Fluorous Drug-Affinity Proteomics for Cancer Drug Discovery

Abstract

Identifying the intracellular targets of small molecules – target ID – is a major problem in chemical biology with broad application to the discovery and development of novel therapies. Traditional target ID studies have relied on drug-affinity chromatography to separate biological mixtures combined with mass spectrometry shotgun sequencing for peptide identification. This workflow is limited, however, by low specificity for unique peptides, high demand for cellular material, unknown depth of profiling, and other problems. To address these problems, we explore and describe here a novel strategy for cell lysis and drug-affinity that we call “fluorous proteomics.” By conjugating a small molecule to a perfluorinated alkane, we hypothesized that we could achieve superior recovery, specificity, and identification, allowing us to identify previously unknown drug targets with drug-affinity methods. We establish the conditions for fluorous proteomics and synthesize fluorinated probes for two drugs as a proof-of-concept. Lenalidomide, a derivative of thalidomide with unknown intracellular targets but widespread clinical use, is investigated and novel binders are identified. A particular derivative, 5HPP33, is singled out for potential future drug development. JQ1, an inhibitor of BET bromodomains in development as a treatment for hematological malignancies, is used to compare biotinylated versus fluorous tags and to identify new binders of possible therapeutic relevance. We conclude that fluorous proteomics retains high potential as an alternative to traditional drug-affinity chromatography strategies and may aid in target ID going forward, but is not without complications.