Small Molecule Hsp90 Inhibitors Research Articles

Abstract 3722: The HSP90 inhibitor ganetespib synergizes with the MET kinase inhibitor crizotinib in both crizotinib-sensitive and crizotinib-resistant MET-driven renal tumor models

Abstract Introduction: The proto-oncogene MET is aberrantly activated via overexpression or mutation in numerous cancers including papillary renal carcinoma, making it a prime anti-cancer molecular target. However, the clinical success of MET-directed tyrosine kinase inhibitors (TKI) has been limited due, in part, to mutations in the MET kinase domain that confer therapeutic resistance. Circumventing this problem remains a key challenge to improving durable responses in patients receiving MET-targeted therapy. Small molecule HSP90 inhibitors cause HSP90 to dissociate from its client proteins, resulting in their destabilization and eventual degradation. HSP90 inhibitors have been validated in numerous preclinical tumor models and have shown promising activity in several clinical trials. Given the distinct mechanisms of action of TKI and HSP90 inhibitors, we evaluated the synergistic effects of these two drugs on both TKI-sensitive and TKI-resistant MET-driven tumor models. Methods: We tested efficacy of the MET TKI crizotinib, the HSP90 inhibitor ganetespib and their combination in MKN45, wild-type MET-overexpressed cell line and MET-Y1248H, TKI-resistant mutant MET- verexpressed cell line in vitro. The antitumor efficacy of HSP90 inhibition in vivo was verified in xenograft models. Results: MET is an HSP90-dependent kinase, and we show that HSP90 preferentially interacts with and stabilizes activated MET, whether the activation is ligand-dependent or is a consequence of kinase domain mutation. In contrast, many MET TKI show a preference for the inactive form of the kinase and activating mutations in MET can confer resistance. Combining the HSP90 inhibitor ganetespib with the MET TKI crizotinib achieves synergistic inhibition of MET, its downstream signaling pathways, and tumor growth in TKI-sensitive MET-driven tumor models. Unexpectedly, low-dose ganetespib also partially restores crizotinib sensitivity, in vitro and in vivo, to cells and tumors expressing TKI-resistant MET mutants. Conclusion: Our findings support the use of HSP90 inhibitors to overcome or delay the initiation of resistance to MET TKI, and they provide the basis for clinical evaluation of this combination in patients with MET-driven papillary renal cancer. Citation Format: Naoto Miyajima, Tomoshige Akino, Kunihiko Tsuchiya, Satoru Maruyama, Takashige Abe, Nobuo Shinohara, Katsuya Nonomura, Len Neckers. The HSP90 inhibitor ganetespib synergizes with the MET kinase inhibitor crizotinib in both crizotinib-sensitive and crizotinib-resistant MET-driven renal tumor models. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3722. doi:10.1158/1538-7445.AM2014-3722

Artificial accelerators of the molecular chaperone Hsp90 facilitate rate-limiting conformational transitions.

The molecular chaperone Hsp90 undergoes an ATP-driven cycle of conformational changes in which large structural rearrangements precede ATP hydrolysis. Well-established small-molecule inhibitors of Hsp90 compete with ATP-binding. We wondered whether compounds exist that can accelerate the conformational cycle. In a FRET-based screen reporting on conformational rearrangements in Hsp90 we identified compounds. We elucidated their mode of action and showed that they can overcome the intrinsic inhibition in Hsp90 which prevents these rearrangements. The mode of action is similar to that of the co-chaperone Aha1 which accelerates the Hsp90 ATPase. However, while the two identified compounds influence conformational changes, they target different aspects of the structural transitions. Also, the binding site determined by NMR spectroscopy is distinct. This study demonstrates that small molecules are capable of triggering specific rate-limiting transitions in Hsp90 by mechanisms similar to those in protein cofactors.

Ganetespib, a Novel Hsp90 Inhibitor in Patients With KRAS Mutated and Wild Type, Refractory Metastatic Colorectal Cancer

BackgroundHeat shock protein 90 (Hsp90) is a cellular chaperone that is required for the maturation and stability of a variety of proteins that play key roles in colon cancer initiation and progression. The primary objective of the current study was to define the safety and efficacy of ganetespib, a novel, selective small-molecule Hsp90 inhibitor, in patients with refractory metastatic colorectal cancer. Patients and MethodsThe study was a single-arm, Simon 2-stage, phase II trial for patients with chemotherapy-refractory, metastatic colorectal cancer. Patients received ganetespib 200 mg/m2 intravenously. Tumor tissue was collected before treatment and 48 hours after treatment for changes in expression of Hsp90 client proteins and other potential pharmacodynamics markers. V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS), v-Raf murine sarcoma viral oncogene homolog B, and phosphatidylinositol-4, 5-bisphosphate 3-kinase, catalytic subunit alpha (PIK3CA) mutational status was also determined. ResultsSeventeen patients were treated (median age, 58; range, 44-79 years). No patients demonstrated objective regression of disease. Two patients had stable disease of 6.8 and 5.1 months duration. Serious adverse events that were potentially attributable to ganetespib included diarrhea (12%, n = 2), fatigue (17%, n = 3), and increased aspartate aminotransferase/alanine aminotransferase (12%, n = 2) and alkaline phosphatase (6%, n = 1) levels. Of the 17 evaluable patients, 9 (53%) including patients with stable disease as best response, had KRAS-mutant tumors. ConclusionIn this first phase II investigation of an Hsp90 inhibitor in colorectal cancer, ganetespib as a single agent did not demonstrate activity in chemotherapy-refractory metastatic colorectal cancer. However, on the basis of the drug's promising preclinical combination data and the relatively mild toxicity profile, further clinical investigation of this agent in combination with standard cytotoxic agents is planned.

Synergetic cytotoxic activity toward breast cancer cells enhanced by the combination of Antp-TPR hybrid peptide targeting Hsp90 and Hsp70-targeted peptide.

BackgroundHeat shock protein (Hsp) 90 and Hsp70 are indispensable for cell survival under conditions of stress. They bind to client proteins to assist in protein stabilization, translocation of polypeptides across the cell membrane, and recovery of proteins from aggregates in the cell. Therefore, these proteins have recently emerged as important targets in the treatment of cancer. We previously reported that the newly designed Antp-TPR hybrid peptide targeting Hsp90 induced cytotoxic activity to cancer cells both in vitro and in vivo.MethodsTo further improve the cytotoxic activity of Antp-TPR toward cancer cells, we investigated the effect of a Hsp70-targeted peptide, which was made cell-permeable by adding the polyarginine with a linker sequence, on the cytotoxic activity of Antp-TPR in breast cancer cell lines.ResultsIt was revealed that Antp-TPR in the presence of a Hsp70-targeted peptide induced effective cytotoxic activity toward breast cancer cells through the descrease of Hsp90 client proteins such as p53, Akt, and cRaf. Moreover, the combined treatment with these peptides did not induce the up-regulation of Hsp70 protein, as determined by western blotting, a promoter assay using a luminometer, and single-cell level imaging with the LV200 system, although a small-molecule inhibitor of Hsp90, 17-allylamino-demethoxygeldanamycin (17-AAG), did induce the up-regulation of this protein. We also found that treatment with Antp-TPR, Hsp70-targeted peptide, or a combination of the two did not induce an increase in the glutathione concentrations in the cancer cells.ConclusionThese findings suggest that targeting both Hsp90 and Hsp70 with Antp-TPR and Hsp70-targeted peptide is an attractive approach for selective cancer cell killing that might provide potent and selective therapeutic options for the treatment of cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2407-14-615) contains supplementary material, which is available to authorized users.

SARC023: Phase I/II trial of ganetespib in combination with sirolimus for refractory sarcomas and malignant peripheral nerve sheath tumors (MPNST).

TPS10603 Background: Combination treatments of Hsp90 inhibitors with the mTOR inhibitor rapamycin produce dramatic tumor shrinkage in a genetically engineered MPNST mouse model of human MPNST (DeRaedt et al. Cancer Cell 2011). Previously, targeted agents have not resulted in substantial tumor regression in these models. Ganetespib is a potent small molecule inhibitor of Hsp90 that enhances endoplasmic reticulum stress with a favorable safety profile and promising activity in a variety of cancers. Sirolimus is a commercially available mTOR inhibitor, which has been tolerated in combination with multiple cytotoxic and targeted agents. These agents have also demonstrated activity in a variety of pre-clinical bone and soft tissue sarcoma models. Methods: SARC023 is a multi-institutional SARC sponsored, Department of Defense funded open label phase I/II trial of ganetespib in combination with sirolimus in patients with refractory sarcoma including MPNST (NCT02008877). The primary objective of the phase I compo...

The double-edged sword: conserved functions of extracellular hsp90 in wound healing and cancer.

Heat shock proteins (Hsps) represent a diverse group of chaperones that play a vital role in the protection of cells against numerous environmental stresses. Although our understanding of chaperone biology has deepened over the last decade, the “atypical” extracellular functions of Hsps have remained somewhat enigmatic and comparatively understudied. The heat shock protein 90 (Hsp90) chaperone is a prototypic model for an Hsp family member exhibiting a duality of intracellular and extracellular functions. Intracellular Hsp90 is best known as a master regulator of protein folding. Cancers are particularly adept at exploiting this function of Hsp90, providing the impetus for the robust clinical development of small molecule Hsp90 inhibitors. However, in addition to its maintenance of protein homeostasis, Hsp90 has also been identified as an extracellular protein. Although early reports ascribed immunoregulatory functions to extracellular Hsp90 (eHsp90), recent studies have illuminated expanded functions for eHsp90 in wound healing and cancer. While the intended physiological role of eHsp90 remains enigmatic, its evolutionarily conserved functions in wound healing are easily co-opted during malignancy, a pathology sharing many properties of wounded tissue. This review will highlight the emerging functions of eHsp90 and shed light on its seemingly dichotomous roles as a benevolent facilitator of wound healing and as a sinister effector of tumor progression.

A repurposing strategy for Hsp90 inhibitors demonstrates their potency against filarial nematodes.

Novel drugs are required for the elimination of infections caused by filarial worms, as most commonly used drugs largely target the microfilariae or first stage larvae of these infections. Previous studies, conducted in vitro, have shown that inhibition of Hsp90 kills adult Brugia pahangi. As numerous small molecule inhibitors of Hsp90 have been developed for use in cancer chemotherapy, we tested the activity of several novel Hsp90 inhibitors in a fluorescence polarization assay and against microfilariae and adult worms of Brugia in vitro. The results from all three assays correlated reasonably well and one particular compound, NVP-AUY922, was shown to be particularly active, inhibiting Mf output from female worms at concentrations as low as 5.0 nanomolar after 6 days exposure to drug. NVP-AUY922 was also active on adult worms after a short 24 h exposure to drug. Based on these in vitro data, NVP-AUY922 was tested in vivo in a mouse model and was shown to significantly reduce the recovery of both adult worms and microfilariae. These studies provide proof of principle that the repurposing of currently available Hsp90 inhibitors may have potential for the development of novel agents with macrofilaricidal properties.

Overcoming Acquired BRAF Inhibitor Resistance in Melanoma via Targeted Inhibition of Hsp90 with Ganetespib

Activating BRAF kinase mutations serve as oncogenic drivers in over half of all melanomas, a feature that has been exploited in the development of new molecularly targeted approaches to treat this disease. Selective BRAF(V600E) inhibitors, such as vemurafenib, typically induce initial, profound tumor regressions within this group of patients; however, durable responses have been hampered by the emergence of drug resistance. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90, in melanoma lines harboring the BRAF(V600E) mutation. Ganetespib exposure resulted in the loss of mutant BRAF expression and depletion of mitogen-activated protein kinase and AKT signaling, resulting in greater in vitro potency and antitumor efficacy compared with targeted BRAF and MAP-ERK kinase (MEK) inhibitors. Dual targeting of Hsp90 and BRAF(V600E) provided combinatorial benefit in vemurafenib-sensitive melanoma cells in vitro and in vivo. Importantly, ganetespib overcame mechanisms of intrinsic and acquired resistance to vemurafenib, the latter of which was characterized by reactivation of extracellular signal-regulated kinase (ERK) signaling. Continued suppression of BRAF(V600E) by vemurafenib potentiated sensitivity to MEK inhibitors after acquired resistance had been established. Ganetespib treatment reduced, but not abolished, elevations in steady-state ERK activity. Profiling studies revealed that the addition of a MEK inhibitor could completely abrogate ERK reactivation in the resistant phenotype, with ganetespib displaying superior combinatorial activity over vemurafenib. Moreover, ganetespib plus the MEK inhibitor TAK-733 induced tumor regressions in vemurafenib-resistant xenografts. Overall these data highlight the potential of ganetespib as a single-agent or combination treatment in BRAF(V600E)-driven melanoma, particularly as a strategy to overcome acquired resistance to selective BRAF inhibitors.

Chronic Treatment with Novel Small Molecule Hsp90 Inhibitors Rescues Striatal Dopamine Levels but Not α-Synuclein-Induced Neuronal Cell Loss

Hsp90 inhibitors such as geldanamycin potently induce Hsp70 and reduce cytotoxicity due to α-synuclein expression, although their use has been limited due to toxicity, brain permeability, and drug design. We recently described the effects of a novel class of potent, small molecule Hsp90 inhibitors in cells overexpressing α-synuclein. Screening yielded several candidate compounds that significantly reduced α-synuclein oligomer formation and cytotoxicity associated with Hsp70 induction. In this study we examined whether chronic treatment with candidate Hsp90 inhibitors could protect against α-synuclein toxicity in a rat model of parkinsonism. Rats were injected unilaterally in the substantia nigra with AAV8 expressing human α-synuclein and then treated with drug for approximately 8 weeks by oral gavage. Chronic treatment with SNX-0723 or the more potent, SNX-9114 failed to reduce dopaminergic toxicity in the substantia nigra compared to vehicle. However, SNX-9114 significantly increased striatal dopamine content suggesting a positive neuromodulatory effect on striatal terminals. Treatment was generally well tolerated, but higher dose SNX-0723 (6–10 mg/kg) resulted in systemic toxicity, weight loss, and early death. Although still limited by potential toxicity, Hsp90 inhibitors tested herein demonstrate oral efficacy and possible beneficial effects on dopamine production in a vertebrate model of parkinsonism that warrant further study.

Retinal toxicity induced by small-molecule Hsp90 inhibitors in beagle dogs

Heat shock protein 90 (Hsp90) is a constitutively expressed molecular chaperone and plays an important role in the folding of client proteins with key regulatory roles in growth, survival, differentiation and metastasis. Because inhibition of Hsp90 degrades multiple oncogenic client proteins, it is considered to be an attractive anticancer therapy, and clinical trials of several Hsp90 inhibitors have been carried out. In the present study, two structurally distinct Hsp90 inhibitors, CH5164840 and CH5449302, were orally administered to beagle dogs to evaluate systemic toxicity. CH5164840 induced symptoms that suggest visual disorder, and ophthalmological observation and electroretinography (ERG) revealed loss of pupillary light reflex and abnormal waveforms, respectively. Histopathological examination showed changes in the photoreceptor cell layer and the outer nuclear layer of retina. On the other hand, while there were no clinical symptoms related to visual disorder, animals treated with CH5449302 showed similar abnormalities of ERG responses and histopathological changes in the photoreceptor cell layer and the outer nuclear layer of retina. The visual symptoms and abnormalities of ERG responses were noted at an earlier stage or lower dose than other toxicities in both compounds. Considering that two structurally distinct Hsp90 inhibitors induced a retinal toxicity in dogs after repeated administration, and that visual disorders were also reported in some clinical trials of Hsp90 inhibitors, it would seem highly likely that Hsp90 inhibition induces retinal toxicity. Also, our study indicated that a detailed ocular examination to evaluate the safety of Hsp90 inhibitors would be useful in both preclinical and clinical studies.

Drugging the cancer genome – discovery of small-molecule targeted therapeutics for personalized, precision medicine

This article describes my research on the discovery of innovative small-molecule targeted therapeutics for use in personalized, precision cancer medicine. This is exemplified by two particular examples. Firstly, I describe our discovery of novel and effective small-molecule inhibitors of the molecular chaperone HSP90 – and especially the leading clinical agent, AUY922, which is showing strong activity in HER2-positive breast cancers that have become refractory to trastuzumab (Herceptin®, Roche Ltd, Basel, Switzerland) and also in non-small cell lung cancers, including those that are resistant to oncogenic driver protein kinase inhibitors – with general potential to overcome or prevent the problem of drug resistance. Secondly, I discuss our discovery of pathfinding small-molecule inhibitors of class I PI3 kinases, especially the first-in-class pan-class I-selective inhibitor GDC-0941, for which clinical responses have been observed in breast and ovarian cancer, gastrointestinal stromal tumours, multiple myeloma and melanoma. More broadly, our Cancer Research UK Cancer Therapeutics Unit has discovered 17 innovative drug candidates since 2005 and progressed seven of these into biomarker-led, hypothesis-driven clinical trials with successful pharmacodynamic biomarker modulation in all cases and clinical responses in most to date, exemplifying the use of our ‘Pharmacologic Audit Trail’ concept for drug discovery and clinical development. Furthermore, our CYP17 inhibitor abiraterone (Zytiga®, Janssen Pharmaceuticals Inc., NJ, USA), designed and synthesized in our unit and initially trialled in our partner hospital, The Royal Marsden Hospital (London, UK), was approved for late-stage castration-resistant prostate cancer in 2011. Thus, we have developed a successful model for high-quality academic drug discovery in the non-profit setting (Anonymous. Drug discovery gets an academic push. Cancer Discov 2011; 1:93–4). Importantly, this provides a creative derisking solution for the innovation gap or ‘valley of death’ that often divides basic research from clinical translation. This new model is especially critical at a time when the adverse economic climate and other pressures have led to downsizing and increased risk aversion in the large pharmaceutical companies together with reduced venture investment in biotechnology companies. Having taken on the early high-risk research of new targets, we have also been very successful in partnering our drug discovery projects with industry at the appropriate stage using a range of commercial models with speed-to-patient benefit as the main driver [Houlton S. Fast, flexible and flourishing. Chemistry World, 2012. URL: www.rsc.org/chemistryworld/more/ ?author=35&date-range=2-5+years (last accessed 30 September 2014)]. Before focusing on our work on what I refer to as ‘drugging the cancer genome’, and particularly the examples of HSP90 and PI3 kinase inhibitors, I provide a short summary of the background to molecularly targeted cancer drug discovery and development together with brief comments on my early research on hypoxia-targeted drugs and signal transduction inhibitors, with the aim of setting the context for what follows. To finish, I offer some concluding remarks and future perspectives on the discovery, development and use of personalized, precision medicines for cancer.

Erratum to: A novel small molecule HSP90 inhibitor, NXD30001, differentially induces heat shock proteins in nervous tissue in culture and in vivo

Erratum to: A novel small molecule HSP90 inhibitor, NXD30001, differentially induces heat shock proteins in nervous tissue in culture and in vivo

A novel small molecule HSP90 inhibitor, NXD30001, differentially induces heat shock proteins in nervous tissue in culture and in vivo.

Heat shock proteins (HSPs) are attractive therapeutic targets for neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), characterized by aberrant formation of protein aggregates. Although motor neurons have a high threshold for activation of HSP genes, HSP90 inhibitors are effective inducers. This study evaluated NXD30001, a novel, small molecule HSP90 inhibitor based on the radicicol backbone, for its ability to induce neuronal HSPs and for efficacy in an experimental model of ALS based on mutations in superoxide-dismutase 1 (SOD1). In motor neurons of dissociated murine spinal cord cultures, NXD30001-induced expression of HSP70/HSPA1 (iHSP70) and its co-chaperone HSP40/DNAJ through activation of HSF1 and exhibited a protective profile against SOD1(G93A) similar to geldanamycin, but with less toxicity. Treatment prevented protein aggregation, mitochondrial fragmentation, and motor neuron death, important features of mutant SOD1 toxicity, but did not effectively prevent aberrant intracellular Ca(2+) accumulation. NXD30001 distributed to brain and spinal cord of wild-type and SOD1(G93A) transgenic mice following intraperitoneal injection; however, unlike in culture, in vivo levels of SOD1 were not reduced. NXD30001-induced expression of iHSP70 in skeletal and cardiac muscle and, to a lesser extent, in kidney, but not in liver, spinal cord, or brain, with either single or repeated administration. NXD30001 is a very useful experimental tool in culture, but these data point to the complex nature of HSP gene regulation in vivo and the necessity for early evaluation of the efficacy of novel HSP inducers in target tissues in vivo.

Network analysis identifies an HSP90-central hub susceptible in ovarian cancer.

Epithelial ovarian cancer (EOC) is usually detected at an advanced stage and is frequently lethal. Although many patients respond to initial surgery and standard chemotherapy consisting of a platinum-based agent and a taxane, most experience recurrence and eventually treatment-resistant disease. Although there have been numerous efforts to apply protein-targeted agents in EOC, these studies have so far documented little efficacy. Our goal was to identify broadly susceptible signaling proteins or pathways in EOC. As a new approach, we conducted data-mining meta-analyses integrating results from multiple siRNA screens to identify gene targets that showed significant inhibition of cell growth. On the basis of this meta-analysis, we established that many genes with such activity were clients of the protein chaperone HSP90. We therefore assessed ganetespib, a clinically promising second-generation small-molecule HSP90 inhibitor, for activity against EOC, both as a single agent and in combination with cytotoxic and targeted therapeutic agents. Ganetespib significantly reduced cell growth, induced cell-cycle arrest and apoptosis in vitro, inhibited growth of orthotopic xenografts and spontaneous ovarian tumors in transgenic mice in vivo, and inhibited expression and activation of numerous proteins linked to EOC progression. Importantly, paclitaxel significantly potentiated ganetespib activity in cultured cells and tumors. Moreover, combined treatment of cells with ganetespib and siRNAs or small molecules inhibiting genes identified in the meta-analysis in several cases resulted in enhanced activity. These results strongly support investigation of ganetespib, a single-targeted agent with effects on numerous proteins and pathways, in augmenting standard EOC therapies.

Hsp70/Hsp90 chaperone machinery is involved in the assembly of the purinosome

The de novo biosynthesis of purines is carried out by a highly conserved metabolic pathway that includes several validated targets for anticancer, immunosuppressant, and anti-inflammatory chemotherapeutics. The six enzymes in humans that catalyze the 10 chemical steps from phosphoribosylpyrophosphate to inosine monophosphate were recently shown to associate into a dynamic multiprotein complex called the purinosome. Here, we demonstrate that heat shock protein 90 (Hsp90), heat shock protein 70 (Hsp70), and several cochaperones functionally colocalize with this protein complex. Knockdown of expression levels of the identified cochaperones leads to disruption of purinosomes. In addition, small molecule inhibitors of Hsp90 and Hsp70 reversibly disrupt purinosomes and are shown to have a synergistic effect with methotrexate, an anticancer agent that targets purine biosynthesis. These data implicate the Hsp90/Hsp70 chaperone machinery in the assembly of the purinosome and provide a strategy for the development of improved anticancer therapies that disrupt purine biosynthesis.

ChemInform Abstract: Discovery of XL888: A Novel Tropane‐Derived Small Molecule Inhibitor of HSP90.

AbstractWith structural guidance, tropane‐derived HTS hits are modified to optimize for HSP90 inhibition and a desirable in vivo profile.

Targeting KRAS-Mutant Non–Small Cell Lung Cancer with the Hsp90 Inhibitor Ganetespib

Mutant KRAS is a feature of more than 25% of non-small cell lung cancers (NSCLC) and represents one of the most prevalent oncogenic drivers in this disease. NSCLC tumors with oncogenic KRAS respond poorly to current therapies, necessitating the pursuit of new treatment strategies. Targeted inhibition of the molecular chaperone Hsp90 results in the coordinated blockade of multiple oncogenic signaling pathways in tumor cells and has thus emerged as an attractive avenue for therapeutic intervention in human malignancies. Here, we examined the activity of ganetespib, a small-molecule inhibitor of Hsp90 currently in clinical trials for NSCLCs in a panel of lung cancer cell lines harboring a diverse spectrum of KRAS mutations. In vitro, ganetespib was potently cytotoxic in all lines, with concomitant destabilization of KRAS signaling effectors. Combinations of low-dose ganetespib with MEK or PI3K/mTOR inhibitors resulted in superior cytotoxic activity than single agents alone in a subset of mutant KRAS cells, and the antitumor efficacy of ganetespib was potentiated by cotreatment with the PI3K/mTOR inhibitor BEZ235 in A549 xenografts in vivo. At the molecular level, ganetespib suppressed activating feedback signaling loops that occurred in response to MEK and PI3K/mTOR inhibition, although this activity was not the sole determinant of combinatorial benefit. In addition, ganetespib sensitized mutant KRAS NSCLC cells to standard-of-care chemotherapeutics of the antimitotic, topoisomerase inhibitor, and alkylating agent classes. Taken together, these data underscore the promise of ganetespib as a single-agent or combination treatment in KRAS-driven lung tumors.

Potent activity of the Hsp90 inhibitor ganetespib in prostate cancer cells irrespective of androgen receptor status or variant receptor expression

Androgen ablation therapy represents the first line of therapeutic intervention in men with advanced or recurrent prostate tumors. However, the incomplete efficacy and lack of durable response to this clinical strategy highlights an urgent need for alternative treatment options to improve patient outcomes. Targeting the molecular chaperone heat shock protein 90 (Hsp90) represents a potential avenue for therapeutic intervention as its inhibition results in the coordinate blockade of multiple oncogenic signaling pathways in cancer cells. Moreover, Hsp90 is essential for the stability and function of numerous client proteins, a number of which have been causally implicated in the pathogenesis of prostate cancer, including the androgen receptor (AR). Here, we examined the preclinical activity of ganetespib, a small molecule inhibitor of Hsp90, in a panel of prostate cancer cell lines. Ganetespib potently decreased viability in all lines, irrespective of their androgen sensitivity or receptor status, and more effectively than the ansamycin inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). Interestingly, while ganetespib exposure decreased AR expression and activation, the constitutively active V7 truncated isoform of the receptor was unaffected by Hsp90 inhibition. Mechanistically, ganetespib exerted concomitant effects on mitogenic and survival pathways, as well as direct modulation of cell cycle regulators, to induce growth arrest and apoptosis. Further, ganetespib displayed robust antitumor efficacy in both AR-negative and positive xenografts, including those derived from the 22Rv1 prostate cancer cell line that co-expresses full-length and variant receptors. Together these data suggest that further investigation of ganetespib as a new therapeutic treatment for prostate cancer patients is warranted.

Induction of Heat Shock Protein 70 (Hsp70) prevents Neuregulin-Induced Demyelination by Enhancing the Proteasomal Clearance of c-Jun

Modulating molecular chaperones is emerging as an attractive approach to treat neurodegenerative diseases associated with protein aggregation, DPN (diabetic peripheral neuropathy) and possibly, demyelinating neuropathies. KU-32 [N-(7-((2R,3R,4S,5R)-3,4-dihydroxy-5-methoxy-6,6-dimethyl-tetrahydro-2H-pyran-2-yloxy)-8-methyl-2-oxo-2H-chromen-3-yl)acetamide] is a small molecule inhibitor of Hsp90 (heat shock protein 90) and reverses sensory deficits associated with myelinated fibre dysfunction in DPN. Additionally, KU-32 prevented the loss of myelinated internodes induced by treating myelinated SC (Schwann cell)-DRG (dorsal root ganglia) sensory neuron co-cultures with NRG1 (neuregulin-1 Type 1). Since KU-32 decreased NRG1-induced demyelination in an Hsp70-dependent manner, the goal of the current study was to clarify how Hsp70 may be mechanistically linked to preventing demyelination. The activation of p42/p44 MAPK (mitogen-activated protein kinase) and induction of the transcription factor c-Jun serve as negative regulators of myelination. NRG1 activated MAPK, induced c-Jun expression and promoted a loss of myelin segments in DRG explants isolated from both WT (wild-type) and Hsp70 KO (knockout) mice. Although KU-32 did not block the activation of MAPK, it blocked c-Jun induction and protected against a loss of myelinated segments in WT mice. In contrast, KU-32 did not prevent the NRG1-dependent induction of c-Jun and loss of myelin segments in explants from Hsp70 KO mice. Overexpression of Hsp70 in myelinated DRG explants prepared from WT or Hsp70 KO mice was sufficient to block the induction of c-Jun and the loss of myelin segments induced by NRG1. Lastly, inhibiting the proteasome prevented KU-32 from decreasing c-Jun levels. Collectively, these data support that Hsp70 induction is sufficient to prevent NRG1-induced demyelination by enhancing the proteasomal degradation of c-Jun.

LC–MS/MS method for determination of geldanamycin derivative GM-AMPL in rat plasma to support preclinical development

A LC–MS/MS method for determining the concentration of the small molecule Hsp90 inhibitor, GM-AMPL, has been developed and validated in rat plasma to support preclinical development. 17-[2-(morpholine-4-yl)ethyl]amino-17-demethoxygeldanamycin (GM-AMPL) and the internal standard 17-allylamino-17-demethoxygeldanamycin (17-AAG) were sufficiently separated on a Venusil MP C18 column that was eluted with 80% methanol in water at 40°C. Quantification studies were performed with a multiple reaction monitoring (MRM) transition of m/z 657.3→614.3 and 584.3→541.3 for GM-AMPL and IS, respectively, in the negative ion mode. The present method exhibited good linearity (R>0.999) over the concentration range of 2–600ng/mL for GM-AMPL in rat plasma with a lower limit of quantification (LLOQ) of 2ng/mL. The intra-batch and inter-batch assay coefficients of variation (CV) were in range of 1.56–6.84% and 1.62–6.98%, respectively. The plasma samples were extracted with methanol to precipitate protein with extraction recovery in range of 84.09–95.25%. The matrix effect was determined as internal substance (IS) normalized matrix factor of 1.09, 1.18 and 1.05 for samples with three concentration levels of 4, 40 and 400ng/mL, respectively. This validated method was further applied to successfully determine the pharmacokinetic parameters and oral availability of GM-AMPL in Sprague-Dawley rats following intravenous injection and oral administration.