Chemically Induced Dimerization Systems for High Throughput Enzyme Assays

Abstract Enzymatic assays are critical for drug discovery: half of approved small molecule drugs inhibit enzymes, and enzymatic HTS (high throughput screening) assays are typically the most frequently used tool during development of a candidate molecule. Even as computational approaches become more prevalent, enzymatic

assays are required to test the activity of molecules following virtual screens and throughout the hit-to-lead process. Enzymes that produce small molecules can present a special challenge from an assay development perspective because selective detection of reaction products that closely resemble substrates in a homogenous

format may exceed the molecular recognition capabilities of known affinity reagents such as antibodies. Examples include epigenetic enzyme families currently under intense focus as therapeutic targets: histone acetyltransferases, sirtuins, and histone demethylases. To overcome these limitations, we will combine the vast ligand binding capabilities of native proteins with in vitro

selection to develop chemically induced dimerization (CID) systems for small molecule enzyme products. In Phase I, we will develop and validate a CID that is capable of detecting nanomolar concentrations of CoA (coenzyme A) in the presence of excess AcCoA (acetyl-CoA), providing a universal assay method for histone

acetyltransferases. Candidate CoA binding domains (BDCoA) will be selected from native proteins, and a library of randomized antibody variable domains will then be screened to identify second binding domains that recognize the CoA- BDCoA interface, providing a substantial enhancement of affinity and selectivity. The resulting CoA CID

will be formatted for TR-FRET and validated with purified HATs in pilot screens, enabling a robust universal HAT enzymatic activity with the sensitivity and specificity needed for HTS. In Phase II, we will develop CID systems for other small molecule enzyme products that have proven intractable to immunodetection, e.g., succinate for histone demethylases and O-acetyl-ADP-ribose for histone

deacetylases. The CID system will accelerate drug discovery by providing a very broad and flexible HTS enzyme assay platform for both established and emerging target families.