Transformation of the Non-Selective Aminocyclohexanol-Based Hsp90 Inhibitor into a Grp94-Seletive Scaffold

Glucose regulated protein 94 kDa, Grp94, is the endoplasmic reticulum (ER) localized isoform of heat shock protein 90 (Hsp90) that is responsible for the trafficking and maturation of toll-like receptors, immunoglobulins, and integrins. As a result, Grp94 has emerged as a therapeutic target to disrupt cellular communication, adhesion, and tumor proliferation, potentially with fewer side effects compared to <i>pan</i>-inhibitors of all Hsp90 isoforms. Although, the N-terminal ATP binding site is highly conserved among all four Hsp90 isoforms, recent cocrystal structures of Grp94 have revealed subtle differences between Grp94 and other Hsp90 isoforms that has been exploited for the development of Grp94-selective inhibitors. In the current study, a structure-based approach has been applied to a Grp94 nonselective compound, SNX 2112, which led to the development of <b>8j</b> (<b>ACO1</b>), a Grp94-selective inhibitor that manifests ∼440 nM affinity and >200-fold selectivity against cytosolic Hsp90 isoforms

Protein Cross-Linking Capillary Electrophoresis for Protein–Protein Interaction Analysis

Capillary electrophoresis (CE) has been identified as a useful platform for detecting, quantifying, and screening for modulators of protein–protein interactions (PPIs). In this method, one protein binding partner is labeled with a fluorophore, the protein binding partners are mixed, and then, the complex is separated from free protein to allow direct determination of bound to free ratios. Although it possesses many advantages for PPI studies, the method is limited by the need to have separation conditions that both prevent protein adsorption to capillary and maintain protein interactions during the separation. In this work, we use protein cross-linking capillary electrophoresis (PXCE) to overcome this limitation. In PXCE, the proteins are cross-linked under binding conditions and then separated. This approach eliminates the need to maintain noncovalent interactions during electrophoresis and facilitates method development. We report PXCE methods for an antibody–antigen interaction and heterodimer and homodimer heat shock protein complexes. Complexes are cross-linked by short treatments with formaldehyde after reaching binding equilibrium. Cross-linked complexes are separated by electrophoretic mobility using free solution CE or by size using sieving electrophoresis of SDS complexes. The method gives good quantitative results; e.g., a lysozyme–antibody interaction was found to have <i>K</i>_d = 24 ± 3 nM by PXCE and <i>K</i>_d = 17 ± 2 nM using isothermal calorimetry (ITC). Heat shock protein 70 (Hsp70) in complex with bcl2 associated athanogene 3 (Bag3) was found to have <i>K</i>_d = 25 ± 5 nM by PXCE which agrees with <i>K</i>_d values reported without cross-linking. Hsp70–Bag3 binding site mutants and small molecule inhibitors of Hsp70–Bag3 were characterized by PXCE with good agreement to inhibitory constants and IC₅₀ values obtained by a bead-based flow cytometry protein interaction assay (FCPIA). PXCE allows rapid method development for quantitative analysis of PPIs

FKBP51 does not affect longevity.

<p>No significant differences were found in the percent survival of wild-type (wt) and <i>FKBP5^−/−</i> mice, p>0.05. wt, n = 34 (18 male and 16 female); <i>FKBP5^−/−</i>, n = 32 (18 male and 14 female).</p

<i>FKBP5^−/−</i> mice display enhanced cognitive flexibility in the radial arm water maze.

<p>(A) No differences were found in acquisition learning between genotypes (p>0.05). (B) There was a main effect of genotype (p<0.05) during reversal training, indicating <i>FKBP5^−/−</i> mice made fewer errors across sessions. Data points represent a session of three trials. *p<0.05. wild-type (wt), n = 9; <i>FKBP5^−/−</i>, n = 10.</p

<i>Fkbp5</i> DNA methylation decreases with age in mice.

<p>Isolated DNA samples from wild-type mice aged 1 (n = 3), 3.5 (n = 3), 4 (n = 6), 5 (n = 7), 6 (n = 6), and 12 (n = 3) months were subjected to bisulfite pyrosequencing. Multiple CpG sites in intron 5 were analyzed for <i>Fkbp5</i> methylation. Significant demethylation was found at CPG_3 (r = −0.3890, p<0.05), CPG_4 (r = −0.4004, p<0.05) and CpG_5 (r = −0.5044, p<0.01) as measured by linear regression analyses.</p

Blood composition and animal mass of wild-type versus FKBP5^−/− mice do not differ.

<p>WBC: white blood cells, RBC: red blood cells, Lym: lymphocytes, Mono: monocytes, Gran: granulocytes, HGB: hemoglobin, PLT: platelets. Wild-type, n = 11; FKBP5^−/−, n = 12. Values are listed as the mean ± the standard error of the mean.</p><p>Blood composition and animal mass of wild-type versus FKBP5^−/− mice do not differ.</p

Deletion of <i>FKBP5</i> does not alter glucose metabolism.

<p><i>FKBP5^−/−</i> mice displayed normal glucose tolerance up to 120 minutes following glucose injection compared to wild-type (wt) mice, p>0.05. wt, n = 7; <i>FKBP5^−/−</i>, n = 10.</p

Ablation of <i>FKBP5</i> does not alter cytokine levels over time.

<p>Serum levels of interleukin-1β (A) and interleukin-5 (D) were decreased at 6 months (p<0.05 via t-test) but not across time (p>0.05 by two-way ANOVA). Levels of interleukin-2 (B), interleukin-4 (C), interleukin-10 (E), granulocyte-macrophage colony-stimulating factor (F), interferon gamma (G), or tumor necrosis factor alpha (H) did not differ between genotypes across lifespan, p>0.05. *p<0.05. wild-type (wt), n = 6 for each age; <i>FKBP5^−/−</i>, n = 7 at 7 and 10 months, n = 6 at 21 months.</p

Inhibition of Both Hsp70 Activity and Tau Aggregation <i>in Vitro</i> Best Predicts Tau Lowering Activity of Small Molecules

Three scaffolds with inhibitory activity against the heat shock protein 70 (Hsp70) family of chaperones have been found to enhance the degradation of the microtubule associated protein tau in cells, neurons, and brain tissue. This is important because tau accumulation is linked to neurodegenerative diseases including Alzheimer’s disease (AD) and chronic traumatic encephalopathy (CTE). Here, we expanded upon this study to investigate the anti-tau efficacy of additional scaffolds with Hsp70 inhibitory activity. Five of the nine scaffolds tested lowered tau levels, with the rhodacyanine and phenothiazine scaffolds exhibiting the highest potency as previously described. Because phenothiazines also inhibit tau aggregation <i>in vitro</i>, we suspected that this activity might be a more accurate predictor of tau lowering. Interestingly, the rhodacyanines did inhibit <i>in vitro</i> tau aggregation to a similar degree as phenothiazines, correlating well with tau-lowering efficacy in cells and <i>ex vivo</i> slices. Moreover, other Hsp70 inhibitor scaffolds with weaker tau-lowering activity in cells inhibited tau aggregation <i>in vitro</i>, albeit at lower potencies. When we tested six well-characterized tau aggregation inhibitors, we determined that this mechanism of action was not a better predictor of tau-lowering than Hsp70 inhibition. Instead, we found that compounds possessing both activities were the most effective at promoting tau clearance. Moreover, cytotoxicity and PAINS activity are critical factors that can lead to false-positive lead identification. Strategies designed around these principles will likely yield more efficacious tau-lowering compounds

Analysis of the Tau-Associated Proteome Reveals That Exchange of Hsp70 for Hsp90 Is Involved in Tau Degradation

The microtubule associated protein tau (MAPT/tau) aberrantly accumulates in 15 neurodegenerative diseases, termed tauopathies. One way to treat tauopathies may be to accelerate tau clearance, but the molecular mechanisms governing tau stability are not yet clear. We recently identified chemical probes that markedly accelerate the clearance of tau in cellular and animal models. In the current study, we used one of these probes in combination with immunoprecipitation and mass spectrometry to identify 48 proteins whose association with tau changes during the first 10 min after treatment. These proteins included known modifiers of tau proteotoxicity, such as ILF-2 (NFAT), ILF-3, and ataxin-2. A striking observation from the data set was that tau binding to heat shock protein 70 (Hsp70) decreased, whereas binding to Hsp90 significantly increased. Both chaperones have been linked to tau homeostasis, but their mechanisms have not been established. Using peptide arrays and binding assays, we found that Hsp70 and Hsp90 appeared to compete for binding to shared sites on tau. Further, the Hsp90-bound complex proved to be important in initiating tau clearance in cells. These results suggest that the relative levels of Hsp70 and Hsp90 may help determine whether tau is retained or degraded. Consistent with this model, analysis of reported microarray expression data from Alzheimer’s disease patients and age-matched controls showed that the levels of Hsp90 are reduced in the diseased hippocampus. These studies suggest that Hsp70 and Hsp90 work together to coordinate tau homeostasis