Hit Validation for Small Molecule Binding Assays on Octet RED

Frank Podlaski, Senior Principal Scientist, Roche Discovery Technologies
Charles Wartchow, Director, Applied R&D, ForteBio Inc.

In the drug discovery process, primary screening of compound libraries with a validated therapeutic protein target generates numerous hits, including false positive results due to non-specific binding of compounds to assay system components as well as assay artifacts associated with the compounds’ physical properties. As a result, validation of the results with a secondary assay is required to verify compound binding to the target protein, and more importantly to determine if the interaction is truly stoichiometric. At the 2008 Society for Biochemical Sciences Symposia Label-free Technologies in Drug Discovery in Dresden, Germany, the use of the Octet RED for hit validation was described.

The Octet RED is an 8-channel instrument that is well-suited for the determination of binding kinetics for small molecules in a 96-well plate format. Advantages of this format include ease of use, fast assay development, the ability to identify aggregating and reactive compounds, and the ability to assay complex or insoluble analytes. In a typical experiment, the binding of compounds in TBS buffer containing 1 mM DTT, 0.005% P20, and 5% DMSO is measured on a disposable fiber-optic tip labeled with the protein target, and the binding events are compared to data from reference sensors without the target. For kinetic determinations, system drift is removed using data from one of the target sensors that obtains responses in buffer only.

To evaluate the Octet RED for hit validation, the interaction of a 16 kDa target protein with test compounds including non-binding compounds, an identified hit, and a reference were analyzed and compared to data from the Biacore S51 instrument. The results obtained on Octet RED were reproducible; the kinetic parameters KD, kon, and koff matched the results obtained on the Biacore instrument. Next, a panel of 35 compounds was analyzed on the Octet RED at 10 and 50 µM, and hits showing proper behavior and a differential response were successfully selected for full dose response assays for KD determination. Non-binders, irreversible binders, and non-specific binders were identified and rejected. Additionally, with the Octet RED, it was also possible to distinguish compounds that show secondary binding events which mimicked the profile seen with the Biacore S51 instrument. These results demonstrate the capability of the Octet RED platform for hit validation in the drug discovery process.