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. Author manuscript; available in PMC: 2024 Aug 3.
Published in final edited form as: Structure. 2023 Jun 20;31(8):987–1004.e8. doi: 10.1016/j.str.2023.05.017

Figure 2. Biochemical and functional evaluation of GRP94 ligands for biological activity via Glyc62GRP94.

Figure 2.

(a) Chemical structure of GRP94 small molecule ligands. EC50GRP94 and EC50HSP90, relative binding affinity constant to recombinant GRP94 and HSP90α, respectively. IC50MDA-MB-468, half maximal inhibitory concentration determined in the MDA-MB-468 EGFR-overexpressing and Glyc62GRP94-expressing breast cancer cells (ATP quantitation assay). Values, mean of three independent experiments.

(b) Schematic showing the conformational changes induced in GRP94 expected from small molecule ligands that act on Glyc62GRP94.

(c,d) Immuno-capture as in (b) of SKBr3 HER2-overexpressing or MDA-MB-468 EGFR-overexpressing (both Glyc62GRP94-expressing) breast cancer cells treated with the indicated ligands in a dose- (for 4 h) and time-dependent (at 2.5 μM) manner. Experiments were repeated trice with similar results.

(e) Western blot analysis for inhibition of Glyc62GRP94 function in MDA-MB-468 cancer cells. Downstream signaling (p-ERK/ERK) and induction of apoptosis (cleaved PARP, cPARP) were analyzed for MDA-MB-468 cancer cells treated for 24 h with 0, 0.5, 1, 2.5, 5 and 10 μM of the indicated inhibitors. GRP94, loading control. Gels are representative of three independent results. See also Figure S1.