Flavins Inhibit Human Cytomegalovirus UL80 Protease via Disulfide Bond Formation

Abstract

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.

Elimination of Retroviral Infectivity byN-Ethylmaleimide with Preservation of Functional Envelope Glycoproteins

“…Beatrice and Wagner reported that malemide-based treatments of VSV inactivated the virus only if the compound could penetrate the viral core (4). Human cytomegalovirus (2,3) and Junin viruses (27,28) were also vulnerable to these types of sulfhydryl reagents. An intact CCCH zinc finger motif was shown to be required for transcription in respiratory syncytial virus (59) and in Ebola virus (46).…”

Selective Nonpeptidic Inhibitors of Herpes Simplex Virus Type 1 and Human Cytomegalovirus Proteases.

“…Several investigations to seek herpesvirus protease inhibitors have recently reported various low molecular weight inhibitors [24][25][26][27][28][29][30][31][32][33] and peptidomimetic inhibitors. 21,34,35) While a low molecular weight inhibitor with in vitro antiviral activity and stability in human plasma has been found, 29) there are likely to be problems involved with the peptidomimetic inhibitors, which display poor cell penetration and are easily subjected to degradation in cell culture.…”

A new one‐step synthesis of pyrimido‐[1,2‐ b ][1,2,4,5]tetrazines

“…Our continued interest in the chemistry of hydrazonoyl halides 4 [2] prompted us to investigate their reactions with N-aminoheterocyclic thiols in an attempt to develop a new strategy for synthesis of the title ring system. The interest in developing new simple syntheses of pyrimido [1,2- megalovirus protease [3]. Here, we wish to report our results utilizing reactions of 4 with 3-amino-2,3-dihydro-6-methyl-2-thioxo-4(1H)-pyrimidinone 5 and 3-amino-6-methyl-2-methylthio-4(3H)-pyrimidinone 6 as a route to 6H-pyrimido [1,2,-b] [1,2,4,5]tetrazin-6-ones 9 (Scheme 1).…”

“…The same results were obtained when the reactions of 4 with 5 were carried out in pyridine at reflux. It was initially anticipated that such reactions would yield the respective pyrimido [2,1-b] [1,3,4]thiadiazines by analogy to the reactions of 4 with 2-aminothiophenol, which were reported to afford benzothiadiazine derivatives [4]. Unexpectedly, the products isolated from the reactions of 5 with 4a-h were found to be free of sulfur.…”