Novel chemical class of pUL97 protein kinase-specific inhibitors with strong anticytomegaloviral activity.

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 (IC(50)s) between 30 and 170 nM. Replication of HCMV in primary human fibroblasts was suppressed with a high efficiency. The IC(50)s of these three quinazoline compounds (2.4 +/- 0.4, 3.4 +/- 0.6, and 3.9 +/- 1.1 microM, respectively) were in the range of the IC(50) of ganciclovir (1.2 +/- 0.2 microM), 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.

The protein kinase pUL97 of human cytomegalovirus interacts with and phosphorylates the DNA polymerase processivity factor pUL44.

The protein kinase pUL97 of human cytomegalovirus plays important but incompletely defined roles in viral replication. Concerning the early phase of infection, it is postulated that pUL97 possesses regulatory functions in gene expression and/or DNA synthesis. Here we report that pUL97 interacts with an essential component of the replication complex, the DNA polymerase processivity factor pUL44. Interaction was determined by yeast two-hybrid and coimmunoprecipitation analyses and was mapped to the pUL97 region 366-459. In vitro kinase assays demonstrated that pUL44, coimmunoprecipitated either from transfected or from infected cells, is phosphorylated by pUL97 (but not by a catalytically inactive pUL97-mutant). In infected fibroblasts, immunofluorescence analysis revealed that pUL97 and pUL44 accumulate in the nucleus and are both incorporated into viral replication centers. The treatment with inhibitors of DNA synthesis or pUL97 kinase activity largely prevented colocalization. Thus, pUL97 may be indirectly involved in viral genome replication by modifying the replication cofactor pUL44.

Direct targeting of human cytomegalovirus protein kinase pUL97 by kinase inhibitors is a novel principle for antiviral therapy.

The protein kinase pUL97, encoded by human cytomegalovirus (HCMV), is an important determinant of virus replication. Recently, indolocarbazoles were identified as a class of substances that inhibit the pUL97 kinase activity in vitro. In parallel, it was shown that indolocarbazoles interfere with HCMV replication; however, the causal relationship between inhibition of pUL97 kinase activity and virus replication has not been clarified. Here evidence is provided that indolocarbazole-mediated inhibition of virus replication is a direct result of diminished pUL97 protein kinase activity. In cell culture infections, a strong and selective antiviral activity was measured with respect to several strains of HCMV in contrast with other related or non-related viruses. For fine quantification, recombinant HCMVs expressing green fluorescent protein were used, demonstrating the high sensitivity towards compounds NGIC-I and Gö6976. Interestingly, a ganciclovir-resistant virus mutant (UL97-M460I) showed increased sensitivity to both compounds. Supporting this concept, transfection experiments with cloned pUL97 revealed that ganciclovir-resistant mutants were characterized by reduced levels of autophosphorylation compared with wild-type and possessed particularly high sensitivity to indolocarbazoles. Moreover, the Epstein-Barr virus-encoded homologous kinase, BGLF4, which showed a similar pattern of autophosphorylation and ganciclovir phosphorylation activities, was not inhibited. Importantly, a cytomegalovirus deletion mutant, lacking a functional UL97 gene and showing a severe impairment of replication, was completely insensitive to indolocarbazoles. Thus, our findings indicate that a specific block in the activity of pUL97 is the critical step in indolocarbazole-mediated inhibition of virus replication and that pUL97 might be targeted very efficiently by a novel antiviral therapy.

Inhibitors of human cytomegalovirus replication drastically reduce the activity of the viral protein kinase pUL97.

The UL97-encoded protein kinase (pUL97) of human cytomegalovirus (HCMV) plays a critical role in the control of virus replication. Deletion of the UL97 gene results in a drastic reduction in the replication efficiency. Although the exact function of pUL97 remains unclear and its sensitivity to specific inhibitors is speculative, protein kinase inhibitors of the indolocarbazole class are effective inhibitors of cytomegalovirus. Based on the phosphorylation of ganciclovir (GCV), a novel quantification system for pUL97 kinase activity was established: the phosphorylated form of GCV exerts an easily quantifiable cytotoxic effect in transfected cells. Importantly, the addition of indolocarbazole compounds, Gö6976 and NGIC-I, which were highly effective at nanomolar concentrations while other protein kinase inhibitors were not, led to a significant reduction of pUL97 kinase activity. It was also demonstrated that a catalytically inactive mutant of pUL97, K355M, and a GCV-resistant mutant, M460I, were both negative for GCV phosphorylation, although protein phosphorylation remained detectable for the latter mutant. In vitro kinase assays were used to confirm the levels of pUL97-mediated phosphorylation recorded. To generate a tool for screening large numbers of putative inhibitors that preferentially interfere with GCV as well as protein phosphorylation, pUL97-expressing cell clones with stable pUL97 kinase activity were selected. This study demonstrates that certain indolocarbazole compounds are potent pUL97 inhibitors and, therefore, represent novel candidates for antiviral drugs that target viral protein kinase functions.