Affinity Chromatography Mass Spectrometry Assays For Small Molecule Screening

Abstract

Enzymes are implicated in many diseases including neurodegenerative, cancer, immune deficiency, and inflammatory disorders. There is a constant need to develop novel drug compounds that target enzymes in order to modulate their function, thus treating the disease state. These compounds are typically small molecules with affinity to the enzyme active site or an allosteric site. In order to discover novel compounds for treating disease, the interaction between an enzyme and a small molecule must first be identified and then characterized. With the target enzyme known, it is beneficial to screen libraries of compounds against the target. Immobilizing the enzyme allows for pre-concentration of ligands on the surface and therefore increased signal enhancement, as well as permitting multiple wash steps and enzyme reuse. Immobilized enzyme columns are optimal for coupling to a variety of detection devices by way of liquid chromatography, including absorbance or mass spectrometric detection. Immobilized enzyme reactors (IMERs) were generated and optimized for two target molecules, acetylcholinesterase (AChE) and adenosine deaminase (ADA), for rapid function-based screening of enzyme inhibitors in mixtures. The IMER mode is useful for increasing throughput and facilitating the identification of hit mixtures, but it is slow and tedious to manually deconvolute hit compounds from mixtures and the IMER method is not amenable to natural product extracts, which are good sources of structurally diverse compounds that are more likely to result in a hit compound. Bio-selective solid-phase extraction (BioSPE) is an orthogonal method of isolating and identifying enzyme inhibitors in a single step, and was used to easily deconvolute complex mixtures, rapidly identifying to key compounds EHNA and MAC-0038732 out of mixtures using ADA columns. A data dependent acquisition MS method was developed and used to screen a set of fungal endophyte extracts, identifying two potentially novel inhibitors that were confirmed by IMER-MS/MS.