Publications by authors named "Samarjit Patnaik"

42 Publications

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

Correction to Remdesivir: A Review of Its Discovery and Development Leading to Human Clinical Trials for Treatment of COVID-19.

ACS Cent Sci 2020 06 16;6(6):1009. Epub 2020 Jun 16.

[This corrects the article DOI: 10.1021/acscentsci.0c00489.].
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http://dx.doi.org/10.1021/acscentsci.0c00747DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318059PMC
June 2020

Remdesivir: A Review of Its Discovery and Development Leading to Emergency Use Authorization for Treatment of COVID-19.

ACS Cent Sci 2020 May 4;6(5):672-683. Epub 2020 May 4.

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

The global pandemic of SARS-CoV-2, the causative viral pathogen of COVID-19, has driven the biomedical community to action-to uncover and develop antiviral interventions. One potential therapeutic approach currently being evaluated in numerous clinical trials is the agent remdesivir, which has endured a long and winding developmental path. Remdesivir is a nucleotide analogue prodrug that perturbs viral replication, originally evaluated in clinical trials to thwart the Ebola outbreak in 2014. Subsequent evaluation by numerous virology laboratories demonstrated the ability of remdesivir to inhibit coronavirus replication, including SARS-CoV-2. Here, we provide an overview of remdesivir's discovery, mechanism of action, and the current studies exploring its clinical effectiveness.
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http://dx.doi.org/10.1021/acscentsci.0c00489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202249PMC
May 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

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

Identification of Activators of Human Fumarate Hydratase by Quantitative High-Throughput Screening.

SLAS Discov 2020 01 14;25(1):43-56. Epub 2019 Sep 14.

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

Fumarate hydratase (FH) is a metabolic enzyme that is part of the Krebs cycle and reversibly catalyzes the hydration of fumarate to malate. Mutations of the gene have been associated with fumarate hydratase deficiency (FHD), hereditary leiomyomatosis and renal cell cancer (HLRCC), and other diseases. Currently, there are no high-quality small-molecule probes for studying human FH. To address this, we developed a quantitative high-throughput screening (qHTS) FH assay and screened a total of 57,037 compounds from in-house libraries in dose-response. While no inhibitors of FH were confirmed, a series of phenyl-pyrrolo-pyrimidine-diones were identified as activators of human FH. These compounds were not substrates of FH, were inactive in a malate dehydrogenase counterscreen, and showed no detectable reduction-oxidation activity. The binding of two compounds from the series to human FH was confirmed by microscale thermophoresis. The low hit rate in this screening campaign confirmed that FH is a "tough target" to modulate, and the small-molecule activators of human FH reported here may serve as a starting point for further optimization and development into cellular probes of human FH and potential drug candidates.
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http://dx.doi.org/10.1177/2472555219873559DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7340117PMC
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

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

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

Metarrestin, a perinucleolar compartment inhibitor, effectively suppresses metastasis.

Sci Transl Med 2018 05;10(441)

Department of Cell and Molecular Biology, Northwestern University, Chicago, IL 60611, USA.

Metastasis remains a leading cause of cancer mortality due to the lack of specific inhibitors against this complex process. To identify compounds selectively targeting the metastatic state, we used the perinucleolar compartment (PNC), a complex nuclear structure associated with metastatic behaviors of cancer cells, as a phenotypic marker for a high-content screen of over 140,000 structurally diverse compounds. Metarrestin, obtained through optimization of a screening hit, disassembles PNCs in multiple cancer cell lines, inhibits invasion in vitro, suppresses metastatic development in three mouse models of human cancer, and extends survival of mice in a metastatic pancreatic cancer xenograft model with no organ toxicity or discernable adverse effects. Metarrestin disrupts the nucleolar structure and inhibits RNA polymerase (Pol) I transcription, at least in part by interacting with the translation elongation factor eEF1A2. Thus, metarrestin represents a potential therapeutic approach for the treatment of metastatic cancer.
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http://dx.doi.org/10.1126/scitranslmed.aap8307DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6176865PMC
May 2018

Inhibition of PIP4Kγ ameliorates the pathological effects of mutant huntingtin protein.

Elife 2017 12 26;6. Epub 2017 Dec 26.

Division of Preclinical Innovation, National Center for Advancing Translational Sciences, Rockville, United States.

The discovery of the causative gene for Huntington's disease (HD) has promoted numerous efforts to uncover cellular pathways that lower levels of mutant huntingtin protein (mHtt) and potentially forestall the appearance of HD-related neurological defects. Using a cell-based model of pathogenic huntingtin expression, we identified a class of compounds that protect cells through selective inhibition of a lipid kinase, PIP4Kγ. Pharmacological inhibition or knock-down of PIP4Kγ modulates the equilibrium between phosphatidylinositide (PI) species within the cell and increases basal autophagy, reducing the total amount of mHtt protein in human patient fibroblasts and aggregates in neurons. In two models of Huntington's disease, genetic knockdown of PIP4K ameliorated neuronal dysfunction and degeneration as assessed using motor performance and retinal degeneration assays respectively. Together, these results suggest that PIP4Kγ is a druggable target whose inhibition enhances productive autophagy and mHtt proteolysis, revealing a useful pharmacological point of intervention for the treatment of Huntington's disease, and potentially for other neurodegenerative disorders.
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http://dx.doi.org/10.7554/eLife.29123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5743427PMC
December 2017

Identification of 4-phenylquinolin-2(1H)-one as a specific allosteric inhibitor of Akt.

Sci Rep 2017 09 15;7(1):11673. Epub 2017 Sep 15.

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

Akt plays a major role in tumorigenesis and the development of specific Akt inhibitors as effective cancer therapeutics has been challenging. Here, we report the identification of a highly specific allosteric inhibitor of Akt through a FRET-based high-throughput screening, and characterization of its inhibitory mechanism. Out of 373,868 compounds screened, 4-phenylquinolin-2(1H)-one specifically decreased Akt phosphorylation at both T308 and S473, and inhibited Akt kinase activity (IC = 6 µM) and downstream signaling. 4-Phenylquinolin-2(1H)-one did not alter the activity of upstream kinases including PI3K, PDK1, and mTORC2 as well as closely related kinases that affect cell proliferation and survival such as SGK1, PKA, PKC, or ERK1/2. This compound inhibited the proliferation of cancer cells but displayed less toxicity compared to inhibitors of PI3K or mTOR. Kinase profiling efforts revealed that 4-phenylquinolin-2(1H)-one does not bind to the kinase active site of over 380 human kinases including Akt. However, 4-phenylquinolin-2(1H)-one interacted with the PH domain of Akt, apparently inducing a conformation that hinders S473 and T308 phosphorylation by mTORC2 and PDK1. In conclusion, we demonstrate that 4-phenylquinolin-2(1H)-one is an exquisitely selective Akt inhibitor with a distinctive molecular mechanism, and a promising lead compound for further optimization toward the development of novel cancer therapeutics.
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http://dx.doi.org/10.1038/s41598-017-11870-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5601486PMC
September 2017

Gastric Acid Secretion from Parietal Cells Is Mediated by a Ca Efflux Channel in the Tubulovesicle.

Dev Cell 2017 05;41(3):262-273.e6

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 3089 Natural Science Building (Kraus), 830 North University, Ann Arbor, MI 48109, USA. Electronic address:

Gastric acid secretion by parietal cells requires trafficking and exocytosis of H/K-ATPase-rich tubulovesicles (TVs) toward apical membranes in response to histamine stimulation via cyclic AMP elevation. Here, we found that TRPML1 (ML1), a protein that is mutated in type IV mucolipidosis (ML-IV), is a tubulovesicular channel essential for TV exocytosis and acid secretion. Whereas ML-IV patients are reportedly achlorhydric, transgenic overexpression of ML1 in mouse parietal cells induced constitutive acid secretion. Gastric acid secretion was blocked and stimulated by ML1 inhibitors and agonists, respectively. Organelle-targeted Ca imaging and direct patch-clamping of apical vacuolar membranes revealed that ML1 mediates a PKA-activated conductance on TV membranes that is required for histamine-induced Ca release from TV stores. Hence, we demonstrated that ML1, acting as a Ca channel in TVs, links transmitter-initiated cyclic nucleotide signaling with Ca-dependent TV exocytosis in parietal cells, providing a regulatory mechanism that could be targeted to manage acid-related gastric diseases.
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http://dx.doi.org/10.1016/j.devcel.2017.04.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5497767PMC
May 2017

Orphan GPR110 (ADGRF1) targeted by N-docosahexaenoylethanolamine in development of neurons and cognitive function.

Nat Commun 2016 10 19;7:13123. Epub 2016 Oct 19.

Laboratory of Molecular Signaling, NIAAA, NIH, 5625 Fishers Lane Room 3N-07, Bethesda, Maryland 20892-9410, USA.

Docosahexaenoic acid (DHA, 22:6n-3) is an omega-3 fatty acid essential for proper brain development. N-docosahexaenoylethanolamine (synaptamide), an endogenous metabolite of DHA, potently promotes neurogenesis, neuritogenesis and synaptogenesis; however, the underlying molecular mechanism is not known. Here, we demonstrate orphan G-protein coupled receptor 110 (GPR110, ADGRF1) as the synaptamide receptor, mediating synaptamide-induced bioactivity in a cAMP-dependent manner. Mass spectrometry-based proteomic characterization and cellular fluorescence tracing with chemical analogues of synaptamide reveal specific binding of GPR110 to synaptamide, which triggers cAMP production with low nM potency. Disruption of this binding or GPR110 gene knockout abolishes while GPR110 overexpression enhances synaptamide-induced bioactivity. GPR110 is highly expressed in fetal brains but rapidly decreases after birth. GPR110 knockout mice show significant deficits in object recognition and spatial memory. GPR110 deorphanized as a functional synaptamide receptor provides a novel target for neurodevelopmental control and new insight into mechanisms by which DHA promotes brain development and function.
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http://dx.doi.org/10.1038/ncomms13123DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5075789PMC
October 2016

Activation of β-Glucocerebrosidase Reduces Pathological α-Synuclein and Restores Lysosomal Function in Parkinson's Patient Midbrain Neurons.

J Neurosci 2016 07;36(29):7693-706

Department of Neurology, Massachusetts General Hospital, Harvard Medical School, MassGeneral Institute for Neurodegeneration, Charlestown, Massachusetts 02129, The Ken and Ruth Davee Department of Neurology, Northwestern University Feinberg School of Medicine, Chicago IL 60611,

Unlabelled: Parkinson's disease (PD) is characterized by the accumulation of α-synuclein (α-syn) within Lewy body inclusions in the nervous system. There are currently no disease-modifying therapies capable of reducing α-syn inclusions in PD. Recent data has indicated that loss-of-function mutations in the GBA1 gene that encodes lysosomal β-glucocerebrosidase (GCase) represent an important risk factor for PD, and can lead to α-syn accumulation. Here we use a small-molecule modulator of GCase to determine whether GCase activation within lysosomes can reduce α-syn levels and ameliorate downstream toxicity. Using induced pluripotent stem cell (iPSC)-derived human midbrain dopamine (DA) neurons from synucleinopathy patients with different PD-linked mutations, we find that a non-inhibitory small molecule modulator of GCase specifically enhanced activity within lysosomal compartments. This resulted in reduction of GCase substrates and clearance of pathological α-syn, regardless of the disease causing mutations. Importantly, the reduction of α-syn was sufficient to reverse downstream cellular pathologies induced by α-syn, including perturbations in hydrolase maturation and lysosomal dysfunction. These results indicate that enhancement of a single lysosomal hydrolase, GCase, can effectively reduce α-syn and provide therapeutic benefit in human midbrain neurons. This suggests that GCase activators may prove beneficial as treatments for PD and related synucleinopathies.

Significance Statement: The presence of Lewy body inclusions comprised of fibrillar α-syn within affected regions of PD brain has been firmly documented, however no treatments exist that are capable of clearing Lewy bodies. Here, we used a mechanistic-based approach to examine the effect of GCase activation on α-syn clearance in human midbrain DA models that naturally accumulate α-syn through genetic mutations. Small molecule-mediated activation of GCase was effective at reducing α-syn inclusions in neurons, as well as associated downstream toxicity, demonstrating a therapeutic effect. Our work provides an example of how human iPSC-derived midbrain models could be used for testing potential treatments for neurodegenerative disorders, and identifies GCase as a critical therapeutic convergence point for a wide range of synucleinopathies.
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http://dx.doi.org/10.1523/JNEUROSCI.0628-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4951575PMC
July 2016

A New Glucocerebrosidase Chaperone Reduces α-Synuclein and Glycolipid Levels in iPSC-Derived Dopaminergic Neurons from Patients with Gaucher Disease and Parkinsonism.

J Neurosci 2016 07;36(28):7441-52

Section of Molecular Neurogenetics, National Human Genome Research Institute,

Unlabelled: Among the known genetic risk factors for Parkinson disease, mutations in GBA1, the gene responsible for the lysosomal disorder Gaucher disease, are the most common. This genetic link has directed attention to the role of the lysosome in the pathogenesis of parkinsonism. To study how glucocerebrosidase impacts parkinsonism and to evaluate new therapeutics, we generated induced human pluripotent stem cells from four patients with Type 1 (non-neuronopathic) Gaucher disease, two with and two without parkinsonism, and one patient with Type 2 (acute neuronopathic) Gaucher disease, and differentiated them into macrophages and dopaminergic neurons. These cells exhibited decreased glucocerebrosidase activity and stored the glycolipid substrates glucosylceramide and glucosylsphingosine, demonstrating their similarity to patients with Gaucher disease. Dopaminergic neurons from patients with Type 2 and Type 1 Gaucher disease with parkinsonism had reduced dopamine storage and dopamine transporter reuptake. Levels of α-synuclein, a protein present as aggregates in Parkinson disease and related synucleinopathies, were selectively elevated in neurons from the patients with parkinsonism or Type 2 Gaucher disease. The cells were then treated with NCGC607, a small-molecule noninhibitory chaperone of glucocerebrosidase identified by high-throughput screening and medicinal chemistry structure optimization. This compound successfully chaperoned the mutant enzyme, restored glucocerebrosidase activity and protein levels, and reduced glycolipid storage in both iPSC-derived macrophages and dopaminergic neurons, indicating its potential for treating neuronopathic Gaucher disease. In addition, NCGC607 reduced α-synuclein levels in dopaminergic neurons from the patients with parkinsonism, suggesting that noninhibitory small-molecule chaperones of glucocerebrosidase may prove useful for the treatment of Parkinson disease.

Significance Statement: Because GBA1 mutations are the most common genetic risk factor for Parkinson disease, dopaminergic neurons were generated from iPSC lines derived from patients with Gaucher disease with and without parkinsonism. These cells exhibit deficient enzymatic activity, reduced lysosomal glucocerebrosidase levels, and storage of glucosylceramide and glucosylsphingosine. Lines generated from the patients with parkinsonism demonstrated elevated levels of α-synuclein. To reverse the observed phenotype, the neurons were treated with a novel noninhibitory glucocerebrosidase chaperone, which successfully restored glucocerebrosidase activity and protein levels and reduced glycolipid storage. In addition, the small-molecule chaperone reduced α-synuclein levels in dopaminergic neurons, indicating that chaperoning glucocerebrosidase to the lysosome may provide a novel therapeutic strategy for both Parkinson disease and neuronopathic forms of Gaucher disease.
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http://dx.doi.org/10.1523/JNEUROSCI.0636-16.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4945664PMC
July 2016

MCOLN1 is a ROS sensor in lysosomes that regulates autophagy.

Nat Commun 2016 06 30;7:12109. Epub 2016 Jun 30.

Department of Molecular, Cellular, and Developmental Biology, University of Michigan, 3089 Natural Science Building (Kraus), 830 North University, Ann Arbor, Michigan 48109, USA.

Cellular stresses trigger autophagy to remove damaged macromolecules and organelles. Lysosomes 'host' multiple stress-sensing mechanisms that trigger the coordinated biogenesis of autophagosomes and lysosomes. For example, transcription factor (TF)EB, which regulates autophagy and lysosome biogenesis, is activated following the inhibition of mTOR, a lysosome-localized nutrient sensor. Here we show that reactive oxygen species (ROS) activate TFEB via a lysosomal Ca(2+)-dependent mechanism independent of mTOR. Exogenous oxidants or increasing mitochondrial ROS levels directly and specifically activate lysosomal TRPML1 channels, inducing lysosomal Ca(2+) release. This activation triggers calcineurin-dependent TFEB-nuclear translocation, autophagy induction and lysosome biogenesis. When TRPML1 is genetically inactivated or pharmacologically inhibited, clearance of damaged mitochondria and removal of excess ROS are blocked. Furthermore, TRPML1's ROS sensitivity is specifically required for lysosome adaptation to mitochondrial damage. Hence, TRPML1 is a ROS sensor localized on the lysosomal membrane that orchestrates an autophagy-dependent negative-feedback programme to mitigate oxidative stress in the cell.
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http://dx.doi.org/10.1038/ncomms12109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931332PMC
June 2016

Progress and potential of non-inhibitory small molecule chaperones for the treatment of Gaucher disease and its implications for Parkinson disease.

Expert Rev Proteomics 2016 05 21;13(5):471-9. Epub 2016 Apr 21.

a Section on Molecular Neurogenetics, Medical Genetics Branch , National Human Genome Research Institute, NIH , Bethesda , MD , USA.

Gaucher disease, caused by pathological mutations GBA1, encodes the lysosome-resident enzyme glucocerebrosidase, which cleaves glucosylceramide into glucose and ceramide. In Gaucher disease, glucocerebrosidase deficiency leads to lysosomal accumulation of substrate, primarily in cells of the reticulo-endothelial system. Gaucher disease has broad clinical heterogeneity, and mutations in GBA1 are a risk factor for the development of different synucleinopathies. Insights into the cell biology and biochemistry of glucocerebrosidase have led to new therapeutic approaches for Gaucher disease including small chemical chaperones. Such chaperones facilitate proper enzyme folding and translocation to lysosomes, thereby preventing premature breakdown of the enzyme in the proteasome. This review discusses recent progress in developing chemical chaperones as a therapy for Gaucher disease, with implications for the treatment of synucleinopathies. It focuses on the development of non-inhibitory glucocerebrosidase chaperones and their therapeutic advantages over inhibitory chaperones, as well as the challenges involved in identifying and validating chemical chaperones.
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http://dx.doi.org/10.1080/14789450.2016.1174583DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5381920PMC
May 2016

Lysosomal storage and impaired autophagy lead to inflammasome activation in Gaucher macrophages.

Aging Cell 2016 Feb 21;15(1):77-88. Epub 2015 Oct 21.

Section of Molecular Neurogenetics, National Human Genome Research Institute, Bethesda, MD, 20892, USA.

Gaucher disease, the inherited deficiency of lysosomal glucocerebrosidase, is characterized by the presence of glucosylcer-amide macrophages, the accumulation of glucosylceramide in lysosomes and the secretion of inflammatory cytokines. However, the connection between this lysosomal storage and inflammation is not clear. Studying macrophages derived from peripheral monocytes from patients with type 1 Gaucher disease with genotype N370S/N370S, we confirmed an increased secretion of interleukins IL-1β and IL-6. In addition, we found that activation of the inflammasome, a multiprotein complex that activates caspase-1, led to the maturation of IL-1β in Gaucher macrophages. We show that inflammasome activation in these cells is the result of impaired autophagy. Treatment with the small-molecule glucocerebrosidase chaperone NCGC758 reversed these defects, inducing autophagy and reducing IL-1β secretion, confirming the role of the deficiency of lysosomal glucocerebrosidase in these processes. We found that in Gaucher macrophages elevated levels of the autophagic adaptor p62 prevented the delivery of inflammasomes to autophagosomes. This increase in p62 led to activation of p65-NF-kB in the nucleus, promoting the expression of inflammatory cytokines and the secretion of IL-1β. This newly elucidated mechanism ties lysosomal dysfunction to inflammasome activation, and may contribute to the massive organomegaly, bone involvement and increased susceptibility to certain malignancies seen in Gaucher disease. Moreover, this link between lysosomal storage, impaired autophagy, and inflammation may have implications relevant to both Parkinson disease and the aging process. Defects in these basic cellular processes may also provide new therapeutic targets.
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http://dx.doi.org/10.1111/acel.12409DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4717273PMC
February 2016

Macrophage models of Gaucher disease for evaluating disease pathogenesis and candidate drugs.

Sci Transl Med 2014 Jun;6(240):240ra73

Section on Molecular Neurogenetics, Medical Genetics Branch, National Institutes of Health, Bethesda, MD 20892, USA.

Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes, particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore, we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition, we created induced pluripotent stem cell (iPSC)-derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages, reduced glycolipid storage, and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development.
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http://dx.doi.org/10.1126/scitranslmed.3008659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4161206PMC
June 2014

Lomofungin and dilomofungin: inhibitors of MBNL1-CUG RNA binding with distinct cellular effects.

Nucleic Acids Res 2014 Jun 5;42(10):6591-602. Epub 2014 May 5.

Department of Neurology, University of Rochester, Rochester, NY 14642, USA

Myotonic dystrophy type 1 (DM1) is a dominantly inherited neuromuscular disorder resulting from expression of RNA containing an expanded CUG repeat (CUG(exp)). The pathogenic RNA is retained in nuclear foci. Poly-(CUG) binding proteins in the Muscleblind-like (MBNL) family are sequestered in foci, causing misregulated alternative splicing of specific pre-mRNAs. Inhibitors of MBNL1-CUG(exp) binding have been shown to restore splicing regulation and correct phenotypes in DM1 models. We therefore conducted a high-throughput screen to identify novel inhibitors of MBNL1-(CUG)12 binding. The most active compound was lomofungin, a natural antimicrobial agent. We found that lomofungin undergoes spontaneous dimerization in DMSO, producing dilomofungin, whose inhibition of MBNL1-(CUG)12 binding was 17-fold more potent than lomofungin itself. However, while dilomofungin displayed the desired binding characteristics in vitro, when applied to cells it produced a large increase of CUG(exp) RNA in nuclear foci, owing to reduced turnover of the CUG(exp) transcript. By comparison, the monomer did not induce CUG(exp) accumulation in cells and was more effective at rescuing a CUG(exp)-induced splicing defect. These results support the feasibility of high-throughput screens to identify compounds targeting toxic RNA, but also demonstrate that ligands for repetitive sequences may have unexpected effects on RNA decay.
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http://dx.doi.org/10.1093/nar/gku275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4041448PMC
June 2014

Allosteric inhibitors of the Eya2 phosphatase are selective and inhibit Eya2-mediated cell migration.

J Biol Chem 2014 Jun 22;289(23):16349-61. Epub 2014 Apr 22.

From the Department of Biochemistry and Molecular Genetics, University of Colorado School of Medicine, Aurora, Colorado 80045,

Eya proteins are essential co-activators of the Six family of transcription factors and contain a unique tyrosine phosphatase domain belonging to the haloacid dehalogenase family of phosphatases. The phosphatase activity of Eya is important for the transcription of a subset of Six1-target genes, and also directs cells to the repair rather than apoptosis pathway upon DNA damage. Furthermore, Eya phosphatase activity has been shown to mediate transformation, invasion, migration, and metastasis of breast cancer cells, making it a potential new drug target for breast cancer. We have previously identified a class of N-arylidenebenzohydrazide compounds that specifically inhibit the Eya2 phosphatase. Herein, we demonstrate that these compounds are reversible inhibitors that selectively inhibit the phosphatase activity of Eya2, but not Eya3. Our mutagenesis results suggest that this class of compounds does not bind to the active site and the binding does not require the coordination with Mg(2+). Moreover, these compounds likely bind within a site on the opposite face of the active site, and function as allosteric inhibitors. We also demonstrate that this class of compounds inhibits Eya2 phosphatase-mediated cell migration, setting the foundation for these molecules to be developed into chemical probes for understanding the specific function of the Eya2 phosphatase and to serve as a prototype for the development of Eya2 phosphatase specific anti-cancer drugs.
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http://dx.doi.org/10.1074/jbc.M114.566729DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4047403PMC
June 2014

Structure-activity relationship of imidazopyridinium analogues as antagonists of neuropeptide s receptor.

J Med Chem 2013 Nov 11;56(22):9045-56. Epub 2013 Nov 11.

National Center for Advancing Translational Sciences, National Institutes of Health , 9800 Medical Center Drive, Rockville, Maryland 20850, United States .

The discovery and characterization of a novel chemical series of phosphorothioyl-containing imidazopyridines as potent neuropeptide S receptor antagonists is presented. The synthesis of analogues and their structure-activity relationship with respect to the Gq, Gs, and ERK pathways is detailed. The pharmacokinetics and in vivo efficacy of a potent analogue in a food intake rodent model are also included, underscoring its potential therapeutic value for the treatment of sleep, anxiety, and addiction disorders.
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http://dx.doi.org/10.1021/jm400904mDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4877059PMC
November 2013

Induction and reversal of myotonic dystrophy type 1 pre-mRNA splicing defects by small molecules.

Nat Commun 2013 ;4:2044

Department of Chemistry, The Scripps Research Institute, Scripps Florida, 130 Scripps Way #3A1, Jupiter, Florida 33458, USA.

The ability to control pre-mRNA splicing with small molecules could facilitate the development of therapeutics or cell-based circuits that control gene function. Myotonic dystrophy type 1 is caused by the dysregulation of alternative pre-mRNA splicing due to sequestration of muscleblind-like 1 protein (MBNL1) by expanded, non-coding r(CUG) repeats (r(CUG)(exp)). Here we report two small molecules that induce or ameliorate alternative splicing dysregulation. A thiophene-containing small molecule (1) inhibits the interaction of MBNL1 with its natural pre-mRNA substrates. Compound (2), a substituted naphthyridine, binds r(CUG)(exp) and displaces MBNL1. Structural models show that 1 binds MBNL1 in the Zn-finger domain and that 2 interacts with UU loops in r(CUG)(exp). This study provides a structural framework for small molecules that target MBNL1 by mimicking r(CUG)(exp) and shows that targeting MBNL1 causes dysregulation of alternative splicing, suggesting that MBNL1 is thus not a suitable therapeutic target for the treatment of myotonic dystrophy type 1.
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http://dx.doi.org/10.1038/ncomms3044DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3710115PMC
December 2013

A novel brain penetrant NPS receptor antagonist, NCGC00185684, blocks alcohol-induced ERK-phosphorylation in the central amygdala and decreases operant alcohol self-administration in rats.

J Neurosci 2013 Jun;33(24):10132-42

Laboratory of Clinical and Translational Studies, National Institute on Alcohol Abuse and Alcoholism, Bethesda, Maryland 20892, USA.

The Neuropeptide S receptor, a Gs/Gq-coupled GPCR expressed in brain regions involved in mediating drug reward, has recently emerged as a candidate therapeutic target in addictive disorders. Here, we describe the in vitro and in vivo pharmacology of a novel, selective and brain penetrant NPSR antagonist with nanomolar affinity for the NPSR, NCGC00185684. In vitro, NCGC00185684 shows biased antagonist properties, and preferentially blocks ERK-phosphorylation over intracellular cAMP or calcium responses to NPS. In vivo, systemic NCGC00185684 blocks alcohol-induced ERK-phosphorylation in the rat central amygdala, a region involved in regulation of alcohol intake. NCGC00185684 also decreases operant alcohol self-administration, and lowers motivation for alcohol reward as measured using progressive ratio responding. These effects are behaviorally specific, in that they are observed at doses that do not influence locomotor activity or reinstatement responding following extinction. Together, these data provide an initial validation of the NPSR as a therapeutic target in alcoholism.
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http://dx.doi.org/10.1523/JNEUROSCI.4742-12.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3682378PMC
June 2013

Discovery, structure-activity relationship, and biological evaluation of noninhibitory small molecule chaperones of glucocerebrosidase.

J Med Chem 2012 Jun 8;55(12):5734-48. Epub 2012 Jun 8.

NIH Chemical Genomic Center, National Center for Advancing Translation Sciences, National Institutes of Health, 9800 Medical Center Drive, Rockville, Maryland, United States.

A major challenge in the field of Gaucher disease has been the development of new therapeutic strategies including molecular chaperones. All previously described chaperones of glucocerebrosidase are enzyme inhibitors, which complicates their clinical development because their chaperone activity must be balanced against the functional inhibition of the enzyme. Using a novel high throughput screening methodology, we identified a chemical series that does not inhibit the enzyme but can still facilitate its translocation to the lysosome as measured by immunostaining of glucocerebrosidase in patient fibroblasts. These compounds provide the basis for the development of a novel approach toward small molecule treatment for patients with Gaucher disease.
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http://dx.doi.org/10.1021/jm300063bDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3400126PMC
June 2012