Fluorescent-Linked Enzyme Chemoproteomic Strategy (FLECS) for Identifying HSP70 Inhibitors.

Activation of the heat shock response, and in particular upregulation of stress-inducible Hsp70, herein referred to as Hsp70i, in newly transformed cells, appears to protect against protein damaging stimuli, induction of premature oncogene-induced terminal senescence (OIS), and apoptosis, thereby enabling tumor initiation and progression to an aggressive phenotype. Expressed at very low or undetectable levels in normal tissue, the cytoprotective effects of Hsp70i appear to be mediated through its activity as a molecular chaperone allowing proper folding of mutated proteins, and by blocking cell signaling pathways that regulate OIS and apoptosis. Identification of small-molecule inhibitors selective for Hsp70i could provide new therapeutic tools for cancer treatment. However, identification of selective inhibitors of Hsp70i has proven challenging largely because of the affinity of the protein for ATP. Additionally, its chaperone functions do not lend the protein amenable to traditional enzymatic high-throughput screens. Here, we describe the use of fluorescence-linked enzyme chemoproteomic strategy (FLECS) to identify Hsp70i inhibitors. The FLECS assay is a simple binding assay that enables proteins tagged with fluorophors to be rapidly and quantitative screened against small-molecule libraries. We show several case history examples of the methodology that led to the discovery of the Fatty acid synthase inhibitor, FASNALL, the DAPK3 inhibitor HS38, and HS72, an allosteric inhibitor selective for Hsp70i.