Different Poses for Ligand and Chaperone in Inhibitor Bound Hsp90 and GRP94: Implications for Paralog-specific Drug Design

Hsp90 chaperones contain an N-terminal ATP binding site that has been effectively targeted by competitive inhibitors. Despite the myriad of inhibitors, none to date have been designed to bind specifically to just one of the four mammalian hsp90 paralogs, which are cytoplasmic Hsp90α and β, ER GRP94, and mitochondrial Trap-1. Given that each of the hsp90 paralogs is responsible for chaperoning a distinct set of client proteins, specific targeting of one hsp90 paralog may result in higher efficacy and therapeutic control. Specific inhibitors may also help elucidate the biochemical roles of each hsp90 paralog. Here we present side by side comparisons of the structures of yeast Hsp90 and mammalian GRP94, bound to the pan-hsp90 inhibitors Geldanamycin and Radamide. These structures reveal paralog specific differences in the Hsp90 and GRP94 conformations in response to Geldanamycin binding. We also report significant variation in the pose and disparate binding affinities for the Geldanamycin-Radicicol chimera Radamide when bound to the two paralogs, which may be exploited in the design of paralog-specific inhibitors

The dependence of LpxH for growth is abrogated by inhibition of LpxC under standard laboratory conditions.

<p>(A) NB48062-JWK0133 was streaked on MHIIB agar supplemented with 1 mM IPTG and grown overnight at 37°C to induce <i>lpxH</i> expression. The following day, cells were washed repeatedly and resuspended to an OD₆₀₀ of 0.01, and 100 μL was plated on MHIIB plates without IPTG. Sterile filter discs containing IPTG, DMSO, or CHIR-090 were placed on the plates which were then incubated 37°C for 24 hours. Left panel; growth of NB48062-JWK0133 was not observed under non-inducing conditions (minus IPTG, DMSO). Center panel; growth of NB48062-JWK0133 is restored in the presence of IPTG. Right panel; NB48062-JWK0133 grew under non-inducing conditions in the presence of the LpxC inhibitor CHIR-090. (B) An overnight culture of NB48062-JWK0133 under inducing conditions (+ IPTG) was diluted to an OD₆₀₀ of 0.1 and then was diluted 100-fold into MHIIB containing 10% Alamar Blue. Next, 100 μL of the inoculum was added to the wells of a 96-well plate containing CHIR-090 to a final assay concentrations ranging from 0.25–64 μg/ml. The plate was incubated for 6 hours at 37°C before fluorescence reading (545ex (nm)– 590em (nm)) on the SpectraMax and analyzed with Softmax® Pro software v 5.4.1.</p

Depletion of LpxH causes accumulation of UDP-Diacyl-GlcN (LpxH substrate).

<p>A) The LCMS-MRM quantification of lipid A precursor UDP-Diacyl-GlcN is shown for <i>A</i>. <i>baumannii</i> ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. The <i>m/z</i> [M–H⁺]^- for UDP-Diacyl-GlcN containing 2 acyl groups, 12:0(3-OH), 12:0(3-OH) is 960.5 and for UDP-Diacyl-GlcN with 1 acyl group 12:0(3-OH) and 1 acyl group 14:0(3-OH) the <i>m/z</i> [M–H⁺]^- is 988.5. Experiments were performed in triplicate and bars show the mean value and SD (two-tailed student t-test, **, P<0.01) between NB46082-JWK0133 in the presence or absence of IPTG. Data shown were normalized to an internal standard (IS) as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160918#pone.0160918.ref040" target="_blank">40</a>]. B) Extracted ion chromatogram (EIC) of NB48062-JWK0133 in the presence or absence of IPTG for 2 acyl groups, 12:0(3-OH), 12:0(3-OH). C) Extracted ion chromatogram of NB48062-JWK0133 in the presence or absence of IPTG for 1 acyl group 12:0(3-OH) and 1 acyl group 14:0(3-OH).</p

Depletion of LpxH causes accumulation of DSMP.

<p>(A) LCMS-MRM quantification of DSMP with three 12:0(3-OH) acyl groups and one 14:0(3-OH) acyl group and with two 12:0(3-OH) acyl groups and two 14:0(3-OH) acyl groups for <i>A</i>. <i>baumannii</i> ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. The experiment was performed in triplicate and bars show the mean value and SD (two-tailed student t-test *, P<0.05 and **, P<0.01) between NB46082-JWK0133 in the presence or absence of IPTG). Data shown was normalized to an internal standard (IS) as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160918#pone.0160918.ref040" target="_blank">40</a>].</p

Transmission EM images of NB48062-JWK0133 plus or minus IPTG.

<p>LpxH depleted cells (-IPTG) display gross morphology changes. The experiment was repeated twice with similar results.</p

Schematic illustration of the <i>lpxH</i> regulated expression strain NB46082-JWK0133 and its dependence on IPTG induction for cell growth.

<p>(A) The chromosomal allele of <i>lpxH</i> is regulated by the P_tac promoter (inducible by IPTG). Plasmid pNOV108 provided extra copies of <i>lacI</i> to enhance repression of <i>lpxH</i> in the absence of IPTG. pNOV108 also contained <i>alaS</i> so that it could be maintained by complementation of an <i>alaS</i> deletion on the chromosome, eliminating the need for antibiotic selection. (B) Growth of NB48062-JWK0133 was IPTG dependent. C) Sub-culture growth curve of NB48062-JWK0133 plus or minus IPTG. Arrows indicate time points of sample collection for LCMS-MRM analysis, CFU determination, TEM images, RT-qPCR, and CHIR-090 rescue. D) A significant loss in viability (*, <i>P</i> ≤ 0.01) was observed at 1.5 OD₆₀₀ under LpxH depletion conditions (-IPTG) compared to inducing conditions (+IPTG).</p

Depletion of LpxH causes accumulation of an alternative LpxC product containing a C14:0(3-OH) acyl chain.

<p>The LCMS-MRM quantification of UDP-3-<i>O</i>-[(<i>R</i>)-3-OH-C_12/14]-GlcN for acyl group 12:0(3-OH) and for acyl group 14:0(3-OH) is shown for <i>A</i>. <i>baumannii</i> ATCC 19606 parent and NB48062-JWK0133 under inducing and non-inducing conditions. The experiment was performed in triplicate and bars show the mean value and SD (two-tailed student t-test ***, P<0.001) between NB46082-JWK0133 in the presence or absence of IPTG. Data shown was normalized to an internal standard (IS) as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0160918#pone.0160918.ref040" target="_blank">40</a>].</p