Publications by authors named "Juan J Marugan"

69 Publications

Anti-apoptotic and Matrix Remodeling Actions of a Small Molecule Agonist of the Human Relaxin Receptor, ML290 in Mice With Unilateral Ureteral Obstruction.

Front Physiol 2021 7;12:650769. Epub 2021 Jul 7.

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

Diseases, such as diabetes and hypertension, often lead to chronic kidney failure. The peptide hormone relaxin has been shown to have therapeutic effects in various organs. In the present study, we tested the hypothesis that ML290, a small molecule agonist of the human relaxin receptor (RXFP1), is able to target the kidney to remodel the extracellular matrix and reduce apoptosis induced by unilateral ureteral obstruction (UUO). UUO was performed on the left kidney of humanized RXFP1 mice, where the right kidneys served as contralateral controls. Mice were randomly allocated to receive either vehicle or ML290 (30 mg/kg) daily intraperitoneal injection, and kidneys were collected for apoptosis, RNA, and protein analyses. UUO significantly increased expression of pro-apoptotic markers in both vehicle- and ML290-treated mice when compared to their contralateral control kidneys. Specifically, expression and Erk1/2 activity were upregulated, accompanied by an increase of TUNEL-positive cells in the UUO kidneys. Additionally, UUO induced marked increase in myofibroblast differentiation and aberrant remodeling on the extracellular matrix. ML290 suppressed these processes by promoting a reduction of pro-apoptotic, fibroblastic, and inflammatory markers in the UUO kidneys. Finally, the potent effects of ML290 to remodel the extracellular matrix were demonstrated by its ability to reduce collagen gene expression in the UUO kidneys. Our data indicate that daily administration of ML290 has renal protective effects in the UUO mouse model, specifically through its anti-apoptotic and extracellular matrix remodeling properties.
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http://dx.doi.org/10.3389/fphys.2021.650769DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8293094PMC
July 2021

The Impact of Assay Design on Medicinal Chemistry: Case Studies.

SLAS Discov 2021 Jul 5:24725552211026238. Epub 2021 Jul 5.

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

Graphical Abstract:
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http://dx.doi.org/10.1177/24725552211026238DOI Listing
July 2021

Discovery and Optimization of a 4-Aminopiperidine Scaffold for Inhibition of Hepatitis C Virus Assembly.

J Med Chem 2021 Jul 29;64(13):9431-9443. Epub 2021 Jun 29.

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

The majority of FDA-approved HCV therapeutics target the viral replicative machinery. An automated high-throughput phenotypic screen identified several small molecules as potent inhibitors of hepatitis C virus replication. Here, we disclose the discovery and optimization of a 4-aminopiperidine (4AP) scaffold targeting the assembly stages of the HCV life cycle. The original screening hit (1) demonstrates efficacy in the HCVcc assay but does not show potency prior to or during viral replication. Colocalization and infectivity studies indicate that the 4AP chemotype inhibits the assembly and release of infectious HCV. Compound 1 acts synergistically with FDA-approved direct-acting antiviral compounds Telaprevir and Daclatasvir, as well as broad spectrum antivirals Ribavirin and cyclosporin A. Following an SAR campaign, several derivatives of the 4AP series have been identified with increased potency against HCV, reduced toxicity, as well as improved and ADME properties.
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http://dx.doi.org/10.1021/acs.jmedchem.1c00696DOI Listing
July 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

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

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

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

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

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

Neural stem cells for disease modeling and evaluation of therapeutics for infantile (CLN1/PPT1) and late infantile (CLN2/TPP1) neuronal ceroid lipofuscinoses.

Orphanet J Rare Dis 2018 04 10;13(1):54. Epub 2018 Apr 10.

National Center for Advancing Translational Sciences, National Institutes of Health, 9800 Medical Center Drive, Bethesda, MD, 20892, USA.

Background: Infantile and late infantile neuronal ceroid lipofuscinoses (NCLs) are lysosomal storage diseases affecting the central nervous system (CNS). The infantile NCL (INCL) is caused by mutations in the PPT1 gene and late-infantile NCL (LINCL) is due to mutations in the TPP1 gene. Deficiency in PPT1 or TPP1 enzyme function results in lysosomal accumulation of pathological lipofuscin-like material in the patient cells. There is currently no small-molecular drug treatment for NCLs.

Results: We have generated induced pluripotent stem cells (iPSC) from three patient dermal fibroblast lines and further differentiated them into neural stem cells (NSCs). Using these new disease models, we evaluated the effect of δ-tocopherol (DT) and hydroxypropyl-β-cyclodextrin (HPBCD) with the enzyme replacement therapy as the control. Treatment with the relevant recombinant enzyme or DT significantly ameliorated the lipid accumulation and lysosomal enlargement in the disease cells. A combination therapy of δ-tocopherol and HPBCD further improved the effect compared to that of either drug used as a single therapy.

Conclusion: The results demonstrate that these patient iPSC derived NCL NSCs are valid cell- based disease models with characteristic disease phenotypes that can be used for study of disease pathophysiology and drug development.
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http://dx.doi.org/10.1186/s13023-018-0798-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891977PMC
April 2018

Preclinical Pharmacological Development of Chlorcyclizine Derivatives for the Treatment of Hepatitis C Virus Infection.

J Infect Dis 2018 05;217(11):1761-1769

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

Hepatitis C virus (HCV) is a small, single-stranded, positive-sense RNA virus that infects more than an estimated 70 million people worldwide. Untreated, persistent HCV infection often results in chronic hepatitis, cirrhosis, or liver failure, with progression to hepatocellular carcinoma. Current anti-HCV regimens comprising direct acting antivirals (DAAs) can provide curative treatment; however, due to high costs there remains a need for effective, shorter-duration, and affordable treatments. Recently, we disclosed anti-HCV activity of the cheap antihistamine chlorcyclizine, targeting viral entry. Following our hit-to-lead optimization campaign, we report evaluation of preclinical in vitro absorption, distribution, metabolism, and excretion properties, and in vivo pharmacokinetic profiles of lead compounds. This led to selection of a new lead compound and evaluation of efficacy in chimeric mice engrafted with primary human hepatocytes infected with HCV. Further development and incorporation of this compound into DAA regimens has the potential to improve treatment efficacy, affordability, and accessibility.
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http://dx.doi.org/10.1093/infdis/jiy039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5946821PMC
May 2018

Discovery of a Positive Allosteric Modulator of the Thyrotropin Receptor: Potentiation of Thyrotropin-Mediated Preosteoblast Differentiation In Vitro.

J Pharmacol Exp Ther 2018 01 31;364(1):38-45. Epub 2017 Oct 31.

Laboratory of Endocrinology and Receptor Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland (S.N., E.E., A.B., S.J.M., M.C.G.); and Division of Pre-Clinical Innovations, National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland (E.B., M.F., N.S., D.K., X.H., J.J.M.).

Recently, we showed that TSH-enhanced differentiation of a human preosteoblast-like cell model involved a -arrestin 1 (-Arr 1)-mediated pathway. To study this pathway in more detail, we sought to discover a small molecule ligand that was functionally selective toward human TSH receptor (TSHR) activation of -Arr 1. High-throughput screening using a cell line stably expressing mutated TSHRs and mutated -Arr 1 (DiscoverX1 cells) led to the discovery of agonists that stimulated translocation of -Arr 1 to the TSHR, but did not activate G-mediated signaling pathways, i.e., cAMP production. D3-Arr (NCGC00379308) was selected. In DiscoverX1 cells, D3-Arr stimulated -Arr 1 translocation with a 5.1-fold greater efficacy than TSH and therefore potentiated the effect of TSH in stimulating -Arr 1 translocation. In human U2OS-TSHR cells expressing wild-type TSHRs, which is a model of human preosteoblast-like cells, TSH upregulated the osteoblast-specific genes osteopontin (OPN) and alkaline phosphatase (ALPL). D3-Arr alone had only a weak effect to upregulate these bone markers, but D3-Arr potentiated TSH-induced upregulation of ALPL and OPN mRNA levels 1.6-fold and 5.5-fold, respectively, at the maximum dose of ligands. Furthermore, the positive allosteric modulator effect of D3-Arr resulted in an increase of TSH-induced secretion of OPN protein. In summary, we have discovered the first small molecule positive allosteric modulator of TSHR. As D3-Arr potentiates the effect of TSH to enhance differentiation of a human preosteoblast in an in vitro model, it will allow a novel experimental approach for probing the role of TSH-induced -Arr 1 signaling in osteoblast differentiation.
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http://dx.doi.org/10.1124/jpet.117.244095DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5729612PMC
January 2018

Mutation of Asn-475 in the Venezuelan Equine Encephalitis Virus nsP2 Cysteine Protease Leads to a Self-Inhibited State.

Biochemistry 2017 11 9;56(47):6221-6230. Epub 2017 Nov 9.

U.S. Naval Research Laboratory , 4555 Overlook Avenue, Washington, D.C. 20375, United States.

The alphaviral nsP2 cysteine protease of the Venezuelan equine encephalitis virus (VEEV) is a validated antiviral drug target. Clan CN proteases contain a cysteine protease domain that is intimately packed with an S-adenosyl-l-methionine-dependent RNA methyltransferase (SAM MTase) domain. Within a cleft formed at the interface of these two domains, the peptide substrate is thought to bind. The nucleophilic cysteine can be found within a conserved motif, NVCWAK, which differs from that of papain (CGSCWAFS). Mutation of the motif residue, N475, to alanine unexpectedly produced a self-inhibited state in which the N-terminal residues flipped into the substrate-binding cleft. Notably, the N-terminal segment was not hydrolyzed-consistent with a catalytically incompetent state. The N475A mutation resulted in a 70-fold decrease in k/K. A side chain-substrate interaction was predicted by the structure; the S701A mutation led to a 17-fold increase in K. An Asn at the n-2 position relative to the Cys was also found in the coronaviral papain-like proteases/deubiquitinases (PLpro) of the SARS and MERS viruses, and in several papain-like human ubiquitin specific proteases (USP). The large conformational change in the N475A variant suggests that Asn-475 plays an important role in stabilizing the N-terminal residues and in orienting the carbonyl during nucleophilic attack but does not directly hydrogen bond the oxyanion. The state trapped in crystallo is an unusual result of site-directed mutagenesis but reveals the role of this highly conserved Asn and identifies key substrate-binding contacts that may be exploited by peptide-like inhibitors.
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http://dx.doi.org/10.1021/acs.biochem.7b00746DOI Listing
November 2017

Development of an Aryloxazole Class of Hepatitis C Virus Inhibitors Targeting the Entry Stage of the Viral Replication Cycle.

J Med Chem 2017 07 13;60(14):6364-6383. Epub 2017 Jul 13.

University of Kansas Specialized Chemistry Center, University of Kansas , Lawrence, Kansas 66047, United States.

Reliance on hepatitis C virus (HCV) replicon systems and protein-based screening assays has led to treatments that target HCV viral replication proteins. The model does not encompass other viral replication cycle steps such as entry, processing, assembly and secretion, or viral host factors. We previously applied a phenotypic high-throughput screening platform based on an infectious HCV system and discovered an aryloxazole-based anti-HCV hit. Structure-activity relationship studies revealed several compounds exhibiting EC values below 100 nM. Lead compounds showed inhibition of the HCV pseudoparticle entry, suggesting a different mode of action from existing HCV drugs. Hit 7a and lead 7ii both showed synergistic effects in combination with existing HCV drugs. In vivo pharmacokinetics studies of 7ii showed high liver distribution and long half-life without obvious hepatotoxicity. The lead compounds are promising as preclinical candidates for the treatment of HCV infection and as molecular probes to study HCV pathogenesis.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00561DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015499PMC
July 2017

Analytical Characterization of Methyl-β-Cyclodextrin for Pharmacological Activity to Reduce Lysosomal Cholesterol Accumulation in Niemann-Pick Disease Type C1 Cells.

Assay Drug Dev Technol 2017 May/Jun;15(4):154-166

1 National Center for Advancing Translational Sciences, National Institutes of Health , Bethesda, Maryland.

Methyl-β-cyclodextrin (MβCD) reduces lysosomal cholesterol accumulation in Niemann-Pick disease type C1 (NPC1) patient fibroblasts. However, the pharmacological activity of MβCD reported by different laboratories varies. To determine the potential causes of this variation, we analyzed the mass spectrum characteristics, pharmacological activity of three preparations of MβCDs, and the protein expression profiles of NPC1 patient fibroblasts after treatment with different sources of MβCDs. Our data revealed varied mass spectrum profiles and pharmacological activities on the reduction of lysosomal cholesterol accumulation in NPC1 fibroblasts for these three preparations of MβCDs obtained from different batches and different sources. Furthermore, a proteomic analysis showed the differences of these three MβCD preparations on amelioration of dysregulated protein expression levels in NPC1 cells. The results demonstrate the importance of prescreening of different cyclodextrin preparations before use as a therapeutic agent. A combination of mass spectrum analysis, measurement of pharmacological activity, and proteomic profiling provides an effective analytical procedure for characterization of cyclodextrins for therapeutic applications.
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http://dx.doi.org/10.1089/adt.2017.774DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5510037PMC
March 2018

ML290 is a biased allosteric agonist at the relaxin receptor RXFP1.

Sci Rep 2017 06 7;7(1):2968. Epub 2017 Jun 7.

Drug Discovery Biology, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Australia.

Activation of the relaxin receptor RXFP1 has been associated with improved survival in acute heart failure. ML290 is a small molecule RXFP1 agonist with simple structure, long half-life and high stability. Here we demonstrate that ML290 is a biased agonist in human cells expressing RXFP1 with long-term beneficial actions on markers of fibrosis in human cardiac fibroblasts (HCFs). ML290 did not directly compete with orthosteric relaxin binding and did not affect binding kinetics, but did increase binding to RXFP1. In HEK-RXFP1 cells, ML290 stimulated cAMP accumulation and p38MAPK phosphorylation but not cGMP accumulation or ERK1/2 phosphorylation although prior addition of ML290 increased p-ERK1/2 responses to relaxin. In human primary vascular endothelial and smooth muscle cells that endogenously express RXFP1, ML290 increased both cAMP and cGMP accumulation but not p-ERK1/2. In HCFs, ML290 increased cGMP accumulation but did not affect p-ERK1/2 and given chronically activated MMP-2 expression and inhibited TGF-β1-induced Smad2 and Smad3 phosphorylation. In vascular cells, ML290 was 10x more potent for cGMP accumulation and p-p38MAPK than for cAMP accumulation. ML290 caused strong coupling of RXFP1 to Gα and Gα but weak coupling to Gα. ML290 exhibited signalling bias at RXFP1 possessing a signalling profile indicative of vasodilator and anti-fibrotic properties.
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http://dx.doi.org/10.1038/s41598-017-02916-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5462828PMC
June 2017

ML372 blocks SMN ubiquitination and improves spinal muscular atrophy pathology in mice.

JCI Insight 2016 11 17;1(19):e88427. Epub 2016 Nov 17.

Department of Anatomy, Physiology and Genetics, Uniformed Services University of the Health Sciences, F. Edward Hebert School of Medicine, Bethesda, Maryland, USA.

Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disease and one of the leading inherited causes of infant mortality. SMA results from insufficient levels of the survival motor neuron (SMN) protein, and studies in animal models of the disease have shown that increasing SMN protein levels ameliorates the disease phenotype. Our group previously identified and optimized a new series of small molecules, with good potency and toxicity profiles and reasonable pharmacokinetics, that were able to increase SMN protein levels in SMA patient-derived cells. We show here that ML372, a representative of this series, almost doubles the half-life of residual SMN protein expressed from the SMN2 locus by blocking its ubiquitination and subsequent degradation by the proteasome. ML372 increased SMN protein levels in muscle, spinal cord, and brain tissue of SMA mice. Importantly, ML372 treatment improved the righting reflex and extended survival of a severe mouse model of SMA. These results demonstrate that slowing SMN degradation by selectively inhibiting its ubiquitination can improve the motor phenotype and lifespan of SMA model mice.
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http://dx.doi.org/10.1172/jci.insight.88427DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5111506PMC
November 2016

Induced Pluripotent Stem Cells for Disease Modeling and Evaluation of Therapeutics for Niemann-Pick Disease Type A.

Stem Cells Transl Med 2016 12 2;5(12):1644-1655. Epub 2016 Aug 2.

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

: Niemann-Pick disease type A (NPA) is a lysosomal storage disease caused by mutations in the SMPD1 gene that encodes acid sphingomyelinase (ASM). Deficiency in ASM function results in lysosomal accumulation of sphingomyelin and neurodegeneration. Currently, there is no effective treatment for NPA. To accelerate drug discovery for treatment of NPA, we generated induced pluripotent stem cells from two patient dermal fibroblast lines and differentiated them into neural stem cells. The NPA neural stem cells exhibit a disease phenotype of lysosomal sphingomyelin accumulation and enlarged lysosomes. By using this disease model, we also evaluated three compounds that reportedly reduced lysosomal lipid accumulation in Niemann-Pick disease type C as well as enzyme replacement therapy with ASM. We found that α-tocopherol, δ-tocopherol, hydroxypropyl-β-cyclodextrin, and ASM reduced sphingomyelin accumulation and enlarged lysosomes in NPA neural stem cells. Therefore, the NPA neural stem cells possess the characteristic NPA disease phenotype that can be ameliorated by tocopherols, cyclodextrin, and ASM. Our results demonstrate the efficacies of cyclodextrin and tocopherols in the NPA cell-based model. Our data also indicate that the NPA neural stem cells can be used as a new cell-based disease model for further study of disease pathophysiology and for high-throughput screening to identify new lead compounds for drug development.

Significance: Currently, there is no effective treatment for Niemann-Pick disease type A (NPA). To accelerate drug discovery for treatment of NPA, NPA-induced pluripotent stem cells were generated from patient dermal fibroblasts and differentiated into neural stem cells. By using the differentiated NPA neuronal cells as a cell-based disease model system, α-tocopherol, δ-tocopherol, and hydroxypropyl-β-cyclodextrin significantly reduced sphingomyelin accumulation in these NPA neuronal cells. Therefore, this cell-based NPA model can be used for further study of disease pathophysiology and for high-throughput screening of compound libraries to identify lead compounds for drug development.
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http://dx.doi.org/10.5966/sctm.2015-0373DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5189647PMC
December 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

Efficacy and Mechanism of Action of Low Dose Emetine against Human Cytomegalovirus.

PLoS Pathog 2016 06 23;12(6):e1005717. Epub 2016 Jun 23.

Department of Pediatrics, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Baltimore, Maryland, United States of America.

Infection with human cytomegalovirus (HCMV) is a threat for pregnant women and immunocompromised hosts. Although limited drugs are available, development of new agents against HCMV is desired. Through screening of the LOPAC library, we identified emetine as HCMV inhibitor. Additional studies confirmed its anti-HCMV activities in human foreskin fibroblasts: EC50-40±1.72 nM, CC50-8±0.56 μM, and selectivity index of 200. HCMV inhibition occurred after virus entry, but before DNA replication, and resulted in decreased expression of viral proteins. Synergistic virus inhibition was achieved when emetine was combined with ganciclovir. In a mouse CMV (MCMV) model, emetine was well-tolerated, displayed long half-life, preferential distribution to tissues over plasma, and effectively suppressed MCMV. Since the in vitro anti-HCMV activity of emetine decreased significantly in low-density cells, a mechanism involving cell cycle regulation was suspected. HCMV inhibition by emetine depended on ribosomal processing S14 (RPS14) binding to MDM2, leading to disruption of HCMV-induced MDM2-p53 and MDM2-IE2 interactions. Irrespective of cell density, emetine induced RPS14 translocation into the nucleus during infection. In infected high-density cells, MDM2 was available for interaction with RPS14, resulting in disruption of MDM2-p53 interaction. However, in low-density cells the pre-existing interaction of MDM2-p53 could not be disrupted, and RPS14 could not interact with MDM2. In high-density cells the interaction of MDM2-RPS14 resulted in ubiquitination and degradation of RPS14, which was not observed in low-density cells. In infected-only or in non-infected emetine-treated cells, RPS14 failed to translocate into the nucleus, hence could not interact with MDM2, and was not ubiquitinated. HCMV replicated similarly in RPS14 knockdown or control cells, but emetine did not inhibit virus replication in the former cell line. The interaction of MDM2-p53 was maintained in infected RPS14 knockdown cells despite emetine treatment, confirming a unique mechanism by which emetine exploits RPS14 to disrupt MDM2-p53 interaction. Summarized, emetine may represent a promising candidate for HCMV therapy alone or in combination with ganciclovir through a novel host-dependent mechanism.
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http://dx.doi.org/10.1371/journal.ppat.1005717DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4919066PMC
June 2016

Disease models for the development of therapies for lysosomal storage diseases.

Ann N Y Acad Sci 2016 05 4;1371(1):15-29. Epub 2016 May 4.

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

Lysosomal storage diseases (LSDs) are a group of rare diseases in which the function of the lysosome is disrupted by the accumulation of macromolecules. The complexity underlying the pathogenesis of LSDs and the small, often pediatric, population of patients make the development of therapies for these diseases challenging. Current treatments are only available for a small subset of LSDs and have not been effective at treating neurological symptoms. Disease-relevant cellular and animal models with high clinical predictability are critical for the discovery and development of new treatments for LSDs. In this paper, we review how LSD patient primary cells and induced pluripotent stem cell-derived cellular models are providing novel assay systems in which phenotypes are more similar to those of the human LSD physiology. Furthermore, larger animal disease models are providing additional tools for evaluation of the efficacy of drug candidates. Early predictors of efficacy and better understanding of disease biology can significantly affect the translational process by focusing efforts on those therapies with the higher probability of success, thus decreasing overall time and cost spent in clinical development and increasing the overall positive outcomes in clinical trials.
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http://dx.doi.org/10.1111/nyas.13052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5287412PMC
May 2016

Structural Insights into the Activation of Human Relaxin Family Peptide Receptor 1 by Small-Molecule Agonists.

Biochemistry 2016 Mar 4;55(12):1772-83. Epub 2016 Mar 4.

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

The GPCR relaxin family peptide receptor 1 (RXFP1) mediates the action of relaxin peptide hormone, including its tissue remodeling and antifibrotic effects. The peptide has a short half-life in plasma, limiting its therapeutic utility. However, small-molecule agonists of human RXFP1 can overcome this limitation and may provide a useful therapeutic approach, especially for chronic diseases such as heart failure and fibrosis. The first small-molecule agonists of RXFP1 were recently identified from a high-throughput screening, using a homogeneous cell-based cAMP assay. Optimization of the hit compounds resulted in a series of highly potent and RXFP1 selective agonists with low cytotoxicity, and excellent in vitro ADME and pharmacokinetic properties. Here, we undertook extensive site-directed mutagenesis studies in combination with computational modeling analysis to probe the molecular basis of the small-molecule binding to RXFP1. The results showed that the agonists bind to an allosteric site of RXFP1 in a manner that closely interacts with the seventh transmembrane domain (TM7) and the third extracellular loop (ECL3). Several residues were determined to play an important role in the agonist binding and receptor activation, including a hydrophobic region at TM7 consisting of W664, F668, and L670. The G659/T660 motif within ECL3 is crucial to the observed species selectivity of the agonists for RXFP1. The receptor binding and activation effects by the small molecule ML290 were compared with the cognate ligand, relaxin, providing valuable insights on the structural basis and molecular mechanism of receptor activation and selectivity for RXFP1.
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http://dx.doi.org/10.1021/acs.biochem.5b01195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5137375PMC
March 2016

Discovery, Optimization, and Characterization of Novel Chlorcyclizine Derivatives for the Treatment of Hepatitis C Virus Infection.

J Med Chem 2016 Feb 1;59(3):841-53. Epub 2016 Feb 1.

Division of Pre-Clinical Innovations, National Center for Advancing Translational Sciences, National Institutes of Health , 9800 Medical Center Drive, Rockville, Maryland 20850, United States.

Recently, we reported that chlorcyclizine (CCZ, Rac-2), an over-the-counter antihistamine piperazine drug, possesses in vitro and in vivo activity against hepatitis C virus. Here, we describe structure-activity relationship (SAR) efforts that resulted in the optimization of novel chlorcyclizine derivatives as anti-HCV agents. Several compounds exhibited EC50 values below 10 nM against HCV infection, cytotoxicity selectivity indices above 2000, and showed improved in vivo pharmacokinetic properties. The optimized molecules can serve as lead preclinical candidates for the treatment of hepatitis C virus infection and as probes to study hepatitis C virus pathogenesis and host-virus interaction.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00752DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753534PMC
February 2016

Activation of Relaxin Family Receptor 1 from Different Mammalian Species by Relaxin Peptide and Small-Molecule Agonist ML290.

Front Endocrinol (Lausanne) 2015 17;6:128. Epub 2015 Aug 17.

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

Relaxin peptide (RLN), which signals through the relaxin family peptide 1 (RXFP1) GPCR receptor, has shown therapeutic effects in an acute heart failure clinical trial. We have identified a small-molecule agonist of human RXFP1, ML290; however, it does not activate the mouse receptor. To find a suitable animal model for ML290 testing and to gain mechanistic insights into the interaction of various ligands with RXFP1, we have cloned rhesus macaque, pig, rabbit, and guinea pig RXFP1s and analyzed their activation by RLN and ML290. HEK293T cells expressing macaque or pig RXFP1 responded to relaxin and ML290 treatment as measured by an increase of cAMP production. Guinea pig RXFP1 responded to relaxin but had very low response to ML290 treatment only at highest concentrations used. The rabbit RXFP1 amino acid sequence was the most divergent, with a number of unique substitutions within the ectodomain and the seven-transmembrane domain (7TM). Two splice variants of rabbit RXFP1 derived through alternative splicing of the fourth exon were identified. In contrast to the other species, rabbit RXFP1s were activated by ML290, but not with human, pig, mouse, or rabbit RLNs. Using FLAG-tagged constructs, we have shown that both rabbit RXFP1 variants are expressed on the cell surface. No binding of human Eu-labeled RLN to rabbit RXFP1 was detected, suggesting that in this species, RXFP1 might be non-functional. We used chimeric rabbit-human and guinea pig-human constructs to identify regions important for RLN or ML290 receptor activation. Chimeras with the human ectodomain and rabbit 7TM domain were activated by RLN, whereas substitution of part of the guinea pig 7TM domain with the human sequence only partially restored ML290 activation, confirming the allosteric mode of action for the two ligands. Our data demonstrate that macaque and pig models can be used for ML290 testing.
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http://dx.doi.org/10.3389/fendo.2015.00128DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4538381PMC
September 2015

High-Throughput Screening, Discovery, and Optimization To Develop a Benzofuran Class of Hepatitis C Virus Inhibitors.

ACS Comb Sci 2015 Oct 17;17(10):641-52. Epub 2015 Sep 17.

Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health , 10 Center Drive, Bethesda, Maryland 20892, United States.

Using a high-throughput, cell-based HCV luciferase reporter assay to screen a diverse small-molecule compound collection (∼ 300,000 compounds), we identified a benzofuran compound class of HCV inhibitors. The optimization of the benzofuran scaffold led to the identification of several exemplars with potent inhibition (EC50 < 100 nM) of HCV, low cytotoxicity (CC50 > 25 μM), and excellent selectivity (selective index = CC50/EC50, > 371-fold). The structure-activity studies culminated in the design and synthesis of a 45-compound library to comprehensively explore the anti-HCV activity. The identification, design, synthesis, and biological characterization for this benzofuran series is discussed.
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http://dx.doi.org/10.1021/acscombsci.5b00101DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6015500PMC
October 2015

Repurposing of the antihistamine chlorcyclizine and related compounds for treatment of hepatitis C virus infection.

Sci Transl Med 2015 Apr;7(282):282ra49

Liver Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.

Hepatitis C virus (HCV) infection affects an estimated 185 million people worldwide, with chronic infection often leading to liver cirrhosis and hepatocellular carcinoma. Although HCV is curable, there is an unmet need for the development of effective and affordable treatment options. Through a cell-based high-throughput screen, we identified chlorcyclizine HCl (CCZ), an over-the-counter drug for allergy symptoms, as a potent inhibitor of HCV infection. CCZ inhibited HCV infection in human hepatoma cells and primary human hepatocytes. The mode of action of CCZ is mediated by inhibiting an early stage of HCV infection, probably targeting viral entry into host cells. The in vitro antiviral effect of CCZ was synergistic with other anti-HCV drugs, including ribavirin, interferon-α, telaprevir, boceprevir, sofosbuvir, daclatasvir, and cyclosporin A, without significant cytotoxicity, suggesting its potential in combination therapy of hepatitis C. In the mouse pharmacokinetic model, CCZ showed preferential liver distribution. In chimeric mice engrafted with primary human hepatocytes, CCZ significantly inhibited infection of HCV genotypes 1b and 2a, without evidence of emergence of drug resistance, during 4 and 6 weeks of treatment, respectively. With its established clinical safety profile as an allergy medication, affordability, and a simple chemical structure for optimization, CCZ represents a promising candidate for drug repurposing and further development as an effective and accessible agent for treatment of HCV infection.
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http://dx.doi.org/10.1126/scitranslmed.3010286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420960PMC
April 2015

Targeting ATM ameliorates mutant Huntingtin toxicity in cell and animal models of Huntington's disease.

Sci Transl Med 2014 Dec;6(268):268ra178

Center for Neurobehavioral Genetics, Semel Institute for Neuroscience & Human Behavior, University of California, Los Angeles (UCLA), Los Angeles, CA 90095, USA. Department of Psychiatry and Biobehavioral Sciences, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA. UCLA Brain Research Institute, Los Angeles, CA 90095, USA.

Age-related neurodegenerative disorders including Alzheimer's disease and Huntington's disease (HD) consistently show elevated DNA damage, but the relevant molecular pathways in disease pathogenesis remain unclear. One attractive gene is that encoding the ataxia-telangiectasia mutated (ATM) protein, a kinase involved in the DNA damage response, apoptosis, and cellular homeostasis. Loss-of-function mutations in both alleles of ATM cause ataxia-telangiectasia in children, but heterozygous mutation carriers are disease-free. Persistently elevated ATM signaling has been demonstrated in Alzheimer's disease and in mouse models of other neurodegenerative diseases. We show that ATM signaling was consistently elevated in cells derived from HD mice and in brain tissue from HD mice and patients. ATM knockdown protected from toxicities induced by mutant Huntingtin (mHTT) fragments in mammalian cells and in transgenic Drosophila models. By crossing the murine Atm heterozygous null allele onto BACHD mice expressing full-length human mHTT, we show that genetic reduction of Atm gene dosage by one copy ameliorated multiple behavioral deficits and partially improved neuropathology. Small-molecule ATM inhibitors reduced mHTT-induced death of rat striatal neurons and induced pluripotent stem cells derived from HD patients. Our study provides converging genetic and pharmacological evidence that reduction of ATM signaling could ameliorate mHTT toxicity in cellular and animal models of HD, suggesting that ATM may be a useful therapeutic target for HD.
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http://dx.doi.org/10.1126/scitranslmed.3010523DOI Listing
December 2014
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