Biological Evaluation of 5′-(N-Ethylcarboxamido)adenosine Analogues as Grp94-Selective Inhibitors

Abstract

The heat shock protein 90 kDa (Hsp90) family of chaperones is highly sought-after for the treatment of cancer and neurodegenerative diseases. Glucose regulated protein 94 (Grp94) is the endoplasmic reticulum localized isoform that is responsible for the maturation of proteins involved in cell adhesion and the immune response, including Toll-like receptors, immunoglobulins, and integrins. Consequently, Grp94 has been implicated in many different diseases including cancer metastasis, glaucoma, and viral infection. 5′-(N-Ethylcarboxamido)­adenosine (NECA) was identified from a high-throughput screen as one of the first molecules to exhibit isoform selectivity toward Grp94, with the ethyl group projecting into a unique pocket within the ATP binding site of Grp94. This pocket has since been exploited by several groups to develop Grp94 selective inhibitors. Despite success in the development of other classes of inhibitors, relatively little work has been done to further develop inhibitors with the NECA scaffold. Unfortunately, NECA is also a potent adenosine receptor agonist, which is likely to confound any biological activity. Therefore, structure–activity relationship studies were performed on the NECA scaffold leading to the discovery of several molecules that displayed similar selectivity and affinity as the parent compound.

How AberrantN-Glycosylation Can Alter Protein Functionality and Ligand Binding: an Atomistic View

“…We found most ligands exhibit a comparable binding affinity for recombinant GRP94 under conditions of equilibrium binding (Figure 1a, see EC50 rGRP94, 120-450 nM for the PU-series and 1.2 µM for BnIm, as reported 15,27,30,31 . Remarkably, even in the structurally closely related PU-type ligands, some but not all, were active in cancer cells dependent on Glyc62 GRP94 for survival, even at the highest tested concentration of 10 µM, which is 1-to 2-log higher than their recorded binding affinity constant (Figure 1a).…”

“…For example, some but not all are toxic to cancer cells characterized and driven by Receptor Tyrosine Kinase (RTK)-overexpression (eg. HER2- and EGFR-overexpressing breast cancer cells) 27,30,31 . In these cells, the N62 glycosylated GRP94 variant (referred to further as Glyc62 GRP94) is enriched at the plasma membrane, where it acts to reduce RTK internalization and to maintain the RTK in a state that is competent for constitutively enhanced downstream signaling 15 .…”

“…We tested each ligand in a battery of assays designed to distinguish active ligands from those inactive on Glyc62 GRP94-related biological functions (see Figure 1a-e and Supplementary Figure 1a-c ). We contrasted these ligands and their activity to BnIm, a ligand reported to productively engage GRP94 in cells, and modulate GRP94-but not Glyc62 GRP94-related functions 15,27,30,31 .…”

“…N-ethylcarboximinoadenosine, cytosine-containing adenosine analogs, PU-WS13 and others) [24][25][26] , resorcinol type compounds (eg. BnIm and KUNG94) and benzamide scaffold derivatives [27][28][29] . These compounds, while tight binders of recombinant GRP94 protein, elicit non-overlapping biological profiles when tested in a variety of disease models.…”

Current Adenosinergic Therapies: What Do Cancer Cells Stand to Gain and Lose?

“…Later, the same research group reported that knockdown of GRP78 facilitated anti-tumour effects of ADO in HepG2 cells [171]. In addition, the non-selective AR agonist NECA directly bound and inhibited GRP94, another GRP member localized in the ER [172]. Interestingly, a growing body of evidence suggests that ER stress signals regulate various downstream pathways (PI3K/AKT, NF-κB, MAPK/ERK, TGF-β, and Wnt/β-catenin) and that these effects are mediated by GPCRs; for a summary, see [173].…”