Guanosine nucleotide analogs as inhibitors of alphavirus mRNA capping enzyme.
The two virus-specific reactions in the capping of alphavirus RNAs, catalyzed by the replicase protein nsP1, are promising targets for developing virus-specific inhibitors. In this report, we have studied the effect of over 50 cap analogs on the guanine-7-methyltransferase and guanylyltransferase activities of Semliki Forest virus nsP1. Recombinant nsP1 was expressed in Escherichia coli and partially purified by flotation in a discontinuous sucrose gradient. The methyltransferase activity had a pH optimum between pH 6.5 and 7.1, and the apparent Km values were 1.9 mM for GTP, 6.0 microM for S-adenosyl-L-methionine and 170 microM for Mg2+. NsP1 methyltransferase was able to methylate efficiently GTP (relative activity 100%), GDP (16%), GpppG (35%), GppppG (50%) and less efficiently GpppA (12%), m2GTP (9%), and m2,2GTP (25%), but not m7GppG. The most potent inhibitors for nsP1 methyltransferase were et2m7GMP (Ki value 42 microM), m2,7GMP, (64 microM), m2,7GpppG (82 microM), m2et7GMP (105 microM), m2(2-phet)7GMP (194 microM) and m2GMP (386 microM). Of these compounds, m2GMP, m2et7GMP and m2(2-phet)7GMP showed competitive inhibition, whereas the others showed mixed type inhibition. All compounds that inhibited the methyltransferase activity inhibited also the guanylyltransferase activity of nsP1.
Lampio
A
A
Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland. anja.lampio@helsinki.fi
Ahola
T
T
Darzynkiewicz
E
E
Stepinski
J
J
Jankowska-Anyszka
M
M
Kääriäinen
L
L
D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D006150:Guanine Nucleotides / Q000494:pharmacology*; D008780:Methyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D012314:RNA Cap Analogs / Q000494:pharmacology*; D012315:RNA Caps / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012672:Semliki forest virus / Q000201:enzymology*; D017361:Viral Nonstructural Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D004791:Enzyme Inhibitors; D006150:Guanine Nucleotides; D012314:RNA Cap Analogs; D012315:RNA Caps; D011994:Recombinant Proteins; D017361:Viral Nonstructural Proteins; D008780:Methyltransferases; C055097:mRNA (guanine(N7))-methyltransferase; D009713:Nucleotidyltransferases; C020842:guanylyltransferase; D012324:RNA-Dependent RNA Polymerase