The Longest Way Round is the Shortest Way Home: HTS Assay Development for Complex Multi-domain Protein Kinases
Poster Oct 10, 2014
Doris Hafenbrandl, Vanessa Nardese, Maria Cristina Sidoli, Valeria Wanke, Mariantonietta Rubino and Daniele Carettoni
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.
P450 Induction in Cryopreserved Hepatocytes from PXR and CAR Nuclear Receptor Knock-out RatsPoster
The nuclear receptors pregnane X receptor (PXR) and constitutive androstane receptor (CAR) are closely related transcription factors that regulate the expression of phase I (cytochrome P450s), phase II metabolizing enzymes and transporter genes in response to xenobiotics, including prescription drugs.READ MORE
Identifying Non-Intentionally Added Substances (NIAS) in Food Contact MaterialsPoster
Concern about the safety of food products has increased dramatically with intentionally and non-intentionally added substances (NIAS) used in packaging being of particular interest. APGC and high resolution MS together with a workflow driven process was used to identify unknown compounds in packaging.READ MORE
Human exposure to cyanotoxins: Exploring in vitro detoxification using atmospheric cold plasma treatment to protect human healthPoster
Recently, there have been increases in freshwater harmful algal blooms. There is therefore a need to try and effectively detoxify drinking water contaminated with cyanotoxins to safeguard human health by use of new and novel techniques.READ MORE