NECA derivatives exploit the paralog-specific properties of the site 3 side pocket of Grp94, the endoplasmic reticulum Hsp90

Abstract

The hsp90 chaperones govern the function of essential client proteins critical for normal cell function as well as cancer initiation and progression. Hsp90 activity is driven by ATP, which binds to the N-terminal domain and induces large conformational changes that are required for client maturation. Inhibitors targeting the ATP-binding pocket of the N-terminal domain have anticancer effects, but most bind with similar affinity to cytosolic Hsp90α and Hsp90β, endoplasmic reticulum Grp94, and mitochondrial Trap1, the four cellular hsp90 paralogs. Paralog-specific inhibitors may lead to drugs with fewer side effects. The ATP-binding pockets of the four paralogs are flanked by three side pockets, termed sites 1, 2, and 3, which differ between the paralogs in their accessibility to inhibitors. Previous insights into the principles governing access to sites 1 and 2 have resulted in development of paralog-selective inhibitors targeting these sites, but the rules for selective targeting of site 3 are less clear. Earlier studies identified 5'N-ethylcarboxamido adenosine (NECA) as a Grp94-selective ligand. Here we use NECA and its derivatives to probe the properties of site 3. We found that derivatives that lengthen the 5' moiety of NECA improve selectivity for Grp94 over Hsp90α. Crystal structures reveal that the derivatives extend further into site 3 of Grp94 compared with their parent compound and that selectivity is due to paralog-specific differences in ligand pose and ligand-induced conformational strain in the protein. These studies provide a structural basis for Grp94-selective inhibition using site 3.

Keywords: Grp94; HSP90B1; X-ray crystallography; allosteric regulation; chaperone; endoplasmin; gp96; heat shock protein 90 (Hsp90); inhibition mechanism; isothermal titration calorimetry (ITC); medicinal chemistry; paralog selectivity.

Figure 1.

Side pockets of the ATP-binding cavity in the Grp94 NTD. Residue numbers of side pocket side chains are indicated.

Figure 2.

NECA and derivatives used in this study. Binding pockets of scaffold and substituent moieties in Hsp90 and Grp94 are indicated schematically.

Figure 3.

ITC analysis of NECA and derivatives binding to Grp94 and Hsp90. Titrations were carried out at 25 °C. Calculated dissociation constants are given on each thermogram. Errors in K_ds are standard error of the mean of two replicate measurements.

Figure 4.

FP binding assay of NECA and derivatives. A and B, the assay contained 10 nm full-length hHsp90α (A) or Grp94 (B) and 6 nm geldanamycin-Cy3b tracer. Graphs represent the average of two independent measurements.

Figure 5.

NECA and derivatives occupy site 3 in Grp94. A, overlay of Grp94:NECA (PDB code 1QY5), Grp94:NPCA, Grp94:NEoCA, and Grp94:NeaCA, showing close overlap of binding pocket residues and ligands. Carbon atoms: yellow, NECA; blue, NPCA; green, NeoCA; magenta, NEaCA. B, surface representation of the site 3 binding pocket. C, the 5′ moieties of NPCA, NEoCA, and NEaCA adopt a gauche conformation at their ends. D, shown in the same orientation as C; the 5′ moiety of NECA adopts an anti conformation.

Figure 6.

NECA and derivatives bound to Hsp90. A, comparison of NECA poses when bound to Hsp90 (NECA^Hsp) and Grp94 (NECA^Grp). The Hsp90-binding pocket is shown. B, modeling NECA^Grp into Hsp90, showing a potential clash with the δ carbon and the carbonyl oxygen of Gly-135. C, structure of Hsp90:NPCA. Two rotamers for the side chain of Tyr-139 were observed in the crystal structure. D, structure of Hsp90:NEoCA. The ϵ oxygen of NEoCA rotates to avoid a clash with Tyr-139, obviating the need to adopt the distal rotamer. E, structure of Hsp90:NEaCA. The ϵ nitrogen adopts two poses and forms an extensive hydrogen bond network with the site 3 binding pocket.