SUPPLEMENTAL INFORMATION Identification and Analysis of Hepatitis C virus NS3 Helicase Inhibitors Using Nucleic Acid Binding Assays Sourav Mukherjee,1 Alicia M. Hanson,1 William R. Shadrick,1 Jean Ndjomou,1 Noreena L. Sweeney,1 John J. Hernandez,1 Diana Bartczak,1 Kelin Li,2 Kevin J. Frankowski,2 Julie A. Heck,3,4 Leggy A. Arnold,1 Frank J. Schoenen,3 and David N. Frick1,* 1Department of Chemistry and Biochemistry, University of Wisconsin-Milwaukee, Milwaukee, WI, 53211, USA. 2University of Kansas Specialized Chemistry Center, University of Kansas, 2034 Becker Dr., Lawrence, KS 66047. 3Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY, 10595. Table S1: Compound effects on the fluorescent polarization (FP)-based NS3h-DNA binding assay and the molecular beacon based helicase assay (MBHAs). Means ± standard deviations (n=2) are reported for normalized inhibition (%) and assay interference (ratio). Compounds colored blue are primuline derivatives, green compounds are known DNA binding agents, and pink compounds are other disclosed NS3 helicase inhibitors. [Data relate to Fig. 4] Table S2: Results of a screen of Sigma’s LOPAC for inhibitors of Cy5-dT15-NS3h complex formation. Pink compounds inhibited complex formation more than 60%, and green compounds interfered with the assay more than 20%. [Data relate to Fig. 5] Table S3: The Cy-15-dT15-SSB binding assay as a screen for specific HCV helicase inhibitors. Means ± standard deviations of IC50 values obtained in titrations (n=3) with each of the shown primuline derivatives. Compounds are ranked based on their ability to inhibit the formation of the Cy-15-dT15-SSB complex, with the least potent compounds on the top. [Data relate to Fig. 8]! Table&S1 $Cy52dT152NS3h$Binding$ $Cy52dT152NS3h$Binding$ DNA$Unwinding$(MBHA)$ DNA$Unwinding$(MBHA)$ CID$No./Compound$Name$ %$Inhibition Interference$ %Inhibition Interference TO*PRO*3 *63&±&0 1.4&±&0.0 115&±&2 0.5&±&0.0 Hoechst&33258 *96&±&2 0.1&±&0.0 104&±&6 0.3&±&0.0 Chromomycin&A3 *4&±&8 1.0&±&0.0 96&±&1 1.0&±&0.0 4049406 62&±&5 0.7&±&0.0 96&±&2 0.7&±&0.0 53308659 17&±&2 1.1&±&0.1 95&±&0 1.0&±&0.0 SYBR&Green&I 24&±&2 0.8&±&0.0 91&±&3 0.7&±&0.0 53255474 46&±&65 0.3&±&0.2 90&±&2 0.4&±&0.0 44251438 74&±&17 1.0&±&0.0 89&±&3 0.9&±&0.0 486270 30&±&4 0.6&±&0.0 87&±&4 0.6&±&0.0 53239937 9&±&2 1.0&±&0.0 87&±&5 1.0&±&0.0 44251434 47&±&10 1.0&±&0.0 82&±&4 1.0&±&0.0 44251428 54&±&4 1.0&±&0.0 81&±&12 1.0&±&0.0 247520 *17&±&2 0.8&±&0.0 80&±&4 0.7&±&0.0 3413238 63&±&2 0.7&±&0.0 78&±&7 0.7&±&0.0 49849282 22&±&3 1.0&±&0.0 77&±&8 1.0&±&0.0 Titan&Yellow 97&±&4 1.1&±&0.0 75&±&3 1.0&±&0.0 483665 *21&±&2 0.9&±&0.0 74&±&4 0.8&±&0.0 4176595 *13&±&3 0.8&±&0.0 69&±&2 0.9&±&0.0 4111820 4&±&2 0.7&±&0.0 68&±&7 0.9&±&0.0 7080057 *17&±&0 0.9&±&0.0 67&±&1 0.9&±&0.0 49849300 73&±&0 1.0&±&0.0 63&±&2 1.0&±&0.0 Soluble&Blue&HT 78&±&0 1.0&±&0.0 63&±&12 0.8&±&0.0 52939982 *9&±&2 0.9&±&0.0 59&±&4 1.0&±&0.0 44251431 73&±&8 1.0&±&0.0 58&±&23 1.0&±&0.0 Ellipticine *6&±&0 1.0&±&0.0 57&±&4 0.6&±&0.0 49849299 *2&±&1 1.0&±&0.0 53&±&14 1.0&±&0.0 Netropsin 8&±&1 0.9&±&0.0 51&±&20 0.8&±&0.1 Thiazole&Orange *4&±&1 0.9&±&0.0 49&±&7 0.7&±&0.0 50930741 56&±&13 1.0&±&0.0 46&±&5 1.0&±&0.0 50930749 *1&±&5 1.0&±&0.0 41&±&13 1.0&±&0.0 50930730 46&±&30 1.0&±&0.0 41&±&25 1.0&±&0.0 49849293 39&±&2 1.0&±&0.0 32&±&12 0.9&±&0.0 49849293 20&±&6 1.0&±&0.0 31&±&4 0.9&±&0.0 50930737 58&±&14 1.0&±&0.0 31&±&11 1.0&±&0.0 50930733 22&±&6 1.0&±&0.0 29&±&4 1.0&±&0.0 50930738 91&±&1 1.1&±&0.0 28&±&2 1.0&±&0.0 DAPI 4&±&0 0.9&±&0.0 28&±&9 0.8&±&0.0 Rebeccamycin *10&±&2 1.0&±&0.0 27&±&11 0.8&±&0.1 46913723 *10&±&4 1.0&±&0.0 27&±&3 0.9&±&0.0 Proflavin *4&±&1 0.9&±&0.0 27&±&4 0.8&±&0.0 53308658 30&±&14 0.9&±&0.2 27&±&2 1.0&±&0.0 50930755 18&±&3 1.0&±&0.0 26&±&1 0.9&±&0.0 49849287 19&±&5 1.0&±&0.0 25&±&1 0.9&±&0.0 Quinacrine *5&±&4 0.9&±&0.0 25&±&2 0.9&±&0.0 49849302 28&±&1 1.0&±&0.0 24&±&6 0.9&±&0.0 16060752 11&±&5 0.9&±&0.0 24&±&3 0.8&±&0.0 5479184 10&±&3 0.9&±&0.0 24&±&1 0.9&±&0.0 50930751 19&±&6 1.0&±&0.0 23&±&0 1.0&±&0.0 53312458 43&±&6 1.0&±&0.0 23&±&2 0.9&±&0.0 49849298 13&±&1 1.0&±&0.0 22&±&3 0.9&±&0.0 50930740 34&±&6 1.0&±&0.0 22&±&1 1.0&±&0.0 Thioflavin&T *8&±&0 1.0&±&0.0 21&±&2 0.9&±&0.0 3084034 30&±&4 1.0&±&0.0 21&±&12 0.8&±&0.0 Eva&Green *3&±&4 1.0&±&0.0 20&±&2 0.9&±&0.0 53255447 18&±&7 1.0&±&0.0 20&±&0 0.9&±&0.1 52914816 10&±&2 1.0&±&0.0 20&±&9 1.0&±&0.0 50930745 11&±&6 1.0&±&0.0 19&±&6 0.9&±&0.0 Berenil *46&±&0 1.0&±&0.0 19&±&3 0.9&±&0.0 49849280 41&±&0 1.0&±&0.0 17&±&3 0.9&±&0.0 46202556 *8&±&0 1.0&±&0.0 17&±&1 1.0&±&0.0 50930743 38&±&2 1.0&±&0.0 17&±&5 0.9&±&0.0 49849276 28&±&11 1.0&±&0.0 16&±&5 1.0&±&0.0 50930732 4&±&0 1.0&±&0.0 16&±&8 1.0&±&0.0 50930756 52&±&10 1.0&±&0.0 15&±&2 1.0&±&0.0 9585555 *4&±&2 0.9&±&0.0 15&±&5 1.0&±&0.0 3832647 *6&±&2 1.0&±&0.0 15&±&2 1.0&±&0.0 Trioxsalen *6&±&2 1.0&±&0.0 15&±&6 0.9&±&0.0 Carrier&Blank/DMSO *7&±&2 1.0&±&0.0 8&±&4 1.0&±&0.0 50930734 18&±&4 1.1&±&0.0 14&±&6 1.0&±&0.0 49849284 26&±&8 1.0&±&0.0 14&±&7 1.0&±&0.0 1 Table&S1 $Cy52dT152NS3h$Binding$ $Cy52dT152NS3h$Binding$ DNA$Unwinding$(MBHA)$ DNA$Unwinding$(MBHA)$ CID$No./Compound$Name$ %$Inhibition Interference$ %Inhibition Interference 53255452 13&±&5 1.0&±&0.0 14&±&0 0.9&±&0.0 5362502 *1&±&5 1.0&±&0.0 13&±&3 0.9&±&0.0 Luciferin *4&±&1 1.0&±&0.0 13&±&2 0.9&±&0.0 9682118 *9&±&1 1.0&±&0.0 13&±&4 1.0&±&0.0 3643004 *5&±&1 1.0&±&0.0 12&±&5 1.0&±&0.0 Carrier&Blank/DMSO *8&±&1 1.0&±&0.0 12&±&4 1.0&±&0.0 46916208 *9&±&2 1.0&±&0.0 11&±&4 1.0&±&0.0 5691857 3&±&0 0.9&±&0.0 11&±&4 1.0&±&0.0 4594423 *5&±&1 1.0&±&0.0 11&±&4 1.0&±&0.0 3496625 *5&±&0 1.0&±&0.0 11&±&3 1.0&±&0.0 2998553 *1&±&2 1.0&±&0.0 11&±&9 0.9&±&0.0 6540194 *9&±&0 1.0&±&0.0 10&±&4 1.0&±&0.0 Primulin 31&±&3 1.0&±&0.0 10&±&4 1.0&±&0.0 978069 1&±&1 0.9&±&0.0 10&±&8 1.0&±&0.0 5447034 *3&±&2 1.0&±&0.0 10&±&14 0.9&±&0.1 45382099 *4&±&1 1.0&±&0.0 10&±&2 1.0&±&0.0 6064372 *6&±&3 0.9&±&0.0 10&±&2 1.0&±&0.0 53255449 0&±&1 1.0&±&0.0 10&±&6 1.0&±&0.0 49849286 27&±&5 1.0&±&0.0 10&±&3 1.0&±&0.0 1293387 *7&±&1 0.9&±&0.0 10&±&7 1.0&±&0.0 6540199 *7&±&3 1.0&±&0.0 9&±&5 1.0&±&0.0 7314627 1&±&1 1.0&±&0.0 9&±&6 1.0&±&0.0 5281571 20&±&2 1.0&±&0.0 9&±&5 1.0&±&0.0 45382098 *4&±&3 1.0&±&0.0 9&±&3 1.0&±&0.0 49795072 *8&±&0 1.0&±&0.0 9&±&9 1.0&±&0.0 4473963 *11&±&1 1.0&±&0.0 8&±&2 1.0&±&0.0 44251428 24&±&6 1.0&±&0.0 8&±&2 1.0&±&0.0 16060753 *10&±&4 1.0&±&0.0 8&±&3 1.0&±&0.0 Carrier&Blank/DMSO *8&±&0 1.0&±&0.0 14&±&6 0.9&±&0.0 46916209 *9&±&0 1.0&±&0.0 8&±&5 1.0&±&0.0 49849289 36&±&2 1.0&±&0.0 8&±&6 1.0&±&0.0 5860596 *4&±&0 1.0&±&0.0 8&±&6 1.0&±&0.0 4611776 *5&±&3 1.0&±&0.0 8&±&2 1.0&±&0.0 16060655 *8&±&2 1.0&±&0.0 7&±&4 1.0&±&0.0 50930748 31&±&2 1.0&±&0.0 7&±&1 1.0&±&0.0 46839370 29&±&5 1.0&±&0.0 7&±&3 1.0&±&0.0 5736971 *3&±&2 0.9&±&0.0 7&±&2 1.1&±&0.0 1601813 *5&±&3 1.0&±&0.0 7&±&3 1.0&±&0.0 2186405 *6&±&0 0.9&±&0.0 7&±&11 1.0&±&0.0 6540163 *10&±&1 0.9&±&0.0 7&±&2 1.0&±&0.0 5293297 *7&±&2 0.9&±&0.0 7&±&0 1.0&±&0.0 45382102 *4&±&2 1.0&±&0.0 7&±&3 1.0&±&0.0 50904396 *10&±&1 1.0&±&0.0 6&±&13 1.0&±&0.0 2571817 *8&±&2 1.0&±&0.0 6&±&4 1.0&±&0.0 4371917 *26&±&4 1.0&±&0.0 6&±&6 1.0&±&0.0 2330223 *47&±&0 0.9&±&0.0 6&±&5 1.0&±&0.0 46897855 37&±&2 1.0&±&0.0 5&±&2 1.0&±&0.0 49795071 2&±&2 1.0&±&0.0 5&±&11 1.0&±&0.0 16238633 *4&±&1 1.0&±&0.0 5&±&4 1.0&±&0.0 1959044 *1&±&2 0.9&±&0.0 4&±&1 1.0&±&0.0 4051948 *7&±&1 1.0&±&0.0 4&±&11 1.0&±&0.0 2214934 *4&±&3 0.9&±&0.0 4&±&3 1.0&±&0.0 4872167 *4&±&2 1.0&±&0.0 4&±&5 1.0&±&0.0 49849294 32&±&6 1.0&±&0.0 4&±&5 1.0&±&0.0 45382104 0&±&1 1.0&±&0.0 4&±&3 1.0&±&0.0 49849279 *5&±&6 1.0&±&0.0 4&±&9 1.0&±&0.0 1126404 *8&±&2 1.0&±&0.0 3&±&6 1.0&±&0.0 53255450 12&±&2 1.0&±&0.0 3&±&4 1.0&±&0.0 49849295 8&±&3 1.0&±&0.0 3&±&1 1.0&±&0.0 45382101 *5&±&2 1.0&±&0.0 2&±&2 1.0&±&0.0 53255448 9&±&1 1.0&±&0.0 2&±&6 1.0&±&0.0 24818137 *3&±&2 1.0&±&0.0 1&±&7 1.0&±&0.0 44251427 8&±&2 1.0&±&0.0 0&±&16 1.1&±&0.0 44251433 7&±&3 1.0&±&0.0 0&±&11 1.1&±&0.0 2115891 7&±&2 0.9&±&0.0 *1&±&6 1.1&±&0.0 45382097 *7&±&2 1.0&±&0.0 *1&±&9 1.0&±&0.0 44251429 18&±&0 1.0&±&0.0 *1&±&3 1.1&±&0.0 46839371 *8&±&2 0.9&±&0.0 *1&±&8 1.0&±&0.0 49795073 *9&±&1 1.0&±&0.0 *1&±&0 1.0&±&0.0 45382103 0&±&2 1.0&±&0.0 *2&±&5 1.1&±&0.0 2 Table&S1 $Cy52dT152NS3h$Binding$ $Cy52dT152NS3h$Binding$ DNA$Unwinding$(MBHA)$ DNA$Unwinding$(MBHA)$ CID$No./Compound$Name$ %$Inhibition Interference$ %Inhibition Interference 3923766 *9&±&2 0.9&±&0.0 *2&±&8 1.0&±&0.0 3291242 *1&±&2 0.9&±&0.0 *4&±&14 1.1&±&0.0 3138378 *9&±&0 0.9&±&0.0 *5&±&4 1.0&±&0.0 49849290 1&±&0 1.0&±&0.0 22&±&7 1.0&±&0.0 3 Table&S2 (Cy53dT153NS3h(Binding( (Cy53dT153NS3h(Binding( Compound(Name( Sigma(Cat.(No.