Flory, E. | Search bioRxiv

bioRxiv2025

Development of a chemical probe to enable characterization of the casein kinase 1γ subfamily

The casein kinase 1{gamma} (CK1{gamma}) subfamily, while severely understudied, is implicated in diverse disease-relevant pathways, including WNT signaling and human cytomegalovirus (HCMV) replication. While genetic tools exist to study CK1{gamma}, the selective inhibition of CK1{gamma} through pharmacological means remains underexplored. Chemical probes, or potent and selective inhibitors, remain one of the most powerful pharmacological tools for uncovering protein biology. Herein, we developed several novel assays for assessing target engagement with the CK1{gamma} subfamily in cells. Enabled by these assays, we conducted a comprehensive structure-activity relationship (SAR) campaign to develop the first chemical probe, SGC-CK1{gamma}-1, for the CK1{gamma} subfamily. SGC-CK1{gamma}-1, which was developed alongside a structurally related negative control compound, potently and selectively inhibited the CK1{gamma} kinases in living cells, plus inhibited both WNT signaling and human cytomegalovirus replication.

pharmacology and toxicology↗

bioRxiv2020

Quantitative Assays Reveal Cell Fusion at Minimal Levels of SARS-CoV-2 Spike Protein and Fusion-from-Without

Cell entry of the pandemic virus SARS-CoV-2 is mediated by its spike protein S. As main antigenic determinant, S protein is in focus of antibody-based prophylactic and therapeutic strategies. Besides particle-cell fusion, S mediates fusion between infected and uninfected cells resulting in syncytia formation. Here we present quantitative assay systems covering not only particle-cell and cell-cell fusion, but also demonstrating fusion-from-without (FFWO), the formation of syncytia induced by S-containing viral particles in absence of newly synthesized S protein. Based on complementation of split {beta}-galactosidase and virus-like-particles (VLPs) displaying S protein, this assay can be performed at BSL-1. All three assays provided readouts with a high dynamic range and signal-to-noise ratios covering several orders of magnitude. The data obtained confirm the enhancing effect of trypsin and overexpression of angiotensin-converting enzyme 2 (ACE2) on membrane fusion. Neutralizing antibodies as well as sera from convalescent patients inhibited particle-cell fusion with high efficiency. Cell-cell fusion, in contrast, was only moderately inhibited despite requiring much lower levels of S protein, which were below the detection limit of flow cytometry and Western blot. The data indicate that syncytia formation as a pathological consequence in tissues of Covid-19 patients can proceed at low levels of S protein and may not be effectively prevented by antibodies.

microbiology↗