In vitro activity: LDC4297 inhibits CDK7 in vitro in the nano-picomolar range. The affinity of LDC4297 for CDK7 proves to be extremely high. The replication of HCMV in cultured primary human fibroblasts (HFFs) is inhibited by LDC4297 in a concentration-dependent manner with a 50% effective concentration (EC50) value of 24.5 ± 1.3 nM. Notably, CDK7 inhibition by LDC4297 is not associated with general cytotoxicity at submicromolar concentrations. In contrast, LDC4297 induces cytotoxicity in a set of tumor cell lines, i.e., already at extremely low, nanomolar concentrations in specific cases. Anti-HCMV activity of LDC4297 is exerted through a multifaceted mode of action that involves an interference with virus-induced Rb phosphorylation.

Kinase Assay: LDC4297 is a novel and potent CDK7 (Cyclin-dependent protein kinase 7) inhibitor with IC50 of 0.13±0.06 nM for CDK7 and IC50s between 10 nM and 10,000 nM for all other analyzed CDKs. Cyclin-dependent protein kinase 7 (CDK7) plays crucial regulatory roles in cell cycle and transcription, both connected with the replication of many viruses. The affinity of LDC4297 for CDK7 proves to be extremely high. The replication of HCMV in cultured primary human fibroblasts (HFFs) is inhibited by LDC4297 in a concentration-dependent manner with a 50% effective concentration (EC50) value of 24.5 ± 1.3 nM. Notably, CDK7 inhibition by LDC4297 is not associated with general cytotoxicity at submicromolar concentrations. Summarized, the CDK7 inhibitor LDC4297 is a promising candidate for further antiviral drug development, possibly offering new options for a comprehensive approach to antiviral therapy.

Cell Assay: A trypan blue exclusion assay is performed with cultured cells seeded in 24-well plates and incubated with increasing concentrations of antiviral compounds (range, 0.1 to 50 μM) for the durations indicated. Cell staining is achieved with 0.1% trypan blue for 10 min at room temperature before the percentage of viable cells is determined by microscopic counting.