Cyclin-Dependent Kinases (CDKs) and the Human Cytomegalovirus-Encoded CDK Ortholog pUL97 Represent Highly Attractive Targets for Synergistic Drug Combinations

Abstract

Human cytomegalovirus (HCMV) is a pathogenic human herpesvirus associated with serious, potentially life-threatening symptoms in the immunocompromised or immunonaïve host. The limitations encountered by antiviral therapy options currently available include a narrow panel of accessible targets, the induction of viral drug resistance as well as severe drug dosage‑mediated side‑effects. Improved drug-targeting strategies to resolve these issues are the focus of our investigations. In particular, pharmaceutical kinase inhibitors (PKIs), either directed to host kinases or directed to the viral protein kinase pUL97, have been considered to overcome these restrictions. Recently, we reported the identification of a synergistic combination of two PKIs directed to host cyclin‑dependent kinase 7 (CDK7) and viral CDK ortholog pUL97. Here, we substantiate these findings with the following results: (i) true drug synergy was exhibited by various chemical classes of PKI pairs directed to pUL97 and CDK7; (ii) no putative amplification of cytotoxicity by these drug combinations was observed; (iii) a reduction in drug dosage levels for synergistic combinations was defined on a quantitative basis and compared to monotreatments; (iv) the quantities of target proteins CDK7 and pUL97 expressed in HCMV-infected cells were assessed by confocal imaging, indicating a strong down-modulation of CDK7 levels as a result of synergistic drug treatment; (v) the functional importance of these target kinases, both binding to cyclin H, was illustrated by assessing HCMV replication under the viral genomic deletion of ORF-UL97 or cellular cyclin knock-out; (vi) new combinations of HCMV-specific drug synergy were demonstrated for solely host-directed treatments using PKIs against CDK2, CDK7, CDK8 and/or CDK9 and (vii) a triple PKI combination provided further support for the synergy approach. With these combined findings, this study highlights the potential of therapeutic drug combinations of approved, developmental and preclinical PKIs for expanding future options for anti‑HCMV therapy.

Highly Conserved Interaction Profiles between Clinically Relevant Mutants of the Cytomegalovirus CDK-like Kinase pUL97 and Human Cyclins: Functional Significance of Cyclin H

“…However, at this point the question remained open whether the strong inhibition of viral replication under cyclin H KO conditions refers directly to pUL97–cyclin H interaction or is linked indirectly with cyclin H-depleted CDK7 deregulation. We previously showed that inhibition of CDK7 strongly inhibits HCMV replication, therefore representing a promising target for antiviral therapy [ 42 , 43 ]. To exclude the possibility that the growth defect induced by cyclin H KO is exclusively caused by indirect CDK7-mediated effects, we next investigated the primary impact of cyclin H on pUL97-specific kinase activity.…”

“…Secondly, this key passage of virus–host interaction may be accessible as a target point for mechanistically new antiviral drugs. Very recently, MBV/LivtencityTM, the selective inhibitor of pUL97 [ 43 ], has been approved for the therapy of HCMV infection and disease refractory to standard treatment [ 58 ]. The mode of MBV inhibition acts as a classical ATP-competitive binder that blocks essential functions within the pUL97 kinase domain.…”

The Oligomeric Assemblies of Cytomegalovirus Core Nuclear Egress Proteins Are Associated with Host Kinases and Show Sensitivity to Antiviral Kinase Inhibitors

“…Inhibitors of both kinase types, i.e., host CDKs, mostly CDK1/2/7/9, as well as the viral CDK ortholog pUL97 (vCDK), exert a strong antiviral effect against HCMV independent of viral strain and host cell type [ 8 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 ]. Moreover, a pronounced level of synergistic interaction has been proven for anti-HCMV drug combination treatments directed to vCDK/pUL97 and CDK inhibitors [ 33 , 34 ]. This finding is further illustrated by the fact that CDK1 and vCDK/pUL97 are able to phosphorylate both HCMV core NEC proteins pUL50 and pUL53 [ 8 , 35 , 36 ].…”

“…Here, we analyzed the putative effect of MBV in this assay. Based on the situation that viral pUL97, representing the target of MBV with proven selectivity [ 33 ], exerts major NEC-regulatory kinase activity [ 7 , 21 , 38 ], we assessed its inhibitory potency using the nuclear egress assay ( Figure 4 ). In fact, the analysis demonstrated a quantitative nuclear egress-inhibitory activity at a concentration of 5 µM in HCMV-infected primary fibroblasts ( Figure 4 A).…”

Abemaciclib restricts HCMV replication by suppressing pUL97-mediated phosphorylation of SAMHD1

“…Pharmacological CDKIs are compounds that impair the activity of CDKs, thus effecting the cell cycle, and they are developed and used as a treatment against various types of cancer [36][37][38] . Since viruses manipulate the cell cycle for efficient replication 34 , CDKIs have been studied as potential anti-viral treatment 17,20,[39][40][41][42][43][44][45] . The FDA approved CDK4/6 inhibitor Palbociclib for instance, is on the one hand used against breast cancer, but on the other hand shown to inhibit HSV-1 and HIV-1 17,44,45 infection in primary macrophages, while a CDK7 inhibitor, LDC4297 has antiviral activity against a broad range of herpesviruses, including HCMV 41,43 .…”