The methyltransferase enzyme (MTase), which catalyzes the transfer of a methyl group from S-adenosyl-methionine (AdoMet) to viral RNA, and generates S-adenosyl-homocysteine (AdoHcy) as a by-product, is essential for the life cycle of many significant human pathogen flaviviruses. Here we investigated inhibition of the flavivirus MTase by several AdoHcy-derivatives. Unexpectedly we found that AdoHcy itself barely inhibits the flavivirus MTase activities, even at high concentrations. AdoHcy was also shown to not inhibit virus growth in cell-culture. Binding studies confirmed that AdoHcy has a much lower binding affinity for the MTase than either the AdoMet co-factor, or the natural AdoMet analog inhibitor sinefungin (SIN). While AdoMet is a positively charged molecule, SIN is similar to AdoHcy in being uncharged, and only has an additional amine group that can make extra electrostatic contacts with the MTase. Molecular Mechanics Poisson-Boltzmann Sovation Area analysis on AdoHcy and SIN binding to the MTase suggests that the stronger binding of SIN may not be directly due to interactions of this amine group, but due to distributed differences in SIN binding resulting from its presence. The results suggest that better MTase inhibitors could be designed by using SIN as a scaffold rather than AdoHcy.
References
[1]
Turtle L, Griffiths MJ, Solomon T (2012) Encephalitis caused by flaviviruses. QJM 105: 219-223. doi:10.1093/qjmed/hcs013. PubMed: 22367423.
[2]
Teruel-López E (1991) Dengue. A review. Invest Clin 32: 201-217. PubMed: 1822723.
[3]
Heinz FX, Stiasny K (2012) Flaviviruses and flavivirus vaccines. Vaccine 30: 4301-4306. doi:10.1016/j.vaccine.2011.09.114. PubMed: 22682286.
[4]
Dong H, Ren S, Zhang B, Zhou Y, Puig-Basagoiti F et al. (2008) West Nile virus methyltransferase catalyzes two methylations of the viral RNA cap through a substrate-repositioning mechanism. J Virol 82: 4295-4307. doi:10.1128/JVI.02202-07. PubMed: 18305027.
[5]
Benarroch D, Egloff MP, Mulard L, Guerreiro C, Romette JL et al. (2004) A structural basis for the inhibition of the NS5 dengue virus mRNA 2'-O-methyltransferase domain by ribavirin 5'-triphosphate. J Biol Chem 279: 35638-35643. doi:10.1074/jbc.M400460200. PubMed: 15152003.
[6]
Bollati M, Alvarez K, Assenberg R, Baronti C, Canard B et al. (2010) Structure and functionality in flavivirus NS-proteins: perspectives for drug design. Antiviral Res 87: 125-148. doi:10.1016/j.antiviral.2009.11.009. PubMed: 19945487.
[7]
Dong H, Zhang B, Shi PY (2008) Flavivirus methyltransferase: a novel antiviral target. Antiviral Res 80: 1-10. doi:10.1016/j.antiviral.2008.05.003. PubMed: 18571739.
[8]
Lim SP, Wen D, Yap TL, Yan CK, Lescar J et al. (2008) A scintillation proximity assay for dengue virus NS5 2'-O-methyltransferase-kinetic and inhibition analyses. Antiviral Res 80: 360-369. doi:10.1016/j.antiviral.2008.08.005. PubMed: 18809436.
[9]
Luzhkov VB, Selisko B, Nordqvist A, Peyrane F, Decroly E et al. (2007) Virtual screening and bioassay study of novel inhibitors for dengue virus mRNA cap (nucleoside-2'O)-methyltransferase. Bioorg Med Chem 15: 7795-7802. doi:10.1016/j.bmc.2007.08.049. PubMed: 17888664.
[10]
Milani M, Mastrangelo E, Bollati M, Selisko B, Decroly E et al. (2009) Flaviviral methyltransferase/RNA interaction: structural basis for enzyme inhibition. Antiviral Res 83: 28-34. doi:10.1016/j.antiviral.2009.03.001. PubMed: 19501254.
[11]
Podvinec M, Lim SP, Schmidt T, Scarsi M, Wen D et al. (2010) Novel inhibitors of dengue virus methyltransferase: discovery by in vitro-driven virtual screening on a desktop computer grid. J Med Chem 53: 1483-1495. doi:10.1021/jm900776m. PubMed: 20108931.
[12]
Puig-Basagoiti F, Qing M, Dong H, Zhang B, Zou G et al. (2009) Identification and characterization of inhibitors of West Nile virus. Antiviral Res 83: 71-79. doi:10.1016/j.antiviral.2009.03.005. PubMed: 19501258.
[13]
Sampath A, Padmanabhan R (2009) Molecular targets for flavivirus drug discovery. Antiviral Res 81: 6-15. doi:10.1016/j.antiviral.2008.08.004. PubMed: 18796313.
[14]
Selisko B, Peyrane FF, Canard B, Alvarez K, Decroly E (2010) Biochemical characterization of the (nucleoside-2'O)-methyltransferase activity of dengue virus protein NS5 using purified capped RNA oligonucleotides. AC: GPPP. (p. 7Me)(n) and AC: GPPP(n). J Gen Virol 91: 112-121.
[15]
Lim SP, Sonntag LS, Noble C, Nilar SH, Ng RH et al. (2011) Small molecule inhibitors that selectively block dengue virus methyltransferase. J Biol Chem 286: 6233-6240. doi:10.1074/jbc.M110.179184. PubMed: 21147775.
[16]
Chen H, Liu L, Jones SA, Banavali N, Kass J et al. (2013) Selective inhibition of the West Nile virus methyltransferase by nucleoside analogs. Antiviral Res 97: 232-239. doi:10.1016/j.antiviral.2012.12.012. PubMed: 23267828.
[17]
Fauman EB, Blumenthal RM, Cheng XD (1999) Structure and evolution of AdoMet-dependent methyltransferases. In: XD ChengRM Blumenthal. S-Adenosylmethionine-dependent methyltransferase: structures and functions. Singapore: World Scientific Publishing Co. pp. 1-38.
[18]
Ray D, Shah A, Tilgner M, Guo Y, Zhao Y et al. (2006) West Nile virus 5'-cap structure is formed by sequential guanine N-7 and ribose 2'-O methylations by nonstructural protein 5. J Virol 80: 8362-8370. doi:10.1128/JVI.00814-06. PubMed: 16912287.
[19]
Zhou Y, Ray D, Zhao Y, Dong H, Ren S et al. (2007) Structure and function of flavivirus NS5 methyltransferase. J Virol 81: 3891-3903. doi:10.1128/JVI.02704-06. PubMed: 17267492.
[20]
Egloff MP, Benarroch D, Selisko B, Romette JL, Canard B (2002) An RNA cap (nucleoside-2'-O-)-methyltransferase in the flavivirus RNA polymerase NS5: crystal structure and functional characterization. EMBO J 21: 2757-2768. doi:10.1093/emboj/21.11.2757. PubMed: 12032088.
[21]
Kroschewski H, Lim SP, Butcher RE, Yap TL, Lescar J et al. (2008) Mutagenesis of the dengue virus type 2 NS5 methyltransferase domain. J Biol Chem 283: 19410-19421. doi:10.1074/jbc.M800613200. PubMed: 18469001.
[22]
Dong H, Chang DC, Hua MH, Lim SP, Chionh YH et al. (2012) 2'-O methylation of internal adenosine by flavivirus NS5 methyltransferase. PLOS Pathog 8: e1002642. PubMed: 22496660.
[23]
Dong H, Liu L, Zou G, Zhao Y, Li Z et al. (2010) Structural and functional analyses of a conserved hydrophobic pocket of flavivirus methyltransferase. J Biol Chem 285: 32586-32595. doi:10.1074/jbc.M110.129197. PubMed: 20685660.
[24]
Dong H, Ren S, Li H, Shi PY (2008) Separate molecules of West Nile virus methyltransferase can independently catalyze the N7 and 2'-O methylations of viral RNA cap. Virology 377: 1-6. doi:10.1016/j.virol.2008.04.026. PubMed: 18501946.
[25]
Bhattacharya D, Hoover S, Falk SP, Weisblum B, Vestling M et al. (2008) Phosphorylation of yellow fever virus NS5 alters methyltransferase activity. Virology 380: 276-284. doi:10.1016/j.virol.2008.07.013. PubMed: 18757072.
[26]
Khromykh AA, Kenney MT, Westaway EG (1998) trans-Complementation of flavivirus RNA polymerase gene NS5 by using Kunjin virus replicon-expressing BHK cells. J Virol 72: 7270-7279. PubMed: 9696822.
[27]
Chung KY, Dong H, Chao AT, Shi PY, Lescar J et al. (2010) Higher catalytic efficiency of N-7-methylation is responsible for processive N-7 and 2'-O methyltransferase activity in dengue virus. Virology 402: 52-60. doi:10.1016/j.virol.2010.03.011. PubMed: 20350738.
[28]
Dong H, Ray D, Ren S, Zhang B, Puig-Basagoiti F et al. (2007) Distinct RNA elements confer specificity to flavivirus RNA cap methylation events. J Virol 81: 4412-4421. doi:10.1128/JVI.02455-06. PubMed: 17301144.
[29]
Li SH, Dong H, Li XF, Xie X, Zhao H et al. (2013) Rational design of a flavivirus vaccine through abolishing viral RNA 2’ -O methylation. J Virol ( in press).
[30]
Liu L, Dong H, Chen H, Zhang J, Ling H et al. (2010) Flavivirus RNA cap methyltransferase: structure, function, and inhibition. Front Biol 5: 286-303. doi:10.1007/s11515-010-0660-y. PubMed: 21927615.
[31]
Loehrer FM, Schwab R, Angst CP, Haefeli WE, Fowler B (1997) Influence of oral S-adenosylmethionine on plasma 5-methyltetrahydrofolate, S-adenosylhomocysteine, homocysteine and methionine in healthy humans. J Pharmacol Exp Ther 282: 845-850. PubMed: 9262350.
[32]
Poirier LA, Wise CK, Delongchamp RR, Sinha R (2001) Blood determinations of S-adenosylmethionine, S-adenosylhomocysteine, and homocysteine: correlations with diet. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology 10: 649-655.
[33]
Perna AF, Ingrosso D, Zappia V, Galletti P, Capasso G et al. (1993) Enzymatic methyl esterification of erythrocyte membrane proteins is impaired in chronic renal failure. Evidence for high levels of the natural inhibitor S-adenosylhomocysteine. J Clin Invest 91: 2497-2503. doi:10.1172/JCI116485. PubMed: 8514862.
[34]
Castro R, Rivera I, Struys EA, Jansen EE, Ravasco P et al. (2003) Increased homocysteine and S-adenosylhomocysteine concentrations and DNA hypomethylation in vascular disease. Clin Chem 49: 1292-1296. doi:10.1373/49.8.1292. PubMed: 12881445.
[35]
Stabler SP, Allen RH, Dolce ET, Johnson MA (2006) Elevated serum S-adenosylhomocysteine in cobalamin-deficient elderly and response to treatment. Am J Clin Nutr 84: 1422-1429. PubMed: 17158426.
[36]
Dall’Acqua W, Goldman ER, Lin W, Teng C, Tsuchiya D et al. (1998) A mutational analysis of binding interactions in an antigen-antibody protein-protein complex. Biochemistry 37: 7981-7991. doi:10.1021/bi980148j. PubMed: 9609690.
[37]
Zheng W, Ibá？ez G, Wu H, Blum G, Zeng H et al. (2012) Sinefungin derivatives as inhibitors and structure probes of protein lysine methyltransferase SETD2. J Am Chem Soc 134: 18004-18014. doi:10.1021/ja307060p. PubMed: 23043551.
[38]
Geiss BJ, Thompson AA, Andrews AJ, Sons RL, Gari HH et al. (2009) Analysis of flavivirus NS5 methyltransferase cap binding. J Mol Biol 385: 1643-1654. doi:10.1016/j.jmb.2008.11.058. PubMed: 19101564.
[39]
Brooks BR, Brooks CL 3rd, Mackerell AD Jr., Nilsson L, Petrella RJ et al. (2009) CHARMM: the biomolecular simulation program. J Comput Chem 30: 1545-1614. doi:10.1002/jcc.21287. PubMed: 19444816.
[40]
Brooks JB, Kuno G, Craven RB, Alley CC, Wycoff BJ (1983) Studies of metabolic changes in cell cultures infected with four serotypes of dengue fever viruses by frequency-pulsed electron-capture gas-liquid chromatography. J Chromatogr 276: 279-288. PubMed: 6630378.
[41]
MacKerell AD, Bashford D, Bellott M, Dunbrack RL, Evanseck JD et al. (1998) All-Atom Empirical Potential for Molecular Modeling and Dynamics Studies of Proteins. J Phys Chem 102: 3586-3616.
[42]
Mackerell AD Jr, Feig M, Brooks CL 3rd (2004) Extending the treatment of backbone energetics in protein force fields: limitations of gas-phase quantum mechanics in reproducing protein conformational distributions in molecular dynamics simulations. J Comput Chem 25: 1400-1415. doi:10.1002/jcc.20065. PubMed: 15185334.
[43]
Jorgensen WJ, Chandrasekhar J, Madura JD, Impey RW, Klein ML (1983) Comparison of simple potential functions for simulating liquid water. J Chem Phys 79: 926-935. doi:10.1063/1.445869.
[44]
BEGLOV D, Roux B (1994) Finite Representation of an Infinite Bulk System: Solvent Boundary Potential for Computer Simulations. J Chem Phys 100: 9050-9063. doi:10.1063/1.466711.
[45]
Wang P, Nicklaus MC, Marquez VE, Brank AS, Christman J et al. (2000) Use of Oligodeoxyribonucleotides with Conformationally Constrained Abasic Sugar Targets to Probe the Mechanism of Base Flipping by HhaI DNA (Cytosine C5)-Methyltransferase. J Am Chem Soc 122: 12422-12434. doi:10.1021/ja001989s.
[46]
Vanommeslaeghe K, Hatcher E, Acharya C, Kundu S, Zhong S et al. (2010) CHARMM general force field: A force field for drug-like molecules compatible with the CHARMM all-atom additive biological force fields. J Comput Chem 31: 671-690. PubMed: 19575467.
[47]
Vanommeslaeghe K, MacKerell AD Jr. (2012) Automation of the CHARMM General Force Field (CGenFF) I: Bond Perception and Atom Typing. J Chem Inf Model 52: 3144-3154. doi:10.1021/ci300363c. PubMed: 23146088.
[48]
Vanommeslaeghe K, Raman EP, MacKerell AD Jr (2012) Automation of the CHARMM General Force Field (CGenFF) II: Assignment of Bonded Parameters and Partial Atomic Charges. J Chem Inf Model 52: 3155-3168. doi:10.1021/ci3003649. PubMed: 23145473.
[49]
Darden T, York D, Pedersen LG (1993) Particle mesh Ewald: An N？log(N) method for Ewald sums in large systems. J Chem Phys 98: 10089-10092. doi:10.1063/1.464397.
[50]
Feller S, Zhang Y, Pastor R, Brooks BR (1995) Constant pressure molecular dynamics simulation: The Langevin piston method J Chem Phys 103: 4613-4621. doi:10.1063/1.470648.
[51]
Kollman PA, Massova I, Reyes C, Kuhn B, Huo S et al. (2000) Calculating structures and free energies of complex molecules: combining molecular mechanics and continuum models. Acc Chem Res 33: 889-897. doi:10.1021/ar000033j. PubMed: 11123888.
