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Antimicrobial Agents and Chemotherapy, November 2004, p. 4154-4162, Vol. 48, No. 11
0066-4804/04/$08.00+0     DOI: 10.1128/AAC.48.11.4154-4162.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Novel Chemical Class of pUL97 Protein Kinase-Specific Inhibitors with Strong Anticytomegaloviral Activity

Thomas Herget,^1, Martina Freitag,² Monika Morbitzer,¹ Regina Kupfer,² Thomas Stamminger,² and Manfred Marschall^2*

Institute for Clinical and Molecular Virology, University of Erlangen-Nürnberg, Erlangen,² Axxima Pharmaceuticals AG, Munich, Germany¹

Received 10 February 2004/ Returned for modification 11 May 2004/ Accepted 14 July 2004

Human cytomegalovirus (HCMV) is a major human pathogen frequently associated with life-threatening disease in immunosuppressed patients and newborns. The HCMV UL97-encoded protein kinase (pUL97) represents an important determinant of viral replication. Recent studies demonstrated that pUL97-specific kinase inhibitors are powerful tools for the control of HCMV replication. We present evidence that three related quinazoline compounds are potent inhibitors of the pUL97 kinase activity and block in vitro substrate phosphorylation, with 50% inhibitory concentrations (IC₅₀s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC₅₀s of these three quinazoline compounds (2.4 ± 0.4, 3.4 ± 0.6, and 3.9 ± 1.1 µM, respectively) were in the range of the IC₅₀ of ganciclovir (1.2 ± 0.2 µM), as determined by the HCMV green fluorescent protein-based antiviral assay. Importantly, the quinazolines were demonstrated to have strong inhibitory effects against clinical HCMV isolates, including ganciclovir- and cidofovir-resistant virus variants. Moreover, in contrast to ganciclovir, the formation of resistance to the quinazolines was not observed. The mechanisms of action of these compounds were confirmed by kinetic analyses with infected cells. Quinazolines specifically inhibited viral early-late protein synthesis but had no effects at other stages of the replication cycle, such as viral entry, consistent with a blockage of the pUL97 function. In contrast to epithelial growth factor receptor inhibitors, quinazolines affected HCMV replication even when they were added hours after virus adsorption. Thus, our findings indicate that quinazolines are highly efficient inhibitors of HCMV replication in vitro by targeting pUL97 protein kinase activity.

Present address: Merck KGaA, 64293 Darmstadt, Germany.

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