Flavins inhibit human cytomegalovirus UL80 protease via disulfide bond formation.
Among the most potent inhibitors of human cytomegalovirus protease identified by random screening of a chemical library was 1,4-dihydro-7,8-dimethyl 6H-pyrimido[1,2-b]-1,2,4,5-tetrazin-6-one (1) (PTH2). The oxidized form (2), PT, which is present in solutions of PTH2, was shown to be the actual inhibitory species which irreversibly inactivates the protease; recycling of PTH2 by dissolved oxygen results in complete inhibition of the protease at substoichiometric amounts of compound. No evidence for a covalent adduct between the protease and the inhibitor was obtained, and protease activity was restored by incubation of the inactivated enzyme with the reducing agent bismercaptoethyl sulfone, suggesting that disulfide bond formation was responsible for the observed inhibition. The five cysteines of the protease are normally in the reduced state; analysis of tryptic peptides from inhibited protease indicated that disulfide bonds Cys84-Cys87 and Cys138-Cys161 were formed. Using site-directed mutagenesis, the disulfide pair induced between Cys138 and Cys161 disulfide is dependent upon interaction of PT with the protease and does not form spontaneously, unlike that of the Cys84-Cys87 pair which can form in the absence of inhibitor. The inhibitor's redox chemistry is analogous to that of flavin, and, in fact, flavin inhibits the protease by the same mechanism, causing formation of a disulfide bond between Cys138 and Cys161. That the cysteines are dispensable, but can regulate protease activity by formation of a unique disulfide pair, suggests a plausible mechanism for control of proteolysis during the viral life cycle.
Baum
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EZ
Wyeth-Ayerst Research, Lederle Laboratories, Pearl River, New York 10965, USA. BAUME@war.wyeth.com
Ding
W D
WD
Siegel
M M
MM
Hulmes
J
J
Bebernitz
G A
GA
Sridharan
L
L
Tabei
K
K
Krishnamurthy
G
G
Carofiglio
T
T
Groves
J T
JT
Bloom
J D
JD
DiGrandi
M
M
Bradley
M
M
Ellestad
G
G
Seddon
A P
AP
Gluzman
Y
Y
D001483:Base Sequence; D001665:Binding Sites; D002851:Chromatography, High Pressure Liquid; D003545:Cysteine / Q000737:chemistry; D003587:Cytomegalovirus / Q000201:enzymology* / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D003606:Dacarbazine / Q000031:analogs & derivatives* / Q000494:pharmacology; D004220:Disulfides / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D005415:Flavins / Q000494:pharmacology*; D006801:Humans; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D017354:Point Mutation; D011480:Protease Inhibitors / Q000494:pharmacology*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
C099593:1,4-dihydro-7,8-dimethyl-6H-pyrimido(1,2-b)-1,2,4,5-tetrazin-6-one; D017931:DNA Primers; D004279:DNA, Viral; D004220:Disulfides; D005415:Flavins; D011480:Protease Inhibitors; D014764:Viral Proteins; D003606:Dacarbazine; D010450:Endopeptidases; C087225:human cytomegalovirus protease UL80; D003545:Cysteine