Publications by authors named "Elena Barnaeva"

17 Publications

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Robotic High-Throughput Biomanufacturing and Functional Differentiation of Human Pluripotent Stem Cells.

bioRxiv 2020 Aug 3. Epub 2020 Aug 3.

Efficient translation of human induced pluripotent stem cells (hiPSCs) depends on implementing scalable cell manufacturing strategies that ensure optimal self-renewal and functional differentiation. Currently, manual culture of hiPSCs is highly variable and labor-intensive posing significant challenges for high-throughput applications. Here, we established a robotic platform and automated all essential steps of hiPSC culture and differentiation under chemically defined conditions. This streamlined approach allowed rapid and standardized manufacturing of billions of hiPSCs that can be produced in parallel from up to 90 different patient-and disease-specific cell lines. Moreover, we established automated multi-lineage differentiation to generate primary embryonic germ layers and more mature phenotypes such as neurons, cardiomyocytes, and hepatocytes. To validate our approach, we carefully compared robotic and manual cell culture and performed molecular and functional cell characterizations (e.g. bulk culture and single-cell transcriptomics, mass cytometry, metabolism, electrophysiology, Zika virus experiments) in order to benchmark industrial-scale cell culture operations towards building an integrated platform for efficient cell manufacturing for disease modeling, drug screening, and cell therapy. Combining stem cell-based models and non-stop robotic cell culture may become a powerful strategy to increase scientific rigor and productivity, which are particularly important during public health emergencies (e.g. opioid crisis, COVID-19 pandemic).
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http://dx.doi.org/10.1101/2020.08.03.235242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7418713PMC
August 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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Discovery and characterization of a G protein-biased agonist that inhibits β-arrestin recruitment to the D2 dopamine receptor.

Mol Pharmacol 2014 Jul 22;86(1):96-105. Epub 2014 Apr 22.

Molecular Neuropharmacology Section, National Institute of Neurologic Disorders and Stroke, National Institutes of Health, Bethesda, Maryland (R.B.F., L.S.C., A.E.M., B.N.M., T.B.D., J.L.C., A.P., J.A.M., D.R.S.); National Center for Advancing Translational Sciences, National Institutes of Health, Rockville, Maryland (J.X., X.H., A.E.D., S.T., M.B.-G., E.B., M.F., N.T.S., J.J.M.); Cellular, Molecular, Developmental Biology & Biophysics Program, Johns Hopkins University, Baltimore, Maryland (L.S.C.); Center for Molecular Recognition and Departments of Psychiatry and Pharmacology, Columbia University College of Physicians and Surgeons, and Division of Molecular Therapeutics, New York State Psychiatric Institute, New York, New York (Y.H., L.D., J.A.J.); Schrödinger Inc., New York, New York (T.B.); and Department of Physiology and Biophysics and Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, New York (L.S.).

A high-throughput screening campaign was conducted to interrogate a 380,000+ small-molecule library for novel D2 dopamine receptor modulators using a calcium mobilization assay. Active agonist compounds from the primary screen were examined for orthogonal D2 dopamine receptor signaling activities including cAMP modulation and β-arrestin recruitment. Although the majority of the subsequently confirmed hits activated all signaling pathways tested, several compounds showed a diminished ability to stimulate β-arrestin recruitment. One such compound (MLS1547; 5-chloro-7-[(4-pyridin-2-ylpiperazin-1-yl)methyl]quinolin-8-ol) is a highly efficacious agonist at D2 receptor-mediated G protein-linked signaling, but does not recruit β-arrestin as demonstrated using two different assays. This compound does, however, antagonize dopamine-stimulated β-arrestin recruitment to the D2 receptor. In an effort to investigate the chemical scaffold of MLS1547 further, we characterized a set of 24 analogs of MLS1547 with respect to their ability to inhibit cAMP accumulation or stimulate β-arrestin recruitment. A number of the analogs were similar to MLS1547 in that they displayed agonist activity for inhibiting cAMP accumulation, but did not stimulate β-arrestin recruitment (i.e., they were highly biased). In contrast, other analogs displayed various degrees of G protein signaling bias. These results provided the basis to use pharmacophore modeling and molecular docking analyses to build a preliminary structure-activity relationship of the functionally selective properties of this series of compounds. In summary, we have identified and characterized a novel G protein-biased agonist of the D2 dopamine receptor and identified structural features that may contribute to its biased signaling properties.
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http://dx.doi.org/10.1124/mol.113.090563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4054005PMC
July 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

Discovery, optimization, and characterization of novel D2 dopamine receptor selective antagonists.

J Med Chem 2014 Apr 10;57(8):3450-63. Epub 2014 Apr 10.

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

The D2 dopamine receptor (D2 DAR) is one of the most validated drug targets for neuropsychiatric and endocrine disorders. However, clinically approved drugs targeting D2 DAR display poor selectivity between the D2 and other receptors, especially the D3 DAR. This lack of selectivity may lead to undesirable side effects. Here we describe the chemical and pharmacological characterization of a novel D2 DAR antagonist series with excellent D2 versus D1, D3, D4, and D5 receptor selectivity. The final probe 65 was obtained through a quantitative high-throughput screening campaign, followed by medicinal chemistry optimization, to yield a selective molecule with good in vitro physical properties, metabolic stability, and in vivo pharmacokinetics. The optimized molecule may be a useful in vivo probe for studying D2 DAR signal modulation and could also serve as a lead compound for the development of D2 DAR-selective druglike molecules for the treatment of multiple neuropsychiatric and endocrine disorders.
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http://dx.doi.org/10.1021/jm500126sDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4315423PMC
April 2014

Thymosin beta4 upregulates the expression of hepatocyte growth factor and downregulates the expression of PDGF-beta receptor in human hepatic stellate cells.

Ann N Y Acad Sci 2007 Sep 21;1112:154-60. Epub 2007 Jun 21.

Department of Clinical Investigation, Walter Reed Army Medical Center, Washington, DC, USA.

Hepatic stellate cells (HSCs) are the main producers of type I collagen in the liver, and therefore are responsible, in part, for the fibrous scar observed in cirrhotic livers. Although there is no approved treatment for this deadly disease, drugs inducing HSC apoptosis in animals (gliotoxin) and hepatocyte regeneration in man (hepatocyte growth factor [HGF]), have been used successfully in ameliorating liver fibrosis. In this communication we investigated whether thymosin beta(4) (Tbeta(4)), an actin-sequestering peptide that prevents scarring of the heart after a myocardial infarction and that prevents kidney fibrosis in animals, has the potential to be used to treat liver fibrosis. To this end we studied whether the administration of Tbeta(4) to HSCs could alter the expression of genes encoding for extracellular matrix components, as well as those required for differentiation of HSCs. Our preliminary findings show that Tbeta(4) had no effect on the expression of alpha2 (I) collagen, tissue inhibitor of metalloproteinases-1, and matrix metalloproteinase-2 mRNAs. However, it upregulated the expression of HGF and downregulated the expression of platelet-derived growth factor-beta receptor mRNAs in these cells. Overall, these findings suggest that Tbeta(4) has antifibrogenic potential.
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http://dx.doi.org/10.1196/annals.1415.035DOI Listing
September 2007