Structure Based Design of a Grp94-Selective Inhibitor: Exploiting a Key Residue in Grp94 To Optimize Paralog-Selective Binding

Grp94 and Hsp90, the ER and cytoplasmic hsp90 paralogs, share a conserved ATP-binding pocket that has been targeted for therapeutics. Paralog-selective inhibitors may lead to drugs with fewer side effects. Here, we analyzed <b>1</b> (BnIm), a benzyl imidazole resorcinylic inhibitor, for its mode of binding. The structures of <b>1</b> bound to Hsp90 and Grp94 reveal large conformational changes in Grp94 but not Hsp90 that expose site 2, a binding pocket adjacent to the central ATP cavity that is ordinarily blocked. The Grp94:<b>1</b> structure reveals a flipped pose of the resorcinylic scaffold that inserts into the exposed site 2. We exploited this flipped binding pose to develop a Grp94-selective derivative of <b>1</b>. Our structural analysis shows that the ability of the ligand to insert its benzyl imidazole substituent into site 1, a different side pocket off the ATP binding cavity, is the key to exposing site 2 in Grp94

Grp94/Hsp90 chimeras interact with Hsp90 co-chaperones.

<p>(A) Cartoon summary of Hsp90:cochaperone binding loci. (B) Ni-NTA pull-down assay shows that His₆-Cdc37 interacts with hspNM₁-grpM₂C and Hsp90 but not grpN-hspMC; His₆-Hop interacts with grpN-hspMC and Hsp90 but not hspNM₁-grpM₂C; His₆-Aha1 interacts with hspNM₁-grpM₂C, Hsp90, and partially with grpN-hspMC. Equal amounts of untagged chaperone and His-tagged cochaperone were mixed together and incubated for 1 h at 4°C followed by overnight incubation with Ni-NTA resin. The Load sample was removed prior to the addition of Ni-NTA resin. Unbound protein was removed by sequential washes of buffer containing 20 mM imidazole. Bound proteins and protein complexes were eluted with buffer containing 300 mM imidazole and resolved in parallel with the Load sample on SDS-PAGE gels. (C) hspNM₁-grpM₂C ATPase activity is stimulated 5-fold by Aha1. Wild-type Hsp90 is stimulated 10-fold whereas grp-hspMC is stimulated a modest 1.5-fold (D). ATPase activity was monitored by a NADH coupled enzymatic assay system, which measures the consumption of NADH as a function of ADP released by Hsp90s. Assays were measured with or without the addition of cochaperone Aha1 at a 1:2 molar ratio of hsp90:Aha1. Wild-type Aha1 alone did not exhibit ATPase activity (not shown).</p

Structure Based Design of a Grp94-Selective Inhibitor: Exploiting a Key Residue in Grp94 To Optimize Paralog-Selective Binding

Grp94 and Hsp90, the ER and cytoplasmic hsp90 paralogs, share a conserved ATP-binding pocket that has been targeted for therapeutics. Paralog-selective inhibitors may lead to drugs with fewer side effects. Here, we analyzed <b>1</b> (BnIm), a benzyl imidazole resorcinylic inhibitor, for its mode of binding. The structures of <b>1</b> bound to Hsp90 and Grp94 reveal large conformational changes in Grp94 but not Hsp90 that expose site 2, a binding pocket adjacent to the central ATP cavity that is ordinarily blocked. The Grp94:<b>1</b> structure reveals a flipped pose of the resorcinylic scaffold that inserts into the exposed site 2. We exploited this flipped binding pose to develop a Grp94-selective derivative of <b>1</b>. Our structural analysis shows that the ability of the ligand to insert its benzyl imidazole substituent into site 1, a different side pocket off the ATP binding cavity, is the key to exposing site 2 in Grp94

Chimeric constructs used in this study.

<p>Schematic representation showing the domain composition of Hsp90, Grp94, and the fragments used to create chimeras of Hsp90 and Grp94. Boundaries are shown with residue numbering for yeast Hsp90 and canine Grp94. The N-terminal chimeras referred to as grpN-hspMC (4), hspN-grpMC (3) and hspNM-grpM₂C (9) were previously described in Ref. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0166271#pone.0166271.ref020" target="_blank">20</a>].</p

Select Grp94 domains support yeast viability.

<p>(A) Plasmid shuffle experiments demonstrate grpN-hspMC and hspNM₁grpM₂C are able to support yeast viability when expressed as the sole Hsp90. Cells were plated on FOA plates as 5-fold serial dilutions and were grown at the indicated temperatures for 3–9 days. Images are from day 4. Green highlights indicate positive complementation. (B) Western blot of yeast whole cell lysates. 20 μg of total protein extracted from the lysis of plasmid-transformed yeast cells grown in SD–Ura -Trp was separated by SDS-PAGE. The expression level of plasmid-expressed hsp90 chimeras was evaluated by Western blotting against the N-terminal His₆ epitope tag. Lysate from Chimera 1 (yeast Hsp90) was included on each blot as a transfer and antibody control. Coomassie blue stained PVDF membranes used for blotting are displayed below their respective luminographs to allow comparison of total protein levels.</p

Domains and functional regions of hsp90 chaperones.

<p>The structure of Grp94 (PDB 2o1v) is shown. A) Schematic organization. B) Cartoon representation of the Grp94 dimer. C) surface representation of the Grp94 monomer.</p

Growth in liquid media of ECU82a expressing yeast Hsp90, grpN-hspMC, human Hsp90a, hspNM₁grpM₂C, and hspNM₁grpM₂C-hspCx as its sole Hsp90.

<p>Cultures were started at a cell density of 3.5 x 10⁶ cells per mL in YPD medium (time 0) and incubated with shaking at 30°C. Data represents an average OD₆₀₀ from two yeast clones.</p

Relative ATPase rates of Grp94/Hsp90 chimeras.

<p>Relative ATPase rates of Grp94/Hsp90 chimeras.</p

Solvent Accessible Surface Area of Domain Interfaces.

<p>Solvent Accessible Surface Area of Domain Interfaces.</p

GRP94 client expression with Grp94/Hsp90 chimeras.

<p>A) Schematic representation of constructs tested. All constructs contained the Grp94 signal sequence (ss), the preN-domain, and the KDEL ER retention sequence found in the Grp94 C-terminal extension (Cx). The FLAG tag is shown as a red bar. B) Detail of the FLAG tag incorporated into each tested construct. C) EGFP gating and intracellular staining of Grp94-null E4.126 cells virally transduced with Grp94 constructs. Top panel: dot plot of the side scattering intensity (SSC-A, Y-axis) of analyzed cells (a measure of cell size and clustering) vs. the fluorescence of the co-expressed EGFP (X-axis). The number of events is represented by the color of the dots. The dots in the magenta box correspond to EGFP positive cells and the numbers show the percentage of all cells that were assigned to this gate. Lower panel: Histogram of the normalized cell count vs. conjugated anti-FLAG (blue) or isotype control (red) fluorescence. Fluorescence intensity is proportional to the copy number of the antibody target. Numbers represent the mean fluorescence intensity of the blue histograms. D) Cell surface expression staining of Grp94 clients as a function of virally transduced Grp94 construct. Cells were gated for EGFP expression as in C). Red = isotype control antibody histogram, blue = anti-client antibody histogram. The mean fluorescence intensity of the blue histogram is indicated.</p