Heat shock protein 90 in neurodegenerative diseases

Hsp90 is a molecular chaperone with important roles in regulating pathogenic transformation. In addition to its well-characterized functions in malignancy, recent evidence from several laboratories suggests a role for Hsp90 in maintaining the functional stability of neuronal proteins of aberrant capacity, whether mutated or over-activated, allowing and sustaining the accumulation of toxic aggregates. In addition, Hsp90 regulates the activity of the transcription factor heat shock factor-1 (HSF-1), the master regulator of the heat shock response, mechanism that cells use for protection when exposed to conditions of stress. These biological functions therefore propose Hsp90 inhibition as a dual therapeutic modality in neurodegenerative diseases. First, by suppressing aberrant neuronal activity, Hsp90 inhibitors may ameliorate protein aggregation and its associated toxicity. Second, by activation of HSF-1 and the subsequent induction of heat shock proteins, such as Hsp70, Hsp90 inhibitors may redirect neuronal aggregate formation, and protect against protein toxicity. This mini-review will summarize our current knowledge on Hsp90 in neurodegeneration and will focus on the potential beneficial application of Hsp90 inhibitors in neurodegenerative diseases

Measuring the Pharmacodynamic Effects of a Novel Hsp90 Inhibitor on HER2/neu Expression in Mice Using 89Zr-DFO-Trastuzumab

The positron-emitting radionuclide (89)Zr (t(1/2) = 3.17 days) was used to prepare (89)Zr-radiolabeled trastuzumab for use as a radiotracer for characterizing HER2/neu-positive breast tumors. In addition, pharmacodynamic studies on HER2/neu expression levels in response to therapeutic doses of PU-H71 (a specific inhibitor of heat-shock protein 90 [Hsp90]) were conducted.Trastuzumab was functionalized with desferrioxamine B (DFO) and radiolabeled with [(89)Zr]Zr-oxalate at room temperature using modified literature methods. ImmunoPET and biodistribution experiments in female, athymic nu/nu mice bearing sub-cutaneous BT-474 (HER2/neu positive) and/or MDA-MB-468 (HER2/neu negative) tumor xenografts were conducted. The change in (89)Zr-DFO-trastuzumab tissue uptake in response to high- and low-specific-activity formulations and co-administration of PU-H71 was evaluated by biodistribution studies, Western blot analysis and immunoPET. (89)Zr-DFO-trastuzumab radiolabeling proceeded in high radiochemical yield and specific-activity 104.3+/-2.1 MBq/mg (2.82+/-0.05 mCi/mg of mAb). In vitro assays demonstrated >99% radiochemical purity with an immunoreactive fraction of 0.87+/-0.07. In vivo biodistribution experiments revealed high specific BT-474 uptake after 24, 48 and 72 h (64.68+/-13.06%ID/g; 71.71+/-10.35%ID/g and 85.18+/-11.10%ID/g, respectively) with retention of activity for over 120 h. Pre-treatment with PU-H71 was followed by biodistribution studies and immunoPET of (89)Zr-DFO-trastuzumab. Expression levels of HER2/neu were modulated during the first 24 and 48 h post-administration (29.75+/-4.43%ID/g and 41.42+/-3.64%ID/g, respectively). By 72 h radiotracer uptake (73.64+/-12.17%ID/g) and Western blot analysis demonstrated that HER2/neu expression recovered to baseline levels.The results indicate that (89)Zr-DFO-trastuzumab provides quantitative and highly-specific delineation of HER2/neu positive tumors, and has potential to be used to measure the efficacy of long-term treatment with Hsp90 inhibitors, like PU-H71, which display extended pharmacodynamic profiles

Assay strategies for the discovery and validation of therapeutics targeting <i>Brugia pahangi</i> Hsp90

The chemotherapy of lymphatic filariasis relies upon drugs such as diethylcarbamazine and ivermectin that largely target the microfilarial stages of the parasite, necessitating continued treatment over the long reproductive life span of the adult worm. The identification of compounds that target adult worms has been a long-term goal of WHO. Here we describe a fluorescence polarization assay for the identification of compounds that target Hsp90 in adult filarial worms. The assay was originally developed to identify inhibitors of Hsp90 in tumor cells, and relies upon the ability of small molecules to inhibit the binding of fluorescently labelled geldanamycin to Hsp90. We demonstrate that the assay works well with soluble extracts of Brugia, while extracts of the free-living nematode C. elegans fail to bind the probe, in agreement with data from other experiments. The assay was validated using known inhibitors of Hsp90 that compete with geldanamycin for binding to Hsp90, including members of the synthetic purine-scaffold series of compounds. The efficacy of some of these compounds against adult worms was confirmed in vitro. Moreover, the assay is sufficiently sensitive to differentiate between binding of purine-scaffold compounds to human and Brugia Hsp90. The assay is suitable for high-throughput screening and provides the first example of a format with the potential to identify novel inhibitors of Hsp90 in filarial worms and in other parasitic species where Hsp90 may be a target

Determinants of Apoptotic Sensitivity to HSP90 Inhibition In Acute Myeloid Leukemia

Abstract Abstract 2159 Background: Acute myeloid leukemia (AML) is a heterogeneous and intrinsically resistant disease group of malignant hematopoietic disorders that accounts for approximately 80% of all adult leukemias. Heat shock proteins (HSPs) are often overexpressed in AML are their expression is associated with poor-prognosis and resistance to chemotherapy. Among HSPs, HSP90 is the main chaperone required for the stabilization of multiple oncogenic kinases, which contribute to AML pathogenesis, providing a rationale for the use of HSP90 inhibitors in the treatment of AML. Hypothesis: To identify patients with AML who will benefit from HSP90 inhibitor therapy there is a need to discover molecules and pathways in AML cells that confer sensitivity and lead to significant apoptosis upon HSP90 inhibition. Study design and Results: To evaluate the spectrum of sensitivities of AML cells to HSP90 inhibitors, and to investigate a possible relationship between their genetic background and apoptotic sensitivity to HSP90 inhibition, we investigated the effects of HSP90 inhibitors in a set of genetically characterized human AML cells. Addition of several HSP90 inhibitors to each of these cell lines potently inhibited cell growth, with a potency reflective of their affinity for HSP90. Normal peripheral blood leukocytes were unaffected at similar concentrations. HSP90 inhibition was associated with destabilization and subsequent degradation of Akt and c-Raf in all tested cells, as well as of several cell-specific onco-proteins such as mutant Flt3 in MOLM-13, TEL-TRKC in M0-91, AML1-ETO and mutant cKit in Kasumi-1 and SKNO-1, and mutant Jak2 in HEL cells, respectively. Notably, the proclivity for these cells to undergo apoptosis upon HSP90 inhibition varied considerably. The most sensitive cell lines were MOLM-13, MV-4-11 and M0-91 cells, and for each these cell lines we observed near 100% killing of the initial cell population after 48–72 h of HSP90 inhibitor treatment. In contrast, only 20% death was seen in HEL and HL-60 cells under these conditions. We next made use of specific inhibitors of known oncogenic signaling pathways known to be dysregulated in AML to demonstrate that apoptotic sensitivity of AML cells to HSP90 inhibition correlated with PI3K-Akt and STAT5 activation, but not with activation of the Raf-MAPK pathway. Importantly, similar results were observed in cells lines, xenograft models and isogenic cell line systems. We also found that dual activation of these two pathways, especially in the context of Bcl-xL overexpression, lowers the apoptotic threshold of AML when HSP90 is inhibited. Conclusions: We found that activation of oncogenic signaling pathways and expression of leukemogenic anti-apoptotic molecules, most importantly p-Akt, predicts for AML sensitivity to HSP90 inhibitors. Importantly, 50– 70% of patients with AML display phosphorylation of both Thr308 and Ser4 Akt. This molecule contributes to proliferation, survival and drug resistance in AML, and is associated with adverse outcome. Taken together, our findings suggest that AML patients with activation of Akt and STAT5 signaling are most likely to benefit from HSP90 inhibitor therapy, and clinical trials should aim to enroll patients with specific activation of these important signaling pathways. Disclosures: No relevant conflicts of interest to declare