BellBrook Labs will be exhibiting at SLAS 2021, January 25-27. Due to the pandemic, this year’s meeting will, of course, be virtual. Although, we are sad that we won’t be able to meet with you in person this year there are still plenty of opportunities to interact. We will have a virtual booth that is all set up and ready to go. It includes some exclusive information and has a product showcase for our two latest products the Transcreener cGAMP TR-FRET cGAS Assay and the Transcreener UDP FI Glycosyltransferase Assay.
There will also be the opportunity to chat with BellBrook directly through video or text via the SLAS platform. This discussion can happen individually or with other attendees as well, just like a real conference! As always there are a variety of programs that SLAS puts on including more than 80 talks, workshops, plenary presenters, and of course posters. BellBrook Labs will present the following posters at the meeting.
Studying GALNT Activity with a Homogenous Transcreener UDP2 FI Glycosyltransferase Assay
Glycosyltransferase enzymes catalyze the transfer of sugar molecules to protein, lipid, and carbohydrate acceptors as well as endogenous and xenobiotic small molecules. More than 200 human glycosyltransferases are known to exist and have diverse metabolic and regulatory roles. Of those, at least 20 distinct GALNTs (Polypeptide N-Acetylgalactosaminyltransferases) are found in the Golgi. Since GALNTs catalyze the initial step of mucin-type O-glycosylation by transferring GalNac to Thr or Ser residues, these enzymes are targets for cancer interrogation. Multiple cancers have been directly linked to GALNT overexpression and dysregulation. Here we use the Transcreener UDP2 Assay with a new fluorescence intensity readout to monitor two glycosyltransferases GALNT2 and GALNT3.
The Transcreener UDP2 assay relies on selective immunodetection of UDP produced by glycosyltransferases that use UDP-sugars as donor substrates, including UDP-glucose, UDP-galactose, UDP-Glucuronic Acid, and in this case UDP-GalNac. Enzymatically generated UDP displaces a UDP-fluor tracer from an antibody-quencher conjugate, resulting in increased fluorescence. The assay is a single step, homogenous assay that is compatible with most multimode plate reader as well as simpler fluorescence plate readers. It can be used in endpoint or continuous modes, making it suitable for HTS as well as kinetic and/or mechanistic studies.
When studying GALNT2 and GALNT3, the assay produced sensitive UDP detection in the presence of UDP-GalNAc down to 100 nM UDP. The method was also used to show both enzymes’ activity under initial velocity conditions and exhibited robust HTS amenable data for GALNTs with a Z’ > 0.7. The Transcreener UDP2 FI Assay illustrated utility for screening campaigns, SAR, and kinetics studies to help further GALNT drug discovery.
Targeting Kinases Involved in the Innate Immune Response Using the Transcreener ADP2 Assay
Protein kinases play an important role in innate immunity signaling, both by transducing signals from pattern recognition receptors and by mediating the downstream effects of type I interferons and cytokines. Therapeutic modulation of these kinases is being investigated clinically to dampen the immune system in autoimmune diseases and to stimulate it for anti-viral or cancer immunotherapy. The use of a common biochemical platform for discovering and characterizing small molecule kinase inhibitors would facilitate these efforts. The Transcreener ADP2 Kinase Assay meets this need as it relies on direct detection of ADP, so it can be applied across diverse kinases, and it has been extensively validated for kinase discovery programs since 2007. Here, we describe how the Transcreener ADP2 Kinase Assay allowed rapid assay development to enable screening and dose-response measurements for five kinases involved in innate immunity: AMP-activated kinase (AMPK), Janus kinase 1 and 3 (JAK1,3), TANK Binding Kinase 1 (TBK1), and IκB Kinase (IKK).
The Transcreener ADP2 Kinase assay relies on selective immunodetection of ADP: enzymatically generated ADP displaces a fluorescent tracer from the antibody resulting in a change in fluorescent properties. The assay has been formatted for fluorescence polarization (FP), time-resolved FRET (TR-FRET), and fluorescence intensity (FI) readouts. A key step in assay development is optimizing the dynamic range of the assay based on the desired concentration of ATP, assuming 2-20% conversion of ATP to ADP. After choosing an ATP concentration close to the reported Km for each kinase, the concentration of ADP2 antibody was titrated to identify the EC80 concentration using reaction conditions specific for each kinase. Next, each kinase was titrated to identify the optimal concentration for producing a robust signal under initial velocity conditions. In some cases, we also performed some optimization of acceptor substrate(s), but these few steps are all that is required to develop an assay for most kinases. We measured Z’ values greater than 0.7 for all assays and dose-response measurements with known inhibitors yielded IC50 values consistent with literature reports. Initial assay development was done using the FP assay, but similar results were observed using the TR-FRET and FI assay. In summary, the Transcreener ADP2 assay provides a universal platform for biochemical kinase assays that should facilitate drug discovery programs targeting protein kinases involved in innate immunity.