Tracy J Worzella

Optimizing Kinase Assays for Ultrahigh-Throughput Profiling using the Kinase-Glo plus Assay

Here we demonstrate the concept of kinase profiling using a panel of four kinases and 10 kinase inhibitors. We used the Kinase-Glo Plus Luminescent Kinase Assay and the Deerac Fluidics Equator HTS noncontact liquid-handling system to design and perform an ultrahigh-throughput profiling screen. We present IC50 values for each test compound and show how such profiling can streamline the initial drug discovery process.

Abstract 5532: A pro-fluorescent membrane integrity dye for the real-time assessment of cytotoxicity.

Cell-based assay models are now universally employed as a routine means of establishing and ranking on- and off-target toxicities. It is well appreciated, however, that individual cytotoxic phenotypes are shaped not only by compound dosage, but by the length of compound-cell exposure. In addition to compound-specific attributes, inherent cellular factors such as biotransformation capacity, cell cycle susceptibility, and receptor expression often dictate the kinetics of cytotoxicity. Unfortunately, the majority of conventional cytotoxicity assays are performed at a terminal endpoint (48-72h) which at worst can underestimate cytotoxicity due to biomarker degradation, or at best, fail to reveal important features of the cytotoxic event which may be important for establishing mechanism-of-action. We have developed a pro-fluorescent, cell-impermeant probe which can be applied to cells at the time of dosing to report changes in membrane integrity as they occur in real-time. The dye enters cells with impaired membrane integrity, greatly enhancing fluorescence which is proportional to non-viable cell number. The dye can be used in traditional plate based formats using standard fluorometry or with image based readers (Essen Bioscience Incucyte™) to establish the dose-dependent kinetics of cytotoxicity. Here we describe the dye9s utility using mechanistically distinct cytotoxins with both cancer cell lines (on-target) and terminally differentiated cells (off-target) and correlate this dead cell fluorescence activity to other cytotoxicity biomarkers by same-well multiplexed methods. Citation Format: Richard L. Somberg, Andrew Niles, Tracy Worzella, Dan Lazar. A pro-fluorescent membrane integrity dye for the real-time assessment of cytotoxicity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5532. doi:10.1158/1538-7445.AM2013-5532

A Flexible Workflow for Automated Bioluminescent Kinase Selectivity Profiling.

Kinase profiling during drug discovery is a necessary process to confirm inhibitor selectivity and assess off-target activities. However, cost and logistical limitations prevent profiling activities from being performed in-house. We describe the development of an automated and flexible kinase profiling workflow that combines ready-to-use kinase enzymes and substrates in convenient eight-tube strips, a bench-top liquid handling device, ADP-Glo Kinase Assay (Promega, Madison, WI) technology to quantify enzyme activity, and a multimode detection instrument. Automated methods were developed for kinase reactions and quantification reactions to be assembled on a Gilson (Middleton, WI) PIPETMAX, following standardized plate layouts for single- and multidose compound profiling. Pipetting protocols were customized at runtime based on user-provided information, including compound number, increment for compound titrations, and number of kinase families to use. After the automated liquid handling procedures, a GloMax Discover (Promega) microplate reader preloaded with SMART protocols was used for luminescence detection and automatic data analysis. The functionality of the automated workflow was evaluated with several compound-kinase combinations in single-dose or dose-response profiling formats. Known target-specific inhibitions were confirmed. Novel small molecule-kinase interactions, including off-target inhibitions, were identified and confirmed in secondary studies. By adopting this streamlined profiling process, researchers can quickly and efficiently profile compounds of interest on site.

Abstract 3883: Miniaturizing the GloSensor™ cAMP assay for MC4R screening using the Echo® acoustic liquid handler

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL As biological assays used for HTS and uHTS are miniaturized, it is critical to maintain assay sensitivity while minimizing reagent volumes and maximizing throughput. The GloSensor™ cAMP Assay provides an extremely sensitive and easy to use, real-time luminescent assay format for the interrogation of over expressed or endogenous GPCRs that signal via changes in the intracellular concentration of cAMP. Tipless, touchless transfers with the Echo® liquid handler eliminate the need for costly disposable tips and greatly simplify assay development efforts. Precise and accurate drop placement eliminates cross-contamination. In this work, we demonstrate optimization of the GloSensor cAMP Assay in low-volume format using a stably transfected HEK293 cell line model expressing the melanocortin 4 receptor (MC4R) and a cAMP-sensing variant of firefly luciferase. We show that the Echo liquid handler is able to titrate small molecule and peptide agonists and antagonists, as well as transfer the GloSensor cAMP Reagent, providing robust assay results that are achieved with significantly reduced volumes of cells and compound. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3883. doi:1538-7445.AM2012-3883