Discovery of XL888: A novel tropane-derived small molecule inhibitor of HSP90

With structural guidance, tropane-derived HTS hits were modified to optimize for HSP90 inhibition and a desirable in vivo profile. Through an iterative SAR development process 12i (XL888) was discovered and shown to reduce HSP90 client protein content in PD studies. Furthermore, efficacy experiments performed in a NCI-N87 mouse xenograft model demonstrated tumor regression in some dosing regimens.     

Alterations in radiation induced cell cycle perturbations by 2-deoxy-D-glucose in human tumor cell lines

In the present studies, effects of glucose analogue, 2-deoxy-D-glucose (2-DG) on radiation-induced cell cycle perturbations were investigated in human tumor cell lines. In unirradiated cells, the levels of cyclin B1 in G2 phase were significantly higher in both the glioma cell lines as compared to squamous carcinoma cells. Upon irradiation with Co60 gamma-rays (2 Gy), the cyclin B1 levels were reduced in U87 cells, while no significant changes could be observed in other cell lines, which correlated well with the transient G2 delay observed under these conditions by the BrdU pulse chase measurements. 2-DG (5 mM, 2 hr) induced accumulation of cells in the G2 phase and a time-dependent increase in the levels of cyclin B1 in both the glioma cell lines, while significant changes could not be observed in any of the squamous carcinoma cell lines. 2-DG enhanced the cyclin B1 level further in all the cell lines following irradiation, albeit to different extents. Interestingly, an increase in the unscheduled expression of B1 levels in G1 phase 48 hr after irradiation was observed in all the cell lines investigated. 2-DG also increased the levels of cyclin D1 at 24 hr in BMG-1 cell line. These observations imply that 2-DG-induced alterations in the cell cycle progression are partly responsible for its radiomodifying effects.     

Proteome-wide changes induced by the Hsp90 inhibitor, geldanamycin in anaplastic large cell lymphoma cells

The molecular chaperone heat shock protein 90 (Hsp90) affects the function of many oncogenic signaling proteins including nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) expressed in anaplastic large cell lymphoma (ALCL). While ALK-positive ALCL cells are sensitive to the Hsp90 inhibitor and the geldanamycin (GA) analog, 17-allylamino-17-demethoxygeldanamycin (17-AAG), the proteomic effects of these drugs on ALK-positive ALCL cells are unpublished. In this study, we investigated the cellular, biologic, and proteomic changes occurring in ALK-positive ALCL cells in response to GA treatment. GA induced G2/M cell cycle arrest and caspase-3-mediated apoptosis. Furthermore, quantitative proteomic changes analyzed by cleavable isotope-coded affinity tag-LC-MS/MS (cICAT-LC-MS/MS) identified 176 differentially expressed proteins. Out of these, 49 were upregulated 1.5-fold or greater and 70 were downregulated 1.5-fold or greater in GA-treated cells. Analysis of biological functions of differentially expressed proteins revealed diverse changes, including induction of proteins involved in the 26S proteasome as well as downregulation of proteins involved in signal transduction and protein and nucleic acid metabolism. Pathway analysis revealed changes in MAPK, WNT, NF-kappaB, TGFbeta, PPAR, and integrin signaling components. Our studies reveal some of the molecular and proteomic consequences of Hsp90 inhibition in ALK-positive ALCL cells and provide novel insights into the mechanisms of its diverse cellular effects.     

Relationships between cdc2 kinase, DNA cross-linking, and cell cycle perturbations induced by nitrogen mustard.

M phase-promoting factor (MPF) consists of a p34cdc2 (cdc2) kinase and cyclin B complex which in its active form promotes G2 to M transition. The role of MPF in G2 arrest following DNA damage, however, has remained largely uncharacterized. We have investigated whether nitrogen mustard (HN2) interfered with either the formation of MPF or its activation. For this purpose, we measured cdc2 kinase activity relative to cdc2 and cyclin B protein turnover and the phosphorylation status of cdc2. Studies were performed in two exceptional human lymphoma cell lines, which differed in HN2 sensitivity by 5-fold (CA46, 50% growth-inhibitory dose = 1.0 microM; JLP119, 50% growth-inhibitory dose = 0.2 microM) but exhibited virtually identical DNA interstrand and DNA-protein cross-link exposure. Following HN2 treatment, CA46 cells ceased to enter mitosis and exhibited a marked delay in G2 phase. Failure to enter mitosis paralleled inhibition of cdc2 kinase. Inhibition was not due to decreased levels of cdc2 or cyclin B protein; rather, G2 arrest correlated with the accumulation of both tyrosine-phosphorylated cdc2 and cyclin B. These findings implied that G2 arrest resulted from a down-regulation of the processes that activate MPF. We also found that JLP119 cells, within a few hours of mitosis at the time of drug treatment, evaded checkpoint control and continued cell division unabated by DNA damage. Furthermore, despite similar DNA cross-link exposure, JLP119 cells within the window of checkpoint control were more susceptible to S phase delay than CA46 cells. Altered cell cycle responses correlated with the greater susceptibility of JLP119 cells to the cytotoxic effects of HN2.     

Method for kinetic analysis of drug-induced cell cycle perturbations.

A method is described for quantitative study of the flux of cells through the cell cycle phases in in vitro systems perturbed by chemicals, such as chemotherapeutic agents. The method utilizes cell count and the flow cytometric technique of bromodeoxyuridine (BrdUrd) labeling, according to an optimized strategy. Cells are exposed to BrdUrd during the last minutes of drug treatment and fixed for analysis at 0, 1/3Ts, 2/3Ts, Ts, and Tc + TG1 recovery times, where Ts, TG1, Tc are the mean durations of phases S and G1 and of the whole cycle of control cells. As an example of application of the proposed procedure, a kinetic study of the effect of 1-(2-chloroethyl)-1-nitrosourea (CNU) on the L1210 cell cycle is described. Simple data analysis, requiring only a pocket calculator, showed that cells in phases G1 and G2M at the end of a 1 h treatment with 1 microgram/ml CNU were fully able to leave these phases but were destined to remain blocked in the following G2M phase (G1 for a minority of them). We also found that cells initially in S phase were slightly delayed in completing their S phase and that 50% of them remained temporarily blocked in the subsequent G2M phase, irrespective of their position in the S phase.     

The decline of porcine sperm motility by geldanamycin, a specific inhibitor of heat-shock protein 90 (HSP90)

Sperm motility is an important parameter for fertility. The molecular mechanisms of mammalian sperm motility are still largely undefined. Our previous observations suggested that heat shock protein 90 (HSP90) may be associated with porcine sperm motility. The aim of the present study was to further characterize the plausible novel function of HSP90 on sperm motility. Semen from normal, sexually mature boars with sperm motility higher than 80% was used. An HSP90-specific inhibitor, geldanamycin (GA), was added to diluted semen at 0.5, 1.0, 2.5 or 5.0 microg/mL and the semen was then incubated at 37 degrees C for 15, 30, 45 or 60 min. Sperm motility was determined by using computer-assisted semen analyzer at the end of incubation. The results indicated that GA significantly reduced sperm motility in a dose and time dependent manner. Moreover, incubation of semen with 5.0 microg/mL GA for 15 min completely stopped sperm motility. To test the reversibility of the GA effect on sperm motility, GA was removed after 30 min incubation and was replaced with fresh extender alone or with extender plus 5 mM caffeine, then incubated for another 15, 30, 45 or 60 min. The results showed that simply removing GA did not reverse the inhibitory effect on sperm motility, while adding caffeine partially reversed this inhibitory effect. However, the effect of 2.5 or 5.0 microg/mL GA was not reversed by caffeine. Considering the specificity of GA targeting to HSP90, the above observations suggested that HSP90 may play a crucial role in regulating porcine sperm motility.     

The anti-myeloma activity of a novel purine scaffold HSP90 inhibitor PU-H71 is via inhibition of both HSP90A and HSP90B1

Background

The ribonucleotide reductase M1 (RRM1) gene encodes the regulatory subunit of ribonucleotide reductase, the molecular target of gemcitabine. The overexpression of RRM1 mRNA in tumor tissues is reported to be associated with gemcitabine resistance. Thus, single nucleotide polymorphisms (SNPs) of the RRM1 gene are potential biomarkers of the response to gemcitabine chemotherapy. We investigated whether RRM1 expression in peripheral blood mononuclear cells (PBMCs) or SNPs were associated with clinical outcome after gemcitabine-based chemotherapy in advanced non-small cell lung cancer (NSCLC) patients.

Methods

PBMC samples were obtained from 62 stage IIIB and IV patients treated with gemcitabine-based chemotherapy. RRM1 mRNA expression levels were assessed by real-time PCR. Three RRM1 SNPs, -37C→A, 2455A→G and 2464G→A, were assessed by direct sequencing.

Results

RRM1 expression was detectable in 57 PBMC samples, and SNPs were sequenced in 56 samples. The overall response rate to gemcitabine was 18%; there was no significant association between RRM1 mRNA expression and response rate (P = 0.560). The median progression-free survival (PFS) was 23.3 weeks in the lower expression group and 26.9 weeks in the higher expression group (P = 0.659). For the -37C→A polymorphism, the median PFS was 30.7 weeks in the C(-)37A group, 24.7 weeks in the A(-)37A group, and 23.3 weeks in the C(-)37C group (P = 0.043). No significant difference in PFS was observed for the SNP 2455A→G or 2464G→A.

Conclusions

The RRM1 polymorphism -37C→A correlated with PFS in NSCLC patients treated with gemcitabine-based chemotherapy. No significant correlation was found between PBMC RRM1 mRNA expression and the efficacy of gemcitabine.     

Extracellular HSP90: Conquering the cell surface

Heat shock protein 90 (HSP90) is a highly conserved molecular chaperone, assisting intracellularly in the folding and conformational regulation of a multitude of client proteins that play a crucial role in growth, cell survival and developmental processes(1). Moreover HSP90 interacts with a great number of molecules that are involved in the development and/or survival of cancer cells, allowing mutant proteins to retain or gain function while permitting cancer cells to tolerate the imbalanced signaling that such oncoproteins create (2,3). Prime examples include the HER-2 receptor, c-Raf-1, Akt/PKB, CDK4, and mutant p53 (4,5). Highly specific inhibitors of HSP90 have been identified and are currently under clinical evaluation. These include geldanamycin and its derivatives 17-allylamino-17-demethoxygeldanamycin and 17-dimethylaminoethylamino-17-demethoxygeldanamycin, which inhibit cancer cell proliferation in vitro and tumor growth in vivo (6-9).     

Uncoupled cell cycle without mitosis induced by a protein kinase inhibitor, K-252a

The staurosporine analogues, K-252a and RK-286C, were found to cause DNA re-replication in rat diploid fibroblasts (3Y1) without an intervening mitosis, producing tetraploid cells. Analysis of cells synchronized in early S phase in the presence of K-252a revealed that initiation of the second S phase required a lag period of 8 h after completion of the previous S phase. Reinitiation of DNA synthesis was inhibited by cycloheximide, actinomycin D, and serum deprivation, but not by Colcemid, suggesting that a functional G1 phase dependent on de novo synthesis of protein and RNA is essential for entry into the next S phase. In a src-transformed 3Y1 cell line, as well as other cell lines, giant cells containing polyploid nuclei with DNA contents of 16C to 32C were produced by continuous treatment with K-252a, indicating that the agent induced several rounds of the incomplete cell cycle without mitosis. Although the effective concentration of K-252a did not cause significant inhibition of affinity-purified p34cdc2 protein kinase activity in vitro, in vivo the full activation of p34cdc2 kinase during the G2/M was blocked by K-252a. On the other hand, the cyclic fluctuation of partially activated p34cdc2 kinase activity peaking in S phase still continued. These results suggest that a putative protein kinase(s) sensitive to K-252a plays an important role in the mechanism for preventing over-replication after completion of previous DNA synthesis. They also suggest that a periodic activation of p34cdc2 is required for S phases in the cell cycle without mitosis.     

低表达HSP90α和高表达HSP90β HepG2细胞株的建立

目的:建立HSP90α低表达和HSP90β高表达HepG2细胞株。方法:通过电转染方法将质粒pSilencerHSP90α和pSmycHSP90β转染入HepG2细胞中,应用Western-blotting和MTT法分别鉴定转染效果及绘制细胞生长曲线。结果:带有HSP90αsiRNA片段的质粒和带有HSP90β片段的质粒成功转入HepG2中,转染细胞与未转染细胞生长情况无差别。结论:电转染方法可以有效地将质粒转染入HepG2中去,转染细胞的生长情况将不会影响后续实验的结果。     

Identification of vibsanin A analog as a novel HSP90 inhibitor

Vibsanin A is the first natural product isolated from Viburnum awabuki and has several biological activities. We have reported that a vibsanin A analog, obtained from process of total synthesis of vibsanin A, has anti-proliferative activity against human cancer cell lines. In this study, we evaluated anti-proliferative effect of the vibsanin A analogs against various human cancer cell lines and examined molecular target of the analog in human cells. Among the vibsanin A analogs, vibsanin A analog C (VAC) showed anti-proliferative effect against various cancer cell lines, and the anti-proliferative activity was strongest among the vibsanin A analogs. Additionally, VAC fluctuated amounts of HSP90-related proteins in cells and inhibited HSP90-mediated protein refolding of luciferase in vitro. These results suggest that the anti-proliferative activity of VAC is due to HSP90 inhibition, and VAC has a potential as novel anti-cancer drug as HSP90 inhibitor.     

NIX protein enhances antioxidant capacity of and reduces the apoptosis induced by HSP90 inhibitor luminespib/NVP-AUY922 in PC12 cells

Pheochromocytomas and paragangliomas (PCPGs) are catecholamine-producing neuroendocrine tumors. Accumulating evidences indicate that the blockade of antioxidative pathways might be a novel therapeutic approach to the treatment of PCPG. NIX has been confirmed to play a key role in maintaining redox homeostasis in tumors, while the function of NIX in PCPG remains unclear. In this study, the analyses of the disease-free survival (DFS) showed that high NIX protein level is related to poor prognosis in patients of PCPG. Consistent with this, high level of NIX protein upregulates the level of p-NF-κB and promotes the migration of PC12 cells. In NIX-over-expressing PC12 cells, the level of reactive oxygen species (ROS) is decreased while trolox-equivalent antioxidant capacity (TEAC) increased. But in NIX-silencing cells, ROS level is increased, while TEAC reversely reduced, consequently antioxidase and phase II enzymes of NRF2 signaling were activated, and elevated endoplasmic reticulum (ER) stress was observed. Additionally, the apoptosis induced by luminespib/NVP-AUY922, an inhibitor of heat shock protein 90 (HSP90, a cellular stress response factor), was enhanced in NIX-silencing cells but reduced in the NIX-over-expressing cells. All of these results indicated that high NIX protein level enhances antioxidant capacity of PC12 cells and reduces the apoptosis caused by cell stress, such as induced by luminespib/NVP-AUY922. Therefore, luminespib/NVP-AUY922 might be effective only for PCPG with low NIX level, while targeting NIX could be a further supplement to the therapeutic treatment strategy for PCPG patients with high NIX protein level.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01193-6.     

Hsp90 phosphorylation,Wee1 and the cell cycle

Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone in eukaryotic cells, and it maintains the functional conformation of a subset of proteins that are typically key components of multiple regulatory and signaling networks mediating cancer cell proliferation, survival, and metastasis. It is possible to selectively inhibit Hsp90 using natural products such as geldanamycin (GA) or radicicol (RD), which have served as prototypes for development of synthetic Hsp90 inhibitors. These compounds bind within the ADP/ATP-binding site of the Hsp90 N-terminal domain to inhibit its ATPase activity. As numerous N-terminal domain inhibitors are currently undergoing extensive clinical evaluation, it is important to understand the factors that may modulate in vivo susceptibility to these drugs. We recently reported that Wee1Swe1-mediated, cell cycle-dependent, tyrosine phosphorylation of Hsp90 affects GA binding and impacts cancer cell sensitivity to Hsp90 inhibition. This phosphorylation also affects Hsp90 ATPase activity and its ability to chaperone a selected group of clients, comprised primarily of protein kinases. Wee1 regulates the G2/M transition. Here we present additional data demonstrating that tyrosine phosphorylation of Hsp90 by Wee1Swe1 is important for Wee1Swe1 association with Hsp90 and for Wee1Swe1 stability. Yeast expressing non-phosphorylatable yHsp90-Y24F, like swe1? yeast, undergo premature nuclear division that is insensitive to G2/M checkpoint arrest. These findings demonstrate the importance of Hsp90 phosphorylation for proper cell cycle regulation.     

HSP90 modulates actin dynamics: Inhibition of HSP90 leads to decreased cell motility and impairs invasion

HSP90, a major molecular chaperone, plays an essential role in the maintenance of several signaling molecules. Inhibition of HSP90 by inhibitors such as 17-allylamino-demethoxy-geldanamycin (17AAG) is known to induce apoptosis in various cancer cells by decreasing the activation or expression of pro-survival molecules such as protein kinase B (Akt). While we did not observe either decrease in expression or activation of pro-survival signaling molecules in human breast cancer cells upon inhibiting HSP90 with 17AAG, we did observe a decrease in cell motility of transformed cells, and cell motility and invasion of cancer cells. We found a significant decrease in the number of filopodia and lamellipodia, and in the F-actin bundles upon HSP90 inhibition. Our results show no change in the active forms or total levels of FAK and Pax, or in the activation of Rac-1 and Cdc-42; however increased levels of HSP90, HSP90α and HSP70 were observed upon HSP90 inhibition. Co-immuno-precipitation of HSP90 reveals interaction of HSP90 with G-actin, which increases upon HSP90 inhibition. FRET results show a significant decrease in interaction between actin monomers, leading to decreased actin polymerization upon HSP90 inhibition. We observed a decrease in the invasion of human breast cancer cells in the matrigel assay upon HSP90 inhibition. Over-expression of αB-crystallin, known to be involved in actin dynamics, did not abrogate the effect of HSP90 inhibition. Our work provides the molecular mechanism by which HSP90 inhibition delays cell migration and should be useful in developing cancer treatment strategies with known anti-cancer drugs such as cisplatin in combination with HSP90 inhibitors.     

Differential regulation of HSC70, HSP70, HSP90 alpha, and HSP90 beta mRNA expression by mitogen activation and heat shock in human lymphocytes

A subset of heat shock proteins, HSP90 alpha, HSP90 beta, and a member of the HSP70 family, HSC70, shows enhanced synthesis following mitogenic activation as well as heat shock in human peripheral blood mononuclear cells. In this study, we have examined expression of mRNA for these proteins, including the major 70-kDa heat shock protein, HSP70, in mononuclear cells following either heat shock or mitogenic activation with phytohemagglutinin (PHA), ionomycin, and the phorbol ester, tetradecanoyl phorbol acetate. The results demonstrate that the kinetics of mRNA expression of these four genes generally parallel the kinetics of enhanced protein synthesis seen following either heat shock or mitogen activation and provide clear evidence that mitogen-induced synthesis of HSC70 and HSP90 is due to increased mRNA levels and not simply to enhanced translation of preexisting mRNA. Although most previous studies have focused on cell cycle regulation of HSP70 mRNA, we found that HSP70 mRNA was only slightly and transiently induced by PHA activation, while HSC70 is the predominant 70-kDa heat shock protein homologue induced by mitogens. Similarly, HSP90 alpha appears more inducible by heat shock than mitogens while the opposite is true for HSP90 beta. These results suggest that, although HSP70 and HSC70 have been shown to contain similar promoter regions, additional regulatory mechanisms which result in differential expression to a given stimulus must exist. They clearly demonstrate that human lymphocytes are an important model system for determining mechanisms for regulation of heat shock protein synthesis in unstressed cells. Finally, based on kinetics of mRNA expression, the results are consistent with the hypothesis that HSC70 and HSP90 gene expression are driven by an IL-2/IL-2 receptor-dependent pathway in human T cells.     

Chemosensitivity of human small cell carcinoma of the lung detected by flow cytometric DNA analysis of drug-induced cell cycle perturbations in vitro

A method based on detection of drug-induced cell cycle perturbation by flow cytometric DNA analysis has previously been described in Ehrlich ascites tumors as a way to estimate chemosensitivity. The method is extended to test human small-cell carcinoma of the lung. Three tumors with different sensitivities to melphalan in nude mice were used. Tumors were disaggregated by a combined mechanical and enzymatic method and thereafter have incubated with different doses of melphalan. After incubation the cells were plated in vitro on agar, and drug induced cell cycle changes were monitored by flow cytometric DNA analysis. Melphalan produced a dose-related S phase accumulation in the two sensitive tumors, whereas no changes in the cell cycle distribution were found in the resistant tumor. The size of S phase accumulation correlated to the chemosensitivity in vivo. For low concentrations of melphalan, the S phase accumulation was accompanied by G2 + M accumulation. The results indicate that the method may be extended to sensitivity testing of human solid tumors, including screening for new agents.     

HSP90高表达细胞株的建立及细胞增殖性状的改变

目的:建立稳定高表达热休克蛋白90(HSP90)细胞株,研究其对细胞增殖的影响.方法:含人HSP90 13全长基因的重组质粒pSmycHSP经亚克隆、纯化、酶切鉴定后,用电穿孔法转染到小鼠成纤维细胞系NIH-3T3细胞内.经G418筛选、克隆分离培养,用免疫细胞化学、免疫印迹鉴定阳性克隆.以转染空质粒的NIH-3T3细胞为对照,用MTT法、流式细胞术测定,分析HSP90高表达对细胞增殖和细胞周期的影响.结果:转染pSmycHSP的NIH-3T3细胞HSP90染色增强,生长速度减慢,S期DNA含量降低.结论:己建立稳定高表达热休克蛋白90(HSP90)NIH-3T3细胞株;转染pSmycHSP的NIH-3T3细胞能够有效地表达HSP90,影响细胞周期,使细胞增殖迟滞.     

DUSP4 promotes esophageal squamous cell carcinoma progression by dephosphorylating HSP90β

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