【病毒外文文獻】2009 Aryl diketoacids (ADK) selectively inhibit duplex DNA-unwinding activity of SARS coronavirus NTPase_helicase

of Korea Korea SARS CoV ADK are good metal chelators we anticipated that ADKs might serve as CoV SCV NTPase helicase Hel by mimicking the binding modes of the bis competes for the Zn 2 ion binding sites in SCV Hel thereby disrupting and helicase activities Phosphate release assay and FRET based assay of that the ADKs selectively inhibit the duplex DNA unwinding activity without binding site in the SCV Hel which warrants further investigations with diverse ADKs to provide valuable nucleotide g of duplex are currently HSV 50 Thus it was of our interest to test if our novel ADK analogues could serve as potential inhibitors of SCV Hel by mimicking the binding modes of the bismuth complexes to the MBD plex formation with malachite green and molybdate AM MG re agent 13 14 Briefly a 25ll solution of SCV helicase 400 nM in 50 mM Tris HCl pH 6 6 buffer was added to each well of the 96 well assay plate which already contained 0 5ll of various con centrations of chemical compounds 15 After incubation for 5 min at rt reactions were started by adding 25ll of reaction solution 50 mM Tris HCl pH 6 6 100 mM NaCl 10 mM MgCl 2 4mM ATP 4 nM circular ssDNA M13 to each well and then further incu bated for 10 min at 37 C176C The reactions were stopped by adding 200ll of AM MG reagent 0 034 malachite green 1 05 ammo nium molybdate 0 04 Tween 20 in 1 M HCl and the color was Corresponding authors Tel 82 2 910 5454 fax 82 2 910 4415 Y J J tel 82 2 2049 6100 fax 82 2 454 8217 Y C E mail addresses jeongyj kookmin ac kr Y J Jeong chongy konkuk ac kr Y Chong Bioorganic Grygon C A Hargrave K D Simoneau B Faucher A M Bolger G Kibler P Liuzzi M Cordingley M G Nat Med 2002 8 386 2 Borowski P Schalinski S Schmitz H Antiviral Res 2002 55 397 C Lee et al Bioorg Med Chem Figure 2 Inhibition of SCV Hel by ADKs 1 d 2 s 3 N 4 D 5 q 6 7 5 8 w a ATPase activity b duplex DNA unwinding activity ities of the ADKs In particular compounds with para relationship between the diketoacid moiety and the OCH 2 Ar group 1 and 2 do not show antiviral activities Also the chlorine substituent on the terminal aromatic ring has completely different effects on the inhibitory activities of the ADKs In comparison with 7 the inhibi tory activity against the target enzyme of 8 is rarely affected by chlorination On the contrary chlorine substitution resulted in sig nificant changes in antiviral activities of 4 and 6 in comparison with their unsubstituted counterparts 3 and 5 Taken together this result suggests a possible specific binding site for the ADKs in the SCV Hel which warrants further investigation of more structurally diverse ADK analogues on their anti SCV activities to provide definitive structure activity relationships as well as the possible binding site Table 1 IC 50 values of ADKs against SCV Hel ATPase activity and duplex DNA unwinding activity O R 1 CO 2 H OH R 4 OCH 2 Ph R 4 OCH 2 4 ClPh R 3 OCH 2 Ph R 3 OCH 2 4 ClPh R 2 OCH 2 Ph R 2 OCH 2 4 ClPh 2 3 4 R 3 NHCH 2 Ph R 3 NHCH 2 4 ClPh 2 3 4 5 6 7 8 Compound IC 50 lM ATPase Duplex DNA unwinding 1 41 3 2 7 39 9 0 5 2 50 50 3 50 50 4 24 4 1 0 13 6 0 3 5 50 50 6 50 28 7 2 3 7 50 5 4 0 1 8 50 11 0 0 6 Lett 19 2009 1636 1638 1637 3 Tanner J A Zheng B J Zhou J Watt R M Jiang J Q Wong K L Lin Y P Lu L Y He M L Kung H F Kesel A J Huang J D Chem Biol 2005 12 303 4 Bernini A Spiga O Venditti V Prischi F Bracci L Huang J D Tanner J A Niccolai N Biochem Biophys Res Commun 2006 343 1101 5 Tanner J A Watt R M Chai Y B Lu L Y Lin M C Peiris J S Poon L L Kung H F Huang J D J Biol Chem 2003 278 39578 6 Seybert A Posthuma C C van Dinten L C Snijder E J Gorbalenya A E Ziebuhr J J Virol 2005 79 696 7 Yang N Tanner J A Wang Z Huang J D Zheng B J Zhu N Zun H Chem Commun 2007 4413 8 Yang N Tanner J A Zheng B J Watt R M He M L Lu L Y Jiang J Q Shum K T Lin Y P Wong K L Lin M C M Kung H F Sun H Huang J D Angew Chem Int Ed 2007 46 6464 9 Hazuda D Felock P Witmer M Wolfe A Stillmock K Grobler J A Espeseth A Gabryelski L Schleif W Blau C Miller M D Science 2000 287 646 10 Summa V Petrocchi A Pace P Matassa V G De Francesco R Altamura S Tomei L Koch U Neuner P J Med Chem 2004 7 14 11 Di Santo R Fermeglia M Ferrone M Paneni M S Costi R Artico M Roux A Gabriele M Tardif K D Siddiqui A Pricl S J Med Chem 2005 48 6304 12 Kim J Kim K S Lee H S Park K S Park S Y Kang S Y Lee S J Park H S Kim D E Chong Y Bioorg Med Chem Lett 2008 18 4661 13 Baykov A A Evtushenko O A Avaeva S M Anal Biochem 1988 171 266 14 Wardell A D Errington W Ciaramella G Merson J McGarvey M J J Gen Virol 1999 80 701 15 Martin G R Yvette M N Chrisotomos P Laurence H P Paul W Wynne A Anal Biochem 2004 327 176 16 Jang K J Lee N R Yeo W S Jeong Y J Kim D E Biochem Biophys Res Commun 2008 366 738 17 Briefly carboxytetramethylrhodamine TAMRA modified 45 base oligomer and fluorescein modified 25 base oligomer were purchased from Integrated DNA Technologies 5 0 20T25Tam 5 0 TTTTTTTTTTTTTTTTTTTTGAGCGGATTA CTATACTACATTAGA TAMRA 3 0 and 3 0 0T25Flu 5 0 Fluorescein TCTAATGTA GTATAGTAATCCGCTC 3 0 The helicase substrate was prepared by annealing the two oligomers which resulted in 25 base pairs of dsDNA with single stranded 20 dT of 5 0 overhang A 80ll solution of SCV helicase 150 nM in 20 mM HEPES pH 7 4 buffer was added to each well of the 96 well assay plate which already contained 1ll of various concentrations of chemical compounds After 5 min incubation at rt the FRET based dsDNA unwinding assay was started by addition of 20ll 5X reaction solution 5 mM MgCl 2 45 mM ATP 25 mM DTT and 100 nM dsDNA substrate in 20 mM HEPES pH 7 4 The reaction mixture was further incubated for 2 min at 37 C176C and stopped with 100 ll of termination solution 0 1 M EDTA and 0 4 lM trap DNA unmodified 25 bases 3 0 0T25 oligomer in 20 mM HEPES pH 7 4 The sample was excited at 485 nm and the fluorescence was measured at 535 nm 18 Kao R Y Tsui W H W Lee T S W Tanner J A Watt R M Huang J D Hu L Chen G Chen Z Zhang L He T Chan K H Tse H To A P C Ng L W Y Wong B C W Tsoi H W Yang D Ho D D Yuen K Y Chem Biol 2004 11 1293 1638 C Lee et al Bioorg Med Chem Lett 19 2009 1636 1638