Cell-Impermeable Hsp90α-Selective Inhibitor NDNA4 Inhibits Cancer Cell Migration and Targets Extracellular Hsp90α's...

Hsp90α is an isoform of the heat shock protein 90 (Hsp90) family of molecular chaperones that mediates the folding and activation of ∼400 client proteins. In addition to its intracellular function, Hsp90α is secreted extracellularly (eHsp90α) and has been shown to modulate processes such as cell motility, inflammation, and wound healing. We previously developed the cell-impermeable and Hsp90α-selective inhibitor, NDNA4. NDNA4 manifested weak antiproliferative activity against various cancer cell lines as assessed by an MTS assay. In addition, NDNA4 inhibited cancer cell invasion at nontoxic concentrations and diminished eHsp90α-activated signaling pathways. A key dual-lysine motif located at the charged linker of Hsp90α, termed the F-5 fragment, is the only necessary portion of eHsp90α required for biological activity including induction of cell migration. Interestingly, NDNA4 inhibited both Hsp90α and F-5-induced cancer cell migration despite only being reported to bind the Hsp90α N-terminal ATP-binding site. Synthesis of a biotinylated analogue, NDNA Biotin, allowed pull-down studies to be conducted, which provided evidence that NDNA4 binds F-5 and revealed that the Hsp90α-selective core is required for this interaction. Circular dichroism experiments revealed that NDNA4 binding induces a decrease in the α-helical character of F-5, indicating that a conformational change takes place upon binding. Furthermore, surface plasmon resonance showed NDNA4 dose-dependently binds F-5 with a K_D = 2.66 ± 1.36 μM. Collectively, these results indicate the existence of a previously unrecognized binding site that may be therapeutically relevant. Small-molecule inhibitors that can inhibit eHsp90α's contribution to cancer progression and metastasis represent a new opportunity in drug discovery.