Abstract
The identification of modulators for proteins without assayable biochemical activity remains a challenge in chemical biology. The presented approach adapts a high-throughput fluorescence binding assay and functional chromatography, two protein-resin technologies, enabling the discovery and isolation of fluorescent natural product probes that target proteins independently of biochemical function. The resulting probes also suggest targetable pockets for lead discovery. Using human survivin as a model, we demonstrate this method with the discovery of members of the prodiginine family as fluorescent probes to the cancer target survivin.
Highlights
The identification of modulators for proteins without assayable biochemical activity remains a challenge in chemical biology
A series of studies including those by our team (Chapman and La Clair)[5] demonstrated that one can employ resin-bound protein as a vehicle to enrich for molecules that bind to a protein of interest
Using a complex mixture of molecules, this method represents a form of reversed-affinity or ‘functional’ chromatography (FC).[6]
Summary
The resulting resins were presented with the fluorescent extracts (step 2, Scheme 1) and evaluated for fluorescence using CONA and image analysis (BREAD).[8,9] Two hits were observed as given by the appearance of fluorescent halos. We confirmed we captured the desired fluorescent material by screening the survivin-bound fractions from the FC resin with the CONA resin (steps 3–4, Scheme 1). The assignment of 1 was further validated by high-resolution mass spectrometry (HRMS) returning m/z for C23H30N3O, [M + H]+ calculated: 364.2383; found: 364.2382 Full characterization of this extract was conducted.[14] While 1 was clearly evident in the FC resin-bound fraction (Fig. 3b), we observed unsaturated fatty acid signatures (*, Fig. 3a) within this fraction, a common non-specific protein binder found during FC.[5]. Such outcome produces a rather exciting proof of concept, while potentially revealing a new binding pocket for this IAP
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