitro ADME characteristics and in vivo pharmacokinetic behavior of metarrestin.

Methods: Solubility, permeability, and efflux ratio as well as in vitro metabolism of metarrestin in hepatocytes, liver microsomes and S9 fractions, recombinant cytochrome P450 (CYP) enzymes, and potential for CYP inhibition were evaluated. Single dose pharmacokinetic profiles after intravenous and oral administration in mice, rat, dog, monkey, and mini-pig were obtained. Simple allometric scaling was applied to predict human pharmacokinetics.

Results: Metarrestin had an aqueous solubility of 150 µM at pH 7.4, high permeability in PAMPA and moderate efflux ratio in Caco-2 assays. The compound was metabolically stable in liver microsomes, S9 fractions, and hepatocytes from six species, including human. Metarrestin is a CYP3A4 substrate and, in mini-pigs, is also directly glucuronidated. Metarrestin did not show cytochrome P450 inhibitory activity. Plasma concentration-time profiles showed low to moderate clearance, ranging from 0.6 mL/min/kg in monkeys to 48 mL/min/kg in mice and moderate to high volume of distribution, ranging from 1.5 L/kg in monkeys to 17 L/kg in mice. Metarrestin has greater than 80% oral bioavailability in all species tested. The excretion of unchanged parent drug in urine was < 5% in dogs and < 1% in monkeys over collection periods of ≥ 144 h; in bile-duct cannulated rats, the excretion of unchanged drug was < 1% in urine and < 2% in bile over a collection period of 48 h.

Conclusions: Metarrestin is a low clearance compound which has good bioavailability and large biodistribution after oral administration. Biotransformation appears to be the major elimination process for the parent drug. In vitro data suggest a low drug-drug interaction potential on CYP-mediated metabolism. Overall favorable ADME and PK properties support metarrestin's progression to clinical investigation.
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http://dx.doi.org/10.1007/s00280-020-04042-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7147825PMC
April 2020

Synaptamide activates the adhesion GPCR GPR110 (ADGRF1) through GAIN domain binding.

Commun Biol 2020 03 6;3(1):109. Epub 2020 Mar 6.

Laboratory of Molecular Signaling, National Institute on Alcohol Abuse and Alcoholism, NIH, 5625 Fishers Lane, Rockville, MD, 20852, USA.

Adhesion G protein-coupled receptors (aGPCR) are characterized by a large extracellular region containing a conserved GPCR-autoproteolysis-inducing (GAIN) domain. Despite their relevance to several disease conditions, we do not understand the molecular mechanism by which aGPCRs are physiologically activated. GPR110 (ADGRF1) was recently deorphanized as the functional receptor of N-docosahexaenoylethanolamine (synaptamide), a potent synaptogenic metabolite of docosahexaenoic acid. Thus far, synaptamide is the first and only small-molecule endogenous ligand of an aGPCR. Here, we demonstrate the molecular basis of synaptamide-induced activation of GPR110 in living cells. Using in-cell chemical cross-linking/mass spectrometry, computational modeling and mutagenesis-assisted functional assays, we discover that synaptamide specifically binds to the interface of GPR110 GAIN subdomains through interactions with residues Q511, N512 and Y513, causing an intracellular conformational change near TM6 that triggers downstream signaling. This ligand-induced GAIN-targeted activation mechanism provides a framework for understanding the physiological function of aGPCRs and therapeutic targeting in the GAIN domain.
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http://dx.doi.org/10.1038/s42003-020-0831-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7060178PMC
March 2020

Small-molecule activation of lysosomal TRP channels ameliorates Duchenne muscular dystrophy in mouse models.

Sci Adv 2020 02 7;6(6):eaaz2736. Epub 2020 Feb 7.

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 4114 Biological Sciences Building, 1105 North University, Ann Arbor, MI 48109, USA.

Duchenne muscular dystrophy (DMD) is a devastating disease caused by mutations in dystrophin that compromise sarcolemma integrity. Currently, there is no treatment for DMD. Mutations in transient receptor potential mucolipin 1 (ML1), a lysosomal Ca channel required for lysosomal exocytosis, produce a DMD-like phenotype. Here, we show that transgenic overexpression or pharmacological activation of ML1 in vivo facilitates sarcolemma repair and alleviates the dystrophic phenotypes in both skeletal and cardiac muscles of mice (a mouse model of DMD). Hallmark dystrophic features of DMD, including myofiber necrosis, central nucleation, fibrosis, elevated serum creatine kinase levels, reduced muscle force, impaired motor ability, and dilated cardiomyopathies, were all ameliorated by increasing ML1 activity. ML1-dependent activation of transcription factor EB (TFEB) corrects lysosomal insufficiency to diminish muscle damage. Hence, targeting lysosomal Ca channels may represent a promising approach to treat DMD and related muscle diseases.
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http://dx.doi.org/10.1126/sciadv.aaz2736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7032923PMC
February 2020

Anxiolytic Drug FGIN-1-27 Ameliorates Autoimmunity by Metabolic Reprogramming of Pathogenic Th17 Cells.

Sci Rep 2020 02 28;10(1):3766. Epub 2020 Feb 28.

Division of Preclinical Innovation, National Center for Advancing Translational Sciences (NCATS), National Institutes of Health, Rockville, MD, USA.

Th17 cells are critical drivers of autoimmune diseases and immunopathology. There is an unmet need to develop therapies targeting pathogenic Th17 cells for the treatment of autoimmune disorders. Here, we report that anxiolytic FGIN-1-27 inhibits differentiation and pathogenicity of Th17 cells in vitro and in vivo using the experimental autoimmune encephalomyelitis (EAE) model of Th17 cell-driven pathology. Remarkably, we found that the effects of FGIN-1-27 were independent of translocator protein (TSPO), the reported target for this small molecule, and instead were driven by a metabolic switch in Th17 cells that led to the induction of the amino acid starvation response and altered cellular fatty acid composition. Our findings suggest that the small molecule FGIN-1-27 can be re-purposed to relieve autoimmunity by metabolic reprogramming of pathogenic Th17 cells.
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http://dx.doi.org/10.1038/s41598-020-60610-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7048748PMC
February 2020

Mannose receptor (CD206) activation in tumor-associated macrophages enhances adaptive and innate antitumor immune responses.

Sci Transl Med 2020 02;12(530)

Rare Tumor Initiative, Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Bethesda, MD 20892, USA.

Solid tumors elicit a detectable immune response including the infiltration of tumor-associated macrophages (TAMs). Unfortunately, this immune response is co-opted into contributing toward tumor growth instead of preventing its progression. We seek to reestablish an antitumor immune response by selectively targeting surface receptors and endogenous signaling processes of the macrophage subtypes driving cancer progression. RP-182 is a synthetic 10-mer amphipathic analog of host defense peptides that selectively induces a conformational switch of the mannose receptor CD206 expressed on TAMs displaying an M2-like phenotype. RP-182-mediated activation of this receptor in human and murine M2-like macrophages elicits a program of endocytosis, phagosome-lysosome formation, and autophagy and reprograms M2-like TAMs to an antitumor M1-like phenotype. In syngeneic and autochthonous murine cancer models, RP-182 suppressed tumor growth, extended survival, and was an effective combination partner with chemo- or immune checkpoint therapy. Antitumor activity of RP-182 was also observed in CD206 patient-derived xenotransplantation models. Mechanistically, via selective reduction of immunosuppressive M2-like TAMs, RP-182 improved adaptive and innate antitumor immune responses, including increased cancer cell phagocytosis by reprogrammed TAMs.
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http://dx.doi.org/10.1126/scitranslmed.aax6337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7832040PMC
February 2020

Agonist-specific voltage-dependent gating of lysosomal two-pore Na channels.

Elife 2019 12 11;8. Epub 2019 Dec 11.

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, Ann Arbor, United States.

Mammalian two-pore-channels (TPC1, 2; ) are ubiquitously- expressed, PI(3,5)P-activated, Na-selective channels in the endosomes and lysosomes that regulate luminal pH homeostasis, membrane trafficking, and viral infection. Whereas the channel activity of TPC1 is strongly dependent on membrane voltage, TPC2 lacks such voltage dependence despite the presence of the presumed 'S4 voltage-sensing' domains. By performing high-throughput screening followed by lysosomal electrophysiology, here we identified a class of tricyclic anti-depressants (TCAs) as small-molecule agonists of TPC channels. TCAs activate both TPC1 and TPC2 in a voltage-dependent manner, referred to as sosomal channel oltage-dependent ctivators (LyNa-VAs). We also identified another compound which, like PI(3,5)P, activates TPC2 independent of voltage, suggesting the existence of agonist-specific gating mechanisms. Our identification of small-molecule TPC agonists should facilitate the studies of the cell biological roles of TPCs and can also readily explain the reported effects of TCAs in the modulation of autophagy and lysosomal functions.
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http://dx.doi.org/10.7554/eLife.51423DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6905855PMC
December 2019

Quantitative High-Throughput Screening Using an Organotypic Model Identifies Compounds that Inhibit Ovarian Cancer Metastasis.

Mol Cancer Ther 2020 01 27;19(1):52-62. Epub 2019 Sep 27.

Department of Obstetrics and Gynecology/Section of Gynecologic Oncology, University of Chicago, Chicago, Illinois.

The tumor microenvironment (TME) is a key determinant of metastatic efficiency. We performed a quantitative high-throughput screen (qHTS) of diverse medicinal chemistry tractable scaffolds (44,420 compounds) and pharmacologically active small molecules (386 compounds) using a layered organotypic, robust assay representing the ovarian cancer metastatic TME. This 3D model contains primary human mesothelial cells, fibroblasts, and extracellular matrix, to which fluorescently labeled ovarian cancer cells are added. Initially, 100 compounds inhibiting ovarian cancer adhesion/invasion to the 3D model in a dose-dependent manner were identified. Of those, eight compounds were confirmed active in five high-grade serous ovarian cancer cell lines and were further validated in secondary and biological assays. Two tyrosine kinase inhibitors, PP-121 and milciclib, and a previously unreported compound, NCGC00117362, were selected because they had potency at 1 μmol/L Specifically, NCGC00117362 and PP-121 inhibited ovarian cancer adhesion, invasion, and proliferation, whereas milciclib inhibited ovarian cancer invasion and proliferation. Using kinase profiling and immunoblotting, we found that milciclib targeted Cdk2 and Cdk6, and PP-121 targeted mTOR. , all three compounds prevented ovarian cancer adhesion/invasion and metastasis, prolonged survival, and reduced omental tumor growth in an intervention study. To evaluate the clinical potential of NCGC00117362, structure-activity relationship studies were performed. Four close analogues of NCGC00117362 efficiently inhibited cancer aggressiveness and metastasis Collectively, these data show that a complex 3D culture of the TME is effective in qHTS. The three compounds identified have promise as therapeutics for prevention and treatment of ovarian cancer metastasis.
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http://dx.doi.org/10.1158/1535-7163.MCT-19-0052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6946883PMC
January 2020

Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.

FASEB J 2019 11 16;33(11):12435-12446. Epub 2019 Aug 16.

Department of Human and Molecular Genetics, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, USA.

Fibrosis is an underlying cause of cirrhosis and hepatic failure resulting in end stage liver disease with limited pharmacological options. The beneficial effects of relaxin peptide treatment were demonstrated in clinically relevant animal models of liver fibrosis. However, the use of relaxin is problematic because of a short half-life. The aim of this study was to test the therapeutic effects of recently identified small molecule agonists of the human relaxin receptor, relaxin family peptide receptor 1 (RXFP1). The lead compound of this series, ML290, was selected based on its effects on the expression of fibrosis-related genes in primary human stellate cells. RNA sequencing analysis of TGF-β1-activated LX-2 cells showed that ML290 treatment primarily affected extracellular matrix remodeling and cytokine signaling, with expression profiles indicating an antifibrotic effect of ML290. ML290 treatment in human liver organoids with LPS-induced fibrotic phenotype resulted in a significant reduction of type I collagen. The pharmacokinetics of ML290 in mice demonstrated its high stability , as evidenced by the sustained concentrations of compound in the liver. In mice expressing human gene treated with carbon tetrachloride, ML290 significantly reduced collagen content, α-smooth muscle actin expression, and cell proliferation around portal ducts. In conclusion, ML290 demonstrated antifibrotic effects in liver fibrosis.-Kaftanovskaya, E. M., Ng, H. H., Soula, M., Rivas, B., Myhr, C., Ho, B. A., Cervantes, B. A., Shupe, T. D., Devarasetty, M., Hu, X., Xu, X., Patnaik, S., Wilson, K. J., Barnaeva, E., Ferrer, M., Southall, N. T., Marugan, J. J., Bishop, C. E., Agoulnik, I. U., Agoulnik, A. I. Therapeutic effects of a small molecule agonist of the relaxin receptor ML290 in liver fibrosis.
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http://dx.doi.org/10.1096/fj.201901046RDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6988856PMC
November 2019

Structural and Functional Analyses of an Allosteric EYA2 Phosphatase Inhibitor That Has On-Target Effects in Human Lung Cancer Cells.

Mol Cancer Ther 2019 09 8;18(9):1484-1496. Epub 2019 Jul 8.

Experimental Drug Discovery Centre, A*STAR, Singapore, Singapore.

EYA proteins (EYA1-4) are critical developmental transcriptional cofactors that contain an EYA domain (ED) harboring Tyr phosphatase activity. EYA proteins are largely downregulated after embryogenesis but are reexpressed in cancers, and their Tyr phosphatase activity plays an important role in the DNA damage response and tumor progression. We previously identified a class of small-molecule allosteric inhibitors that specifically inhibit the Tyr phosphatase activity of EYA2. Herein, we determined the crystal structure of the EYA2 ED in complex with NCGC00249987 (a representative compound in this class), revealing that it binds to an induced pocket distant from the active site. NCGC00249987 binding leads to a conformational change of the active site that is unfavorable for Mg binding, thereby inhibiting EYA2's Tyr phosphatase activity. We demonstrate, using genetic mutations, that migration, invadopodia formation, and invasion of lung adenocarcinoma cells are dependent on EYA2 Tyr phosphatase activity, whereas growth and survival are not. Further, we demonstrate that NCGC00249987 specifically targets migration, invadopodia formation, and invasion of lung cancer cells, but that it does not inhibit cell growth or survival. The compound has no effect on lung cancer cells carrying an EYA2 F290Y mutant that abolishes compound binding, indicating that NCGC00249987 is on target in lung cancer cells. These data suggest that the NCGC00249987 allosteric inhibitor can be used as a chemical probe to study the function of the EYA2 Tyr phosphatase activity in cells and may have the potential to be developed into an antimetastatic agent for cancers reliant on EYA2's Tyr phosphatase activity.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-1239DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6726557PMC
September 2019

-Tocopherol Effect on Endocytosis and Its Combination with Enzyme Replacement Therapy for Lysosomal Disorders: A New Type of Drug Interaction?

J Pharmacol Exp Ther 2019 09 17;370(3):823-833. Epub 2019 May 17.

Fischell Department of Bioengineering (R.L.M., J.A.R., V.V., M.H.) and Institute for Bioscience and Biotechnology Research (M.S., S.M.), University of Maryland, College Park, Maryland; National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, Maryland (Y.L., J.M., W.Z.); Institute for Bioengineering of Catalonia of the Barcelona Institute of Science and Technology, Barcelona, Spain (D.G., S.M.); and Institution of Catalonia for Research and Advanced Studies, Barcelona, Spain (S.M.)

Induction of lysosomal exocytosis alleviates lysosomal storage of undigested metabolites in cell models of lysosomal disorders (LDs). However, whether this strategy affects other vesicular compartments, e.g., those involved in endocytosis, is unknown. This is important both to predict side effects and to use this strategy in combination with therapies that require endocytosis for intracellular delivery, such as lysosomal enzyme replacement therapy (ERT). We investigated this using -tocopherol as a model previously shown to induce lysosomal exocytosis and cell models of type A Niemann-Pick disease, a LD characterized by acid sphingomyelinase (ASM) deficiency and sphingomyelin storage. -Tocopherol and derivative CF3-T reduced net accumulation of fluid phase, ligands, and polymer particles via phagocytic, caveolae-, clathrin-, and cell adhesion molecule (CAM)-mediated pathways, yet the latter route was less affected due to receptor overexpression. In agreement, -tocopherol lowered uptake of recombinant ASM by deficient cells (known to occur via the clathrin pathway) and via targeting intercellular adhesion molecule-1 (associated to the CAM pathway). However, the net enzyme activity delivered and lysosomal storage attenuation were greater via the latter route. Data suggest stimulation of exocytosis by tocopherols is not specific of lysosomes and affects endocytic cargo. However, this effect was transient and became unnoticeable several hours after tocopherol removal. Therefore, induction of exocytosis in combination with therapies requiring endocytic uptake, such as ERT, may represent a new type of drug interaction, yet this strategy could be valuable if properly timed for minimal interference.
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http://dx.doi.org/10.1124/jpet.119.257345DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6806345PMC
September 2019

Identification, design and synthesis of novel pyrazolopyridine influenza virus nonstructural protein 1 antagonists.

Bioorg Med Chem Lett 2019 05 26;29(9):1113-1119. Epub 2019 Feb 26.

Division of Pre-Clinical Innovation, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD 20850, United States. Electronic address:

Nonstructural protein 1 (NS1) plays a crucial function in the replication, spread, and pathogenesis of influenza virus by inhibiting the host innate immune response. Here we report the discovery and optimization of novel pyrazolopyridine NS1 antagonists that can potently inhibit influenza A/PR/8/34 replication in MDCK cells, rescue MDCK cells from cytopathic effects of seasonal influenza A strains, reverse NS1-dependent inhibition of IFN-β gene expression, and suppress the slow growth phenotype in NS1-expressing yeast. These pyrazolopyridines will enable researchers to investigate NS1 function during infection and how antagonists can be utilized in the next generation of treatments for influenza infection.
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http://dx.doi.org/10.1016/j.bmcl.2019.02.027DOI Listing
May 2019

Sunitinib promotes myogenic regeneration and mitigates disease progression in the mdx mouse model of Duchenne muscular dystrophy.

Hum Mol Genet 2019 07;28(13):2120-2132

Department of Pharmacology, University of Nevada, Reno School of Medicine, , Reno, NV, USA.

Duchenne muscular dystrophy (DMD) is a lethal, muscle degenerative disease causing premature death of affected children. DMD is characterized by mutations in the dystrophin gene that result in a loss of the dystrophin protein. Loss of dystrophin causes an associated reduction in proteins of the dystrophin glycoprotein complex, leading to contraction-induced sarcolemmal weakening, muscle tearing, fibrotic infiltration and rounds of degeneration and failed regeneration affecting satellite cell populations. The α7β1 integrin has been implicated in increasing myogenic capacity of satellite cells, therefore restoring muscle viability, increasing muscle force and preserving muscle function in dystrophic mouse models. In this study, we show that a Food and Drug Administration (FDA)-approved small molecule, Sunitinib, is a potent α7 integrin enhancer capable of promoting myogenic regeneration by stimulating satellite cell activation and increasing myofiber fusion. Sunitinib exerts its regenerative effects via transient inhibition of SHP-2 and subsequent activation of the STAT3 pathway. Treatment of mdx mice with Sunitinib demonstrated decreased membrane leakiness and damage owing to myofiber regeneration and enhanced support at the extracellular matrix. The decreased myofiber damage translated into a significant increase in muscle force production. This study identifies an already FDA-approved compound, Sunitinib, as a possible DMD therapeutic with the potential to treat other muscular dystrophies in which there is defective muscle repair.
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http://dx.doi.org/10.1093/hmg/ddz044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6586148PMC
July 2019

A family of PIKFYVE inhibitors with therapeutic potential against autophagy-dependent cancer cells disrupt multiple events in lysosome homeostasis.

Autophagy 2019 10 8;15(10):1694-1718. Epub 2019 Mar 8.

Division of Developmental Biology, National Institute of Child Health & Human Development, National Institutes of Health , Bethesda , MD , USA.

High-throughput screening identified 5 chemical analogs (termed the WX8-family) that disrupted 3 events in lysosome homeostasis: (1) lysosome fission via tubulation without preventing homotypic lysosome fusion; (2) trafficking of molecules into lysosomes without altering lysosomal acidity, and (3) heterotypic fusion between lysosomes and autophagosomes. Remarkably, these compounds did not prevent homotypic fusion between lysosomes, despite the fact that homotypic fusion required some of the same machinery essential for heterotypic fusion. These effects varied 400-fold among WX8-family members, were time and concentration dependent, reversible, and resulted primarily from their ability to bind specifically to the PIKFYVE phosphoinositide kinase. The ability of the WX8-family to prevent lysosomes from participating in macroautophagy/autophagy suggested they have therapeutic potential in treating autophagy-dependent diseases. In fact, the most potent family member (WX8) was 100-times more lethal to 'autophagy-addicted' melanoma A375 cells than the lysosomal inhibitors hydroxychloroquine and chloroquine. In contrast, cells that were insensitive to hydroxychloroquine and chloroquine were also insensitive to WX8. Therefore, the WX8-family of PIKFYVE inhibitors provides a basis for developing drugs that could selectively kill autophagy-dependent cancer cells, as well as increasing the effectiveness of established anti-cancer therapies through combinatorial treatments. : ACTB: actin beta; Baf: bafilomycin A; BECN1: beclin 1; BODIPY: boron-dipyrromethene; BORC: BLOC-1 related complex; BRAF: B-Raf proto-oncogene, serine/threonine kinase; BSA: bovine serum albumin; CTSD: cathepsin D; CQ: chloroquine; DNA: deoxyribonucleic acid; EC: half maximal effective concentration; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; HCQ: hydroxychloroquine; HOPS complex: homotypic fusion and protein sorting complex; Kd: equilibrium binding constant; IC50: half maximal inhibitory concentration; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MAP1LC3A: microtubule associated protein 1 light chain 3 alpha; MES: 2-(N-morpholino)ethanesulphonic acid; MTOR: mechanistic target of rapamycin kinase; μM: micromolar; NDF: 3-methylbenzaldehyde (2,6-dimorpholin-4-ylpyrimidin-4-yl)hydrazine;NEM: N-ethylmaleimide; NSF: N-ethylmaleimide sensitive factor; PBS: phosphate-buffered saline; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PIP4K2C: phosphatidylinositol-5-phosphate 4-kinase type 2 gamma; PtdIns3P: phosphatidylinositol 3-phosphate; PtdIns(3,5)P: phosphatidylinositol 3,5-biphosphate; RFP: red fluorescent protein; RPS6: ribosomal protein S6; RPS6KB1: ribosomal protein S6 kinase B1; SQSTM1: sequestosome 1; TWEEN 20: polysorbate 20; V-ATPase: vacuolar-type H-translocating ATPase; VPS39: VPS39 subunit of HOPS complex; VPS41: VPS41 subunit of HOPS complex; WWL: benzaldehyde [2,6-di(4-morpholinyl)-4-pyrimidinyl]hydrazone; WX8: 1H-indole-3-carbaldehyde [4-anilino-6-(4-morpholinyl)-1,3,5-triazin-2-yl]hydrazine; XBA: N-(3-chloro-4-fluorophenyl)-4,6-dimorpholino-1,3,5-triazin-2-amine hydrochloride; XB6: N-(4-ethylphenyl)-4,6-dimorpholino-1,3,5-triazin-2-amine hydrochloride.
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http://dx.doi.org/10.1080/15548627.2019.1586257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6735543PMC
October 2019

Proteolytic cleavage of host proteins by the Group IV viral proteases of Venezuelan equine encephalitis virus and Zika virus.

Antiviral Res 2019 04 10;164:106-122. Epub 2019 Feb 10.

Center for Bio/molecular Science and Engineering, U.S. Naval Research Laboratory, Washington, DC 20375, USA. Electronic address:

The alphaviral nonstructural protein 2 (nsP2) cysteine proteases (EC 3.4.22.-) are essential for the proteolytic processing of the nonstructural (ns) polyprotein and are validated drug targets. A common secondary role of these proteases is to antagonize the effects of interferon (IFN). After delineating the cleavage site motif of the Venezuelan equine encephalitis virus (VEEV) nsP2 cysteine protease, we searched the human genome to identify host protein substrates. Here we identify a new host substrate of the VEEV nsP2 protease, human TRIM14, a component of the mitochondrial antiviral-signaling protein (MAVS) signalosome. Short stretches of homologous host-pathogen protein sequences (SSHHPS) are present in the nonstructural polyprotein and TRIM14. A 25-residue cyan-yellow fluorescent protein TRIM14 substrate was cleaved in vitro by the VEEV nsP2 protease and the cleavage site was confirmed by tandem mass spectrometry. A TRIM14 cleavage product also was found in VEEV-infected cell lysates. At least ten other Group IV (+)ssRNA viral proteases have been shown to cleave host proteins involved in generating the innate immune responses against viruses, suggesting that the integration of these short host protein sequences into the viral protease cleavage sites may represent an embedded mechanism of IFN antagonism. This interference mechanism shows several parallels with those of CRISPR/Cas9 and RNAi/RISC, but with a protease recognizing a protein sequence common to both the host and pathogen. The short host sequences embedded within the viral genome appear to be analogous to the short phage sequences found in a host's CRISPR spacer sequences. To test this algorithm, we applied it to another Group IV virus, Zika virus (ZIKV), and identified cleavage sites within human SFRP1 (secreted frizzled related protein 1), a retinal G alpha subunit, NT5M, and Forkhead box protein G1 (FOXG1) in vitro. Proteolytic cleavage of these proteins suggests a possible link between the protease and the virus-induced phenotype of ZIKV. The algorithm may have value for selecting cell lines and animal models that recapitulate virus-induced phenotypes, predicting host-range and susceptibility, selecting oncolytic viruses, identifying biomarkers, and de-risking live virus vaccines. Inhibitors of the proteases that utilize this mechanism may both inhibit viral replication and alleviate suppression of the innate immune responses.
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http://dx.doi.org/10.1016/j.antiviral.2019.02.001DOI Listing
April 2019

A large scale high-throughput screen identifies chemical inhibitors of phosphatidylinositol 4-kinase type II alpha.

J Lipid Res 2019 03 9;60(3):683-693. Epub 2019 Jan 9.

Section on Molecular Signal Transduction, Program for Developmental Neuroscience, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892

The minor phospholipid, phosphatidylinositol 4-phosphate (PI4P), is emerging as a key regulator of lipid transfer in ER-membrane contact sites. Four different phosphatidylinositol 4-kinase (PI4K) enzymes generate PI4P in different membrane compartments supporting distinct cellular processes, many of which are crucial for the maintenance of cellular integrity but also hijacked by intracellular pathogens. While type III PI4Ks have been targeted by small molecular inhibitors, thus helping decipher their importance in cellular physiology, no inhibitors are available for the type II PI4Ks, which hinders investigations into their cellular functions. Here, we describe the identification of small molecular inhibitors of PI4K type II alpha (PI4K2A) by implementing a large scale small molecule high-throughput screening. A novel assay was developed that allows testing of selected inhibitors against PI4K2A in intact cells using a bioluminescence resonance energy transfer approach adapted to plate readers. The compounds disclosed here will pave the way to the optimization of PI4K2A inhibitors that can be used in cellular and animal studies to better understand the role of this enzyme in both normal and pathological states.
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http://dx.doi.org/10.1194/jlr.D090159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6399489PMC
March 2019

Identification of Chemotype Agonists for Human Resolvin D1 Receptor DRV1 with Pro-Resolving Functions.

Cell Chem Biol 2019 02 13;26(2):244-254.e4. Epub 2018 Dec 13.

Center for Experimental Therapeutics and Reperfusion Injury, Department of Anesthesiology, Perioperative and Pain Medicine, Brigham and Women's Hospital and Harvard Medical School, 60 Fenwood Road BTM 3-016, Boston, MA 02115, USA. Electronic address:

Resolution of acute inflammation is governed, in part, by specialized pro-resolving mediators, including lipoxins, resolvins, protectins, and maresins. Among them, resolvin D1 (RvD1) exhibits potent pro-resolving functions via activating human resolvin D1 receptor (DRV1/GPR32). RvD1 is a complex molecule that requires challenging organic synthesis, diminishing its potential as a therapeutic. Therefore, we implemented a high-throughput screening of small-molecule libraries and identified several chemotypes that activated recombinant DRV1, represented by NCGC00120943 (C1A), NCGC00135472 (C2A), pMPPF, and pMPPI. These chemotypes also elicited rapid impedance changes in cells overexpressing recombinant DRV1. With human macrophages, they each stimulated phagocytosis of serum-treated zymosan at concentrations comparable with that of RvD1, the endogenous DRV1 ligand. In addition, macrophage phagocytosis of live E. coli was significantly increased by these chemotypes in DRV1-transfected macrophages, compared with mock-transfected cells. Taken together, these chemotypes identified by unbiased screens act as RvD1 mimetics, exhibiting pro-resolving functions via interacting with human DRV1.
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http://dx.doi.org/10.1016/j.chembiol.2018.10.023DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405215PMC
February 2019

DPTIP, a newly identified potent brain penetrant neutral sphingomyelinase 2 inhibitor, regulates astrocyte-peripheral immune communication following brain inflammation.

Sci Rep 2018 12 7;8(1):17715. Epub 2018 Dec 7.

Johns Hopkins Drug Discovery, Johns Hopkins School of Medicine, Baltimore, Maryland, 21205, USA.

Brain injury and inflammation induces a local release of extracellular vesicles (EVs) from astrocytes carrying proteins, RNAs, and microRNAs into the circulation. When these vesicles reach the liver, they stimulate the secretion of cytokines that mobilize peripheral immune cell infiltration into the brain, which can cause secondary tissue damage and impair recovery. Recent studies suggest that suppression of EV biosynthesis through neutral sphingomyelinase 2 (nSMase2) inhibition may represent a new therapeutic strategy. Unfortunately, currently available nSMase2 inhibitors exhibit low potency (IC ≥ 1 μM), poor solubility and/or limited brain penetration. Through a high throughput screening campaign of >365,000 compounds against human nSMase2 we identified 2,6-Dimethoxy-4-(5-Phenyl-4-Thiophen-2-yl-1H-Imidazol-2-yl)-Phenol (DPTIP), a potent (IC 30 nM), selective, metabolically stable, and brain penetrable (AUC/AUC = 0.26) nSMase2 inhibitor. DPTIP dose-dependently inhibited EV release in primary astrocyte cultures. In a mouse model of brain injury conducted in GFAP-GFP mice, DPTIP potently (10 mg/kg IP) inhibited IL-1β-induced astrocyte-derived EV release (51 ± 13%; p < 0.001). This inhibition led to a reduction of cytokine upregulation in liver and attenuation of the infiltration of immune cells into the brain (80 ± 23%; p < 0.01). A structurally similar but inactive analog had no effect in vitro or in vivo.
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http://dx.doi.org/10.1038/s41598-018-36144-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6286365PMC
December 2018

Pharmacokinetic evaluation of the PNC disassembler metarrestin in wild-type and Pdx1-Cre;LSL-Kras;Tp53 (KPC) mice, a genetically engineered model of pancreatic cancer.

Cancer Chemother Pharmacol 2018 12 10;82(6):1067-1080. Epub 2018 Oct 10.

Rare Tumor Initiative (RTI), Pediatric Oncology Branch, Center for Cancer Research, National Cancer Institute, Hatfield Center, 10 Center Drive, 9000 Rockville Pike, Bethesda, MD, 20892, USA.

Purpose: Metarrestin is a first-in-class small molecule clinical candidate capable of disrupting the perinucleolar compartment, a subnuclear structure unique to metastatic cancer cells. This study aims to define the pharmacokinetic (PK) profile of metarrestin and the pharmacokinetic/pharmacodynamic relationship of metarrestin-regulated markers.

Methods: PK studies included the administration of single or multiple dose of metarrestin at 3, 10, or 25 mg/kg via intravenous (IV) injection, gavage (PO) or with chow to wild-type C57BL/6 mice and KPC mice bearing autochthonous pancreatic tumors. Metarrestin concentrations were analyzed by UPLC-MS/MS. Pharmacodynamic assays included mRNA expression profiling by RNA-seq and qRT-PCR for KPC mice.

Results: Metarrestin had a moderate plasma clearance of 48 mL/min/kg and a large volume of distribution of 17 L/kg at 3 mg/kg IV in C57BL/6 mice. The oral bioavailability after single-dose (SD) treatment was > 80%. In KPC mice treated with SD 25 mg/kg PO, plasma AUC of 14400 ng h/mL, C of 810 ng/mL and half-life (t) of 8.5 h were observed. At 24 h after SD of 25 mg/kg PO, the intratumor concentration of metarrestin was high with a mean value of 6.2 µg/g tissue (or 13 µM), well above the cell-based IC of 0.4 µM. At multiple dose (MD) 25 mg/kg/day PO in KPC mice, mean tissue/plasma AUC ratio for tumor, spleen and liver was 37, 30 and 31, respectively. There was a good linear relationship of dosage to AUC and C. AUC MD to AUC SD ratios ranged from two for liver to five for tumor indicating additional accumulation in tumors. Dose-dependent normalization of FOXA1 and FOXO6 mRNA expression was observed in KPC tumors.

Conclusions: Metarrestin is an effective therapeutic candidate with a favorable PK profile achieving excellent intratumor tissue levels in a disease with known poor drug delivery.
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http://dx.doi.org/10.1007/s00280-018-3699-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6267684PMC
December 2018

Neural stem cells for disease modeling and evaluation of therapeutics for Tay-Sachs disease.

Orphanet J Rare Dis 2018 09 17;13(1):152. Epub 2018 Sep 17.

National Center for Advancing Translational Sciences, National Institutes of Health, Bethesda, MD, USA.

Background: Tay-Sachs disease (TSD) is a rare neurodegenerative disorder caused by autosomal recessive mutations in the HEXA gene on chromosome 15 that encodes β-hexosaminidase. Deficiency in HEXA results in accumulation of GM2 ganglioside, a glycosphingolipid, in lysosomes. Currently, there is no effective treatment for TSD.

Results: We generated induced pluripotent stem cells (iPSCs) from two TSD patient dermal fibroblast lines and further differentiated them into neural stem cells (NSCs). The TSD neural stem cells exhibited a disease phenotype of lysosomal lipid accumulation. The Tay-Sachs disease NSCs were then used to evaluate the therapeutic effects of enzyme replacement therapy (ERT) with recombinant human Hex A protein and two small molecular compounds: hydroxypropyl-β-cyclodextrin (HPβCD) and δ-tocopherol. Using this disease model, we observed reduction of lipid accumulation by employing enzyme replacement therapy as well as by the use of HPβCD and δ-tocopherol.

Conclusion: Our results demonstrate that the Tay-Sachs disease NSCs possess the characteristic phenotype to serve as a cell-based disease model for study of the disease pathogenesis and evaluation of drug efficacy. The enzyme replacement therapy with recombinant Hex A protein and two small molecules (cyclodextrin and tocopherol) significantly ameliorated lipid accumulation in the Tay-Sachs disease cell model.
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http://dx.doi.org/10.1186/s13023-018-0886-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139903PMC
September 2018

Optimization of the first small-molecule relaxin/insulin-like family peptide receptor (RXFP1) agonists: Activation results in an antifibrotic gene expression profile.

Eur J Med Chem 2018 Aug 7;156:79-92. Epub 2018 Jun 7.

NIH Chemical Genomics Center, National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, MD, 20850, USA. Electronic address:

A dose responsive quantitative high throughput screen (qHTS) of >350,000 compounds against a human relaxin/insulin-like family peptide receptor (RXFP1) transfected HEK293 cell line identified 2-acetamido-N-phenylbenzamides 1 and 3 with modest agonist activity. An extensive structure-activity study has been undertaken to optimize the potency, efficacy, and physical properties of the series, resulting in the identification of compound 65 (ML-290), which has excellent in vivo PK properties with high levels of systemic exposure. This series, exemplified by 65, has produced first-in-class small-molecule agonists of RXFP1 and is a potent activator of anti-fibrotic genes.
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http://dx.doi.org/10.1016/j.ejmech.2018.06.008DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6102074PMC
August 2018

A widely-applicable high-throughput cellular thermal shift assay (CETSA) using split Nano Luciferase.

Sci Rep 2018 06 21;8(1):9472. Epub 2018 Jun 21.

National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland, 20850, USA.

Assessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space. The Cellular Thermal Shift Assay (CETSA) enables such an assessment by quantifying the changes in the thermal stability of proteins upon ligand binding in intact cells. Here, we present the development and validation of a homogeneous, standardized, target-independent, and high-throughput (384- and 1536-well formats) CETSA platform that uses a split Nano Luciferase approach (SplitLuc CETSA). The broad applicability of the assay was demonstrated for diverse targets, and its performance was compared with independent biochemical and cell-based readouts using a set of well-characterized inhibitors. Moreover, we investigated the utility of the platform as a primary assay for high-throughput screening. The SplitLuc CETSA presented here enables target engagement studies for medium and high-throughput applications. Additionally, it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available.
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http://dx.doi.org/10.1038/s41598-018-27834-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6013488PMC
June 2018

High-throughput screening identified selective inhibitors of exosome biogenesis and secretion: A drug repurposing strategy for advanced cancer.

Sci Rep 2018 05 25;8(1):8161. Epub 2018 May 25.

Departments of Urology, Tulane University School of Medicine, New Orleans, LA, 70112, United States.

Targeting exosome biogenesis and release may have potential clinical implications for cancer therapy. Herein, we have optimized a quantitative high throughput screen (qHTS) assay to identify compounds that modulate exosome biogenesis and/or release by aggressive prostate cancer (PCa) CD63-GFP-expressing C4-2B cells. A total of 4,580 compounds were screened from the LOPAC library (a collection of 1,280 pharmacologically active compounds) and the NPC library (NCGC collection of 3,300 compounds approved for clinical use). Twenty-two compounds were found to be either potent activators or inhibitors of intracellular GFP signal in the CD63-GFP-expressing C4-2B cells. The activity of lead compounds in modulating the secretion of exosomes was validated by a tunable resistive pulse sensing (TRPS) system (qNano-IZON) and flow cytometry. The mechanism of action of the lead compounds in modulating exosome biogenesis and/or secretion were delineated by immunoblot analysis of protein markers of the endosomal sorting complex required for transport (ESCRT)-dependent and ESCRT-independent pathways. The lead compounds tipifarnib, neticonazole, climbazole, ketoconazole, and triademenol were validated as potent inhibitors and sitafloxacin, forskolin, SB218795, fenoterol, nitrefazole and pentetrazol as activators of exosome biogenesis and/or secretion in PC cells. Our findings implicate the potential utility of drug-repurposing as novel adjunct therapeutic strategies in advanced cancer.
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http://dx.doi.org/10.1038/s41598-018-26411-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5970137PMC
May 2018