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The Longest Way Round is the Shortest Way Home: HTS Assay Development for Complex Multi-domain Protein Kinases

Lead discovery programmes often rely on recombinant proteins encompassing solely their catalytic domain. However, the catalytic activity of multi-domain enzymes is modulated by ancillary regions, which provide potential and underexplored allosteric sites to target the enzymatic activity.
To address this issue, we applied our expertise in recombinant protein production in support to HTS on two multi-domain protein kinases: Citron Rho-Interacting Kinase and Traf2- and Nck-Interacting protein Kinase. CIT is a 2017-aa protein used in compound testing as 449-aa kinase domain version, while TNIK is a 1360-aa protein produced as 367-aa version encompassing exclusively the catalytic domain. To support drug discovery programs with longer kinase versions, we expressed different recombinant forms of CIT and TNIK in insect cells with the baculovirus system and we identified the permissive conditions to obtain them in the active form. CIT was successfully expressed as 1461-aa version (170 kDa), while TNIK was expressed as full-length enzyme (160 kDa). Their catalytic activity was configured with a HTS-suitable fluorescence-based assay in 384 well/plate format. Effective substrates for both kinases were identified by screening a library of peptides and the kinetic properties of the enzymatic reactions were fully characterized. Finally, the respective expression-purification procedures were up-scaled to support HTS campaigns on 2,000,000 compounds, leading to homogeneous batches of active kinases.