Inhibition of human cytomegalovirus UL80 protease by specific intramolecular disulfide bond formation.
A symmetrically substituted disulfide compound, CL13933, was identified as a potent inhibitor of human cytomegalovirus UL80 protease. Two types of inhibited protease were observed, depending on inhibitor concentration. At high concentrations, CL13933 formed a covalent adduct with the protease on Cys residues. At lower concentrations, this compound induced specific intramolecular disulfide formation between Cys84 and Cys87, and between Cys138 and Cys161. In contrast, Cys202 did not form disulfide bonds. Inhibition was reversed upon reduction of the protease. Each of the five cysteines of the UL80 protease was individually mutated to Ala. Each of the mutant proteases retained enzymatic activity, but mutants C138A and C161A were resistant to inhibition by CL13933, suggesting that disulfide bond formation between Cys138 and Cys161 is responsible for inhibition. This disulfide is apparently not induced by air oxidation. Examination of the CL13933 loading patterns of wild type and the five mutant proteases by mass spectrometry revealed that residues Cys87, Cys138, and Cys161 react with CL13933, and that the disulfide pair partner of each (Cys84, Cys161, and Cys138, respectively) is able to displace the compound via thiol-disulfide exchange. The possible significance of these reactive thiols in the protease is discussed.
Baum
E Z
EZ
Molecular Biology Research Section, Wyeth-Ayerst Research, Lederle Laboratories, Pearl River, New York 10965, USA. BAUME@war.wyeth.com
Siegel
M M
MM
Bebernitz
G A
GA
Hulmes
J D
JD
Sridharan
L
L
Sun
L
L
Tabei
K
K
Johnston
S H
SH
Wildey
M J
MJ
Nygaard
J
J
Jones
T R
TR
Gluzman
Y
Y
D000595:Amino Acid Sequence; D001645:Biguanides / Q000494:pharmacology*; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000737:chemistry; D003587:Cytomegalovirus / Q000201:enzymology* / Q000235:genetics; D004220:Disulfides / Q000737:chemistry; D010450:Endopeptidases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D011480:Protease Inhibitors / Q000494:pharmacology*; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D001645:Biguanides; C099592:CL 13933; D004220:Disulfides; D011480:Protease Inhibitors; D014764:Viral Proteins; D010450:Endopeptidases; C087225:human cytomegalovirus protease UL80; D003545:Cysteine