Bruce Sherf

Athersys Inc., Cleveland, Ohio, United States

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Publications (9)57.78 Total impact

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    ABSTRACT: CRTh2 (DP(2)) is a prostaglandin D(2) receptor implicated in the recruitment of eosinophils and basophils within the asthmatic lung. Here we report the discovery of a novel series of 3-indolyl sultam antagonists with low nM affinity for CRTh2. These compounds proved to be selective over the other primary prostaglandin D(2) receptor (DP1) as well as the thromboxane A(2) receptor (TP).
    Bioorganic & medicinal chemistry letters 06/2010; 20(11):3287-90. · 2.65 Impact Factor
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    ABSTRACT: The hERG (human ether-a-go-go related gene) potassium channel is required for normal cardiac repolarization, is susceptible to inhibition by a wide variety of compounds, and its blockage can lead to cardiac QT interval prolongation and life threatening arrhythmias. The present report examines the ability of hERG binding and functional assays to identify compounds with potential cardiovascular liabilities at the earliest stages of drug discovery. Competitive binding assays were developed using (3)H-dofetilide and membranes from HEK293EBNA cells stably expressing recombinant hERG (HEK293-hERG) and IMR-32 cells expressing hERG endogenously. hERG functional assays were also developed using membrane potential indicator dye and rubidium efflux. The ability of these assays to identify compounds with potential adverse cardiac effects was examined using drugs with known cardiac effects ranging from those with no known adverse effects to drugs that were withdrawn from the market due to increased risk of sudden death associated with Torsades de Points. Binding assays using HEK293-hERG membranes and (3)H-dofetilide were robust (Z'=0.69+/-0.015, mean+/-S.E.M.), highly reproducible (test-retest slope=1.04, r(2)=0.98), and correlated well with IC(50) values obtained by patch clamp (slope=0.98, r(2)=0.89). Binding assays using IMR-32 membranes were less sensitive (Z'=0.4+/-0.03, mean+/-S.E.M., false negative rate=0.4) but still correlated well with patch clamp data (slope=1.06, r(2)=0.83). The hERG membrane potential assay could detect potent hERG inhibitors (defined by hERG patch clamp IC(50)<0.1 muM) using HEK293-hERG cells, but were prone to generate false-negative results with less potent inhibitors (false negative rate=0.5). Finally, the rubidium efflux assay gave highly reproducible results (Z'=0.80+/-0.02, mean+/-S.E.M.) that correlated with patch clamp IC(50) values (slope=0.87, r(2)=0.73). The hERG binding and rubidium efflux assays are robust, predictive of patch clamp results, and can be used at the earliest stages of drug discovery.
    Journal of Pharmacological and Toxicological Methods 01/2006; 54(1):42-55. · 2.15 Impact Factor
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    ABSTRACT: The serotonin (5-hydroxytryptamine) 5-HT2 receptor subfamily consists of three members, 5-HT2A, 5-HT2B, and 5-HT2C. These receptors share high homology in their amino acid sequence, have similar signaling pathways, and have been indicated to play important roles in feeding, anxiety, aggression, sexual behavior, mood, and pain. Subtype-selective agonists and antagonists have been explored as drugs for hypertension, Parkinson's disease, sleep disorders, anxiety, depression, schizophrenia, and obesity. In this study, we report the development of homogeneous agonist binding assays in a scintillation proximity assay (SPA) format to determine the high-affinity binding state of agonist compounds for the human 5-HT2C, 5-HT2A, and 5-HT2B receptors. The 5-HT2 agonist 1-(4- [125I]iodo-2,5-dimethoxyphenyl)-2-aminopropane ([125I]DOI) was used to label the high-affinity sites for the 5-HT2A and 5-HT2C receptors. The high-affinity sites for the 5-HT2B receptor were labeled with [3H]lysergic acid diethylamide. Total receptor expression was determined with the 5-HT2 antagonist [3H]mesulergine for the 5-HT2B and 5-HT2C receptors, and [3H]ketanserin for the 5-HT2A receptor. The agonist high-affinity binding sites accounted for 2.3% (5-HT(2C) receptor), 4.0% (5-HT2A receptor), and 22% (5-HT2B receptor) of the total receptor population. Competition binding studies using known agonists indicated high Z' values of the agonist binding assays in SPA format (Z' > 0.70). The Ki values of 5-HT, (R)(-)DOI, and VER-3323 for the 5-HT2A, 5-HT2B, and 5-HT2C receptors by SPA format were equivalent to published data determined by filtration binding assays. These results indicate that agonist binding assays in SPA format can be easily adapted to a high throughput assay to screen for selective 5-HT2C receptor agonists, as well as for selectivity profiling of the compounds.
    Assay and Drug Development Technologies 12/2005; 3(6):649-59. · 1.90 Impact Factor
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    ABSTRACT: Target-based high-throughput screening (HTS) plays an integral role in drug discovery. The implementation of HTS assays generally requires high expression levels of the target protein, and this is typically accomplished using recombinant cDNA methodologies. However, the isolated gene sequences to many drug targets have intellectual property claims that restrict the ability to implement drug discovery programs. The present study describes the pharmacological characterization of the human histamine H3 receptor that was expressed using random activation of gene expression (RAGE), a technology that over-expresses proteins by up-regulating endogenous genes rather than introducing cDNA expression vectors into the cell. Saturation binding analysis using [125I]iodoproxyfan and RAGE-H3 membranes revealed a single class of binding sites with a K(D) value of 0.77 nM and a B(max) equal to 756 fmol/mg of protein. Competition binding studies showed that the rank order of potency for H3 agonists was N(alpha)-methylhistamine approximately (R)-alpha- methylhistamine > histamine and that the rank order of potency for H3 antagonists was clobenpropit > iodophenpropit > thioperamide. The same rank order of potency for H3 agonists and antagonists was observed in the functional assays as in the binding assays. The Fluorometic Imaging Plate Reader assays in RAGE-H3 cells gave high Z' values for agonist and antagonist screening, respectively. These results reveal that the human H3 receptor expressed with the RAGE technology is pharmacologically comparable to that expressed through recombinant methods. Moreover, the level of expression of the H3 receptor in the RAGE-H3 cells is suitable for HTS and secondary assays.
    Assay and Drug Development Technologies 07/2005; 3(3):309-18. · 1.90 Impact Factor
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    ABSTRACT: The chemoattractant receptor-homologous molecule expressed on T(H)2 cells (CRTH-2), also found on eosinophils and basophils, is a prostaglandin D2 receptor involved in the recruitment of these cell types during an inflammatory response. In this report, we describe the synthesis and optimization of a ramatroban isostere that is a selective and potent antagonist of CRTH-2 which may be useful in the treatment of certain diseases.
    Bioorganic & Medicinal Chemistry Letters 04/2005; 15(6):1749-53. · 2.34 Impact Factor
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    ABSTRACT: Flap endonuclease-1 (FEN1) is a key enzyme involved in base excision repair (BER), a primary pathway utilized by mammalian cells to repair DNA damage. Sensitization to DNA damaging agents is a potential method for the improvement of the therapeutic window of traditional chemotherapeutics. In this paper, we describe the identification and SAR of a series of low nanomolar FEN1 inhibitors. Over 1000-fold specificity was achieved against a related endonuclease, xeroderma pigmentosum G (XPG). Two compounds from this series significantly potentiate the action of methyl methanesulfonate (MMS) and temozolamide in a bladder cancer cell line (T24). To our knowledge, these are the most potent endonuclease inhibitors reported to date.
    Bioorganic & Medicinal Chemistry Letters 02/2005; 15(2):277-81. · 2.34 Impact Factor
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    ABSTRACT: There have been several recent reports of chemopotentiation via inhibition of DNA repair processes. Flap endonuclease 1 (FEN1) is a key enzyme involved in base excision repair (BER), a primary pathway utilized by mammalian cells to repair DNA damage. In this report, we describe the identification and SAR of a series of 2,4-diketobutyric acid FEN1 inhibitors.
    Bioorganic & Medicinal Chemistry Letters 11/2004; 14(19):4915-8. · 2.34 Impact Factor
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    ABSTRACT: The NF-kappaB pathway plays a critical role in regulating cellular processes such as immune responses, stress responses, apoptosis, proliferation and differentiation, whereas dysfunction of this pathway has been associated with numerous cancer and immune disorders. We have applied our Random Activation of Gene Expression technology to an NF-kappaB reporter cell line to facilitate the discovery of positive regulators of NF-kappaB activation. A small protein expression library, corresponding to approximately 0.1x genome coverage, was generated and screened for clones exhibiting constitutive activation of NF-kappaB. After isolation of cellular clones displaying the relevant phenotypes, we identified two known components of the NF-kappaB pathway and a hypothetical gene that we have designated the human ortholog of Xenopus TAK1-binding protein 3 (TAB3). Overexpression of human TAB3 was found to activate both NF-kappaB and AP-1 transcription factors. Furthermore, the activation of NF-kappaB by TAB3 was blocked by the NF-kappaB inhibitor, SN50, and by expression of dominant-negative forms of tumor necrosis factor alpha-associated factor 6 and transforming growth factor beta-activated kinase. Taken together, these data demonstrate that TAB3 transforming growth factor is a constituent of the NF-kappaB pathway functioning upstream of tumor necrosis factor alpha-associated factor 6/transforming growth factor beta-activated kinase. Interestingly, increased expression of TAB3 was found in some cancer tissues, and its overexpression in NIH 3T3 cells resulted in cellular transformation, thus establishing a causative link between elevated TAB3 expression, constitutive NF-kappaB activation, and oncogenesis.
    Proceedings of the National Academy of Sciences 03/2004; 101(7):2028-33. · 9.74 Impact Factor
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    ABSTRACT: Here we report the use of random activation of gene expression (RAGE) to create genome-wide protein expression libraries. RAGE libraries containing only 5 x 10(6) individual clones were found to express every gene tested, including genes that are normally silent in the parent cell line. Furthermore, endogenous genes were activated at similar frequencies and expressed at similar levels within RAGE libraries created from multiple human cell lines, demonstrating that RAGE libraries are inherently normalized. Pools of RAGE clones were used to isolate 19,547 human gene clusters, approximately 53% of which were novel when tested against public databases of expressed sequence tag (EST) and complementary DNA (cDNA). Isolation of individual clones confirmed that the activated endogenous genes can be expressed at high levels to produce biologically active proteins. The properties of RAGE libraries and RAGE expression clones are well suited for a number of biotechnological applications including gene discovery, protein characterization, drug development, and protein manufacturing.
    Nature Biotechnology 06/2001; 19(5):440-5. · 32.44 Impact Factor