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Sun Suk Kim,1,4,
Sirinya Matchacheep,2,3
Xiaoguang Lei,5
Shun Su,5
Wenyu Lin,1
Weerawat Runguphan,2,3
Won-Hyeok Choe,1
Naoya Sakamoto,6
Masanori Ikeda,7
Nobuyuki Kato,7
Aaron B. Beeler,5
John A. Porco Jr.,5
Stuart L. Schreiber,2,3,8 and
Raymond T. Chung1*
GI Unit, Department of Medicine, Massachusetts General Hospital, 55 Fruit Street, Boston, Massachusetts 02114,1 Department of Chemistry and Chemical Biology, Harvard University, 12 Oxford Street, Cambridge, Massachusetts 02138,2 The Broad Institute of Harvard and MIT, 7 Cambridge Center, Cambridge, Massachusetts 02142,3 Department of Gastroenterology and Hepatology, Gachon University Gil Medical Center, 1198 Guwol-dong, Namdong-gu, Incheon, 405-760 Korea,4 Department of Chemistry and Center for Chemical Methodology and Library Development (CMLD-BU), Boston University, 590 Commonwealth Avenue, Boston, Massachusetts 02215,5 Department of Gastroenterology and Hepatology, Tokyo Medical and Dental University, Tokyo, Japan,6 Department of Molecular Biology, Okayama University Graduate School of Medicine, Dentistry, and Pharmaceutical Sciences, Okayama, Japan,7 Howard Hughes Medical Institute, Chevy Chase, Maryland8
Received 15 February 2007/ Returned for modification 7 May 2007/ Accepted 28 July 2007
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ABSTRACT |
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INTRODUCTION |
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We have recently reported the successful adaptation of the Huh7/Rep-Feo replicon cell line (18) to a high-throughput screening assay system (8). Using this system, we previously screened a library of 2,568 well-known compounds whose biological activity is fully characterized (8). In order to discover novel regulators of HCV replication, we then screened a library of 8,064 diversity-oriented synthesis (DOS) compounds (15, 16). This library, known as the DOS set, is ameta-library comprised of DOS libraries from chemists throughout the United States and Canada. Information about the DOS set is available at http://www.broad.harvard.edu/chembio/platform/screening/compound_libraries/index.htm.
The high-throughput primary screen and the secondary validation assays were performed as described in our previous publication (8).
Computational data analysis of the primary screening results was performed as previously described (8) except for the hit criteria. As the characteristics of this data set are different from those generated by our previous screen (8), different threshold values were chosen to assure optimal hit selection. Compounds were considered hits for inhibiting replication if they had a composite Z score of <–2.57 in the reporter gene screen, a reproducibility of >0.9 or <–0.9 in that screen, and a composite Z score of >–2.00 in the cell viability screen. Compounds were considered hits for stimulating luciferase production if they had a composite Z score of >2.50 in the reporter gene screen, a reproducibility of >0.9 or <–0.9 in that screen, and a composite Z score of <1.00 in the cell viability screen.
Full synthetic experimental procedures and spectroscopic data for the SM library compounds discussed in this publication are provided in the supplemental material. The synthesis of the full SM library, including compounds not discussed here, will be the subject of an upcoming report.
The synthesis of the BUCMLD epoxyquinol library has been previously described (10, 17).
Full experimental details regarding the JFH1 HCVcc system (11) are provided in the supplemental material. We identified 41 antiviral compounds that inhibited HCV replication and 20 proviral compounds that increased luciferase production (Table 1). In our analysis of the antiviral hit compounds from the DOS set, a striking finding was that 21 of the 41 compounds contained an epoxide moiety. Moreover, the most potent of these compounds were epoxides. Further analysis revealed that these epoxides came from only two DOS libraries, SM and BUCMLD epoxyquinol (10, 17), with very high sublibrary hit rates of 35% and 33%, respectively (Table 1). Of note, the non-hit members of these two libraries did exhibit antiviral activity but failed to meet the formal hit criteria.
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SM_A14B5, which incorporates all of these elements, was therefore synthesized, as it was reasoned to be the most active SM library compound. Indeed, SM_A14B5 had a 50% effective concentration (EC50) of approximately 3.5 µM, which is about half that of SM_A6B5_2P100 (Table 2 and Fig. 2).
The most potent compounds from each library, SM_A14B5 and BUCMLD-B10A11, underwent further validation in the infectious JFH1 HCVcc system (11). They were tested at concentrations of 5 µM and 1 µM, respectively, and inhibited HCV replication 48.4% ± 5.9% and 45.1% ± 5.2%, respectively, relative to the level of inhibition achieved by interferon at a concentration of 1 ng/ml. These data roughly approximate the EC50 validation data derived from the OR6 system (7) in which inhibition was also measured relative to that of interferon at a concentration of 1 ng/ml.
Our observations suggest that the epoxide moiety is essential for potent antiviral activity. Analyzing the BUCMLD compounds, those compounds that bear an epoxide moiety are, in general, more-potent antivirals than those that do not (Table 2 and Fig. 1). Furthermore, all of the compounds from the SM library bear epoxides. SM_A12B3, an analog of SM_A5B3_2P141, which bears a tetrahydrofuran moiety in place of an epoxide, was therefore synthesized to further test this hypothesis. SM_A12B3 had negligible antiviral activity (Table 2), while SM_A5B3_2P141 displayed modest antiviral activity. Other analogs of SM compounds bearing tetrahydrofuran rings in place of epoxides showed similar attenuation of antiviral activity relative to their parent compounds. Unfortunately, attempts to synthesize the tetrahydrofuran analog of the most potent SM compound, SM_A14B5, have so far been unsuccessful.
It is interesting to note that it is the urazole-containing epoxyquinol constituents of the BUCMLD epoxyquinol library, rather than the maleimide-derived ones, that demonstrated anti-HCV activity in the primary screen. It is therefore likely that the combination of a urazole with the epoxide is necessary for the activity of the BUCMLD epoxyquinol compounds.
Although none of our most potent antiviral DOS compounds showed significant cytotoxicity at their EC50s, all of them ultimately proved to be cytotoxic at higher concentrations (Table 2 and Fig. 2). Therefore, future modifications should not only aim to improve anti-HCV activity but should also attempt to decrease cytotoxicity, in order to widen the therapeutic window.
It is tempting to hypothesize that these epoxides exert their antiviral effects through a common pathway. Presumably, they act as electrophiles, with the nucleophilic target making a covalent bond by attacking and opening the epoxides. Studies to elucidate their mechanism of action are under way.
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ACKNOWLEDGMENTS |
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The project has been funded in whole or in part with federal funds from the National Cancer Institute's Initiative for Chemical Genetics, National Institutes of Health, under contract no. N01-CO-12400.
The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. government.
Financial support was provided by the following: The American Gastroenterological Association FDHN/TAP Pharmaceuticals FFT Award (L.F.P.), The GlaxoSmithKline Research Fund of the Korean Association for The Study of The Liver (S.S.K.), NIH 5T32DK07191-31 (L.F.P.), NIH NS050854-01 (R.T.C.), and the NIGMS CMLD Initiative P50 GM067041 (J.A.P.).
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FOOTNOTES |
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Published ahead of print on 6 August 2007.
This publication is dedicated to Yoshito Kishi on the occasion of his 70th birthday.
Supplemental material for this article may be found at http://aac.asm.org/.
L.F.P. and S.S.K. contributed equally to this project.
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