Uncage hijacked kinases from SARS-CoV-2 interactions

Kinases are versatile signaling nodes and thus represent a central target for viruses to reprogram the host cell for their needs. SARS-CoV-2 efficiently hijacks this crucial cellular control mechanism for ensuring their optimal propagation. We assume that targeting druggable kinase activities may alter SARS-CoV-2:host-protein interactions and functions. We bear the hope that targeting a hijacked host kinase may reduce virus assembly and/or pathological implications for cellular functions. We aim to validate and chemically target interdependencies emanating from two kinase cascades with viral proteins directly in human pulmonary cell lines. First, we apply a cell-based kinase conformation (KinCon) reporter for determining enzyme activity changes upon transient and viral expression of selected viral SARS-Cov-2 proteins. Second, we define the impact of kinase-directed small molecules (activators/inhibitors) on cellular SARS-CoV-2 functions. Third, we focus on modulating the cAMP-PKA and MAPK pathways to determine consequences on the effectiveness of virus assembly and function. Vaccines for combating SARS-CoV-2 infections save thousands of lives. Mutations may hamper their efficacies. We bear the hope that targeting host kinases may become part of COVID-19 and mutation-decoupled therapy approaches.

In this AKUT project, we investigated how human protein functions and SARS-CoV-2 biology are interlinked in distinct human cell lines using a cell-culture-based approach. The project was carried out in collaboration between the Medical University of Innsbruck (MUI) and the University of Innsbruck (UIBK), with a central focus on how small molecules modulate enzyme activity and thereby directly or indirectly influence viral replication and related cellular processes. Building on our earlier work on kinase functions, mutations, and modes of blockade, we gained new insights into how small molecules alter cell and host cell functions through unexpected on- and off-target effects. An initial outcome of the project at the beginning was the identification of two small molecules that alter enzyme functions and affect the SARS-CoV-2 replication cycle. The first target: previous findings suggested that the small molecule kinetin acted through kinase-related functions, including the PINK/Parkin mitophagy pathway, which we then analyzed in depth. Later evidence from another group indicated that its antiviral effect is likely independent of kinase activation and may instead involve RNA polymerase activity. This finding redirected the project toward a deeper analysis of on-target and off-target effects of kinase blockers and molecular interactions of SARS-CoV-2 proteins and of host enzymes. The second target: to empower the unveiling of drug candidate specificities, we expanded our enzyme activity conformation reporter platform to include both kinase complexes and viral MPro. Moreover, we established additional readouts to study MPro activity, mutation effects, and drug resistance. These assays allowed us to monitor specificities and efficacies of well and less validated small molecules. In parallel, we placed strong emphasis on the off-target effects of host-directed compounds, particularly their impact on mitochondrial functions, phosphorylation dynamics, and the metabolome. In this context, a newly established PINK/Parkin activity platform opens new avenues for identifying internal and external triggers of mitophagy. Another major achievement of the MUI team was the optimization and validation of SARS-CoV-2 infection assays. An optimized VSV-based pseudovirus assay was established which resolved a previously observed discrepancy between authentic virus and pseudovirus readouts.