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Research ArticleArticle

Synthesis and Evaluation of a Novel Deguelin Derivative, L80, which Disrupts ATP Binding to the C-terminal Domain of Heat Shock Protein 90

Su-Chan Lee, Hye-Young Min, Hoon Choi, Ho Shin Kim, Kyong-Cheol Kim, So-Jung Park, Myeong A Seong, Ji Hae Seo, Hyun-Ju Park, Young-Ger Suh, Kyu-Won Kim, Hyun-Seok Hong, Hee Kim, Min-Young Lee, Jeewoo Lee and Ho-Young Lee
Molecular Pharmacology August 2015, 88 (2) 245-255; DOI: https://doi.org/10.1124/mol.114.096883

Abstract

The clinical benefit of current anticancer regimens for lung cancer therapy is still limited due to moderate efficacy, drug resistance, and recurrence. Therefore, the development of effective anticancer drugs for first-line therapy and for optimal second-line treatment is necessary. Because the 90-kDa molecular chaperone heat shock protein (Hsp90) contributes to the maturation of numerous mutated or overexpressed oncogenic proteins, targeting Hsp90 may offer an effective anticancer therapy. Here, we investigated antitumor activities and toxicity of a novel deguelin-derived C-terminal Hsp90 inhibitor, designated L80. L80 displayed significant inhibitory effects on the viability, colony formation, angiogenesis-stimulating activity, migration, and invasion of a panel of non–small cell lung cancer cell lines and their sublines with acquired resistance to paclitaxel with minimal toxicity to normal lung epithelial cells, hippocampal cells, vascular endothelial cells, and ocular cells. Biochemical analyses and molecular docking simulation revealed that L80 disrupted Hsp90 function by binding to the C-terminal ATP-binding pocket of Hsp90, leading to the disruption of the interaction between hypoxia-inducible factor (HIF)-1α and Hsp90, downregulation of HIF-1α and its target genes, including vascular endothelial growth factor (VEGF) and insulin-like growth factor 2 (IGF2), and decreased the expression of various Hsp90 client proteins. Consistent with these in vitro findings, L80 exhibited significant antitumor and antiangiogenic activities in H1299 xenograft tumors. These results suggest that L80 represents a novel C-terminal Hsp90 inhibitor with effective anticancer activities with minimal toxicities.

Introduction

Lung cancer is the leading cause of cancer-related human deaths worldwide. Despite several decades of intense efforts to develop therapeutic interventions, the 5-year survival rate for lung cancer is still less than 20% (Jemal et al., 2011; Siegel et al., 2013). Lung cancer is briefly classified into non–small cell lung cancer (NSCLC) and small cell lung cancer, and about 85–90% of lung cancers are diagnosed as NSCLC. Based on chemotherapy resistance and toxicity, the focus of anticancer drug development has moved to therapies that target specific cellular molecules (Dobbelstein and Moll, 2014). Some therapies have resulted in remarkable tumor reductions in some types of lung cancers (Sharma et al., 2007; Lee et al., 2013); however, many patients do not respond to these drugs, and initially responsive patients ultimately develop resistance after prolonged, continuous treatment. Therefore, the development of novel therapeutic drugs that involve identified molecular mechanisms and promising antitumor activities in both chemotherapy-naive and resistant cancer cells with suitable therapeutic indices needs to be investigated.

Deguelin, a naturally occurring rotenoid, might be considered as a promising lead compound for the rapid production of structural analogs for lung cancer treatment. In previous studies, we and others have demonstrated that deguelin exhibits profound antiproliferative effects on human premalignant and malignant bronchial epithelial cells and a variety of cancer cells without cytotoxicity to normal cells (Chun et al., 2003; Murillo et al., 2009; Lee et al., 2010; Thamilselvan et al., 2011; Kang et al., 2012; Mehta et al., 2013a,b; Suh et al., 2013). Furthermore, deguelin has shown in vivo effectiveness in suppressing the growth of xenograft tumors and lung tumor formation in tobacco carcinogen-treated A/J mice and in genetically engineered mice with mutant K-Ras (Chun et al., 2003; Lee et al., 2005; Kim et al., 2008; Woo et al., 2009). Structural biology and molecular modeling techniques have revealed that deguelin exhibits antitumor and antiangiogenic activities by targeting heat shock protein 90 (Hsp90), which functions as a molecular chaperone and is required for the stability and proper function of many oncogenic proteins, including hypoxia inducible factor (HIF)-1α; mutated p53, mitogen-activated protein kinase kinase (MEK1/2); cyclin-dependent kinase 4; erbB2; and Akt (Oh et al., 2007, 2008).

A concern on the use of deguelin as an anticancer drug is its potential toxicities, which could be an obstacle to its clinical development. A previous study reported a Parkinson’s disease–like syndrome in rats treated with deguelin at high doses (Caboni et al., 2004). In addition, it was reported that Hsp90 inhibitors may induce ocular toxicity (Kanamaru et al., 2014). Although it is not clear whether therapeutic doses of deguelin would induce the side effects and whether the side effects can be relieved after drug withdrawal, the potential toxicities of deguelin can be a considerable obstacle to its clinical use. To circumvent these issues, we investigated to develop deguelin analogs that harbor less or no potential toxicity to various normal cells.

The aim of the current study was to evaluate the antitumor effects and potential toxicity of a novel deguelin analog, designated (6,7-dimethoxyquinolin-4-yl)(5-methoxy-2,2-dimethyl-2H-chromen-6-yl)methyl acetate (L80; Fig. 1A) and to investigate the mechanisms underlying the anticancer activities of this analog in lung cancer. We found that L80 has significant antitumor and antiangiogenic activities in vitro in multiple lines of NSCLC cells and their sublines carrying acquired chemoresistance and in vivo in xenograft tumors. L80 was found to possess minimal toxicities to normal lung epithelial cells, hippocampal cells, retinal pigment epithelial cells, and vascular endothelial cells, whereas deguelin induced significant cytotoxicities in these cells. L80 appeared to disrupt Hsp90 function by directly interacting with the C-terminal domain of Hsp90 and the concomitant destabilization of multiple Hsp90 client proteins, including HIF-1α. Collectively, these results suggest the potential use of L80 as a novel C-terminal Hsp90 inhibitor with effective antitumor activity without severe side effects.

Fig. 1.
Fig. 1.

Minimal toxic effect of L80 in normal cells compared with deguelin. (A) Chemical structure of deguelin and L80. (B) Blood-brain barrier permeability assay was conducted by using the PAMPA Explorer kit (pION Inc., Billerica, MA). Progesterone and theophylline were used as positive and negative controls. Pro, progesterone; Theo, theophylline; Deg, deguelin. (C–E) Mouse hippocampal (HT-22), HUVEC, retinal pigment epithelial (RPE), and normal immortalized lung epithelial (HBE and BEAS-2B) cells were treated with increasing concentrations of L80 or deguelin for 3 days. Values represent mean ± S.D. of experiments conducted in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way analysis of variance or Student’s t test compared with control group.

Materials and Methods

Detailed experimental procedures are described in the Supplemental Material.

Reagents.

Antibodies against cleaved caspase-3, β-tubulin, Akt, and ErbB2 were purchased from Cell Signaling Technology (Danvers, MA). Antibodies against cleaved poly-(ADP-ribose) polymerase and HIF-1α were purchased from BD Biosciences (San Jose, CA). Ni-NTA agarose was purchased from Invitrogen (Carlsbad, CA). The first-strand cDNA synthesis kit was purchased from Dakara Korea Biomedical, Inc. (Seoul, Republic of Korea). The Econotaq DNA polymerase was purchased from Lucigen Corp. (Middleton, WI). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), propidium iodide, crystal violet, and other chemicals were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise specified.

Cell Culture.

H1299, A549, and BEAS-2B cells were purchased from the American Type Culture Collection (Manassas, VA). Human bronchial epithelial (HBE) cells were kindly provided by Dr. John Minna (The University of Texas Southwestern Medical Center, Dallas, TX). Human umbilical endothelial cells (HUVECs) were purchased from Invitrogen. H226B cells were kindly provided by Dr. Jack A. Roth (The University of Texas M. D. Anderson Cancer Center, Houston, TX). H460/R and H226B/R cells were generated by continuous exposure to increasing concentrations of paclitaxel for more than 6 months (S.-H. Park et al., manuscript in preparation). NSCLC cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and antibiotics. BEAS-2B and HBE cells were maintained in K-SFM (Invitrogen) supplemented with epidermal growth factor and bovine pituitary extracts. Cells were incubated at 37°C with 5% CO2 in a humidified atmosphere. These cell lines were authenticated by Genetic Resources Core Facility of Johns Hopkins University or by the Korean Cell Line Bank using AmplFLSTR identifiler PCR Amplification kit (Applied Biosystems, Foster, CA).

MTT Assay.

To evaluate the effect of L80 on the proliferation of lung cancer cells, MTT assays were performed as described previously (Oh et al., 2007; Chang et al., 2012). Cells (1–2 × 103 cells/well in 96-well plates) were treated with increasing concentrations of L80 for 3 days. The drug-containing medium was replaced at 2 days after drug treatment. Cells were treated with MTT (final concentration of 200 μg/ml) and incubated for 2–4 hours at 37°C. The formazan products were dissolved in dimethylsulfoxide, and the absorbance was measured at 570 nm. The data are presented as a percentage of the control group.

Immunoprecipitation and Pull-Down Assay.

Immunoprecipitation, the purification of Hsp90 proteins, and a pull-down assay to determine the competitive binding to the ATP-binding pocket using ATP-agarose (Innova Biosciences, Cambridge, UK) were performed according to our previous report (Oh et al., 2007).

Animal Studies.

All animal procedures were performed using a protocol approved by the Seoul National University Institutional Animal Care and Use Committee (approval No. SNU-130820-6), which was in line with the National Institutes of Health guideline. For xenograft experiments, H1299 cells (4 × 106 cells/mouse) were subcutaneously injected into the right flank of a 6-week-old NOD/SCID mouse. After the tumor volume reached 50–150 mm3, mice were randomly grouped and treated with vehicle (10% dimethylsulfoxide in corn oil) or L80 (20 mg/kg) six times per week for 3 weeks by intraperitoneal injection. Tumor growth was determined by measuring the short and long diameters of the tumor with a caliper, and body weight was measured twice per week to monitor toxicity. The tumor volume was calculated using the following formula: tumor volume (mm3) = (short diameter)2 × (long diameter) × 0.5.

Immunohistochemistry.

Immunohistochemical analysis to detect CD31 and cleaved caspase-3 expression in the tumors were performed as described previously (Oh et al., 2007).

Statistics.

Data are presented as the mean ± S.D. The results not included in the main figures are shown in the Supplemental Material. The statistical significance was determined using either two-sided Student’s t test or one-way analysis of variance followed by Dunnett’s post hoc test using GraphPad Prism 6 (GraphPad Software Inc., La Jolla, CA). A P value less than 0.05 was considered to be statistically significant.

Results

Synthesis of L80.

As mentioned previously, we synthesized a series of structural deguelin analogs to develop lead compounds with enhanced efficacy and/or reduced toxicity compared with deguelin (Kim et al., 2008; Chang et al., 2012). A detailed scheme and the L80 synthesis procedure are described in the Supplemental Material. L80 has a dimethoxy quinoline ring and a 1,2-unsaturated benzopyran ring linked by one carbon with an acetyl group. 4-Hydroxy quinolone (compound 2) was synthesized from 2′-amino-4′,5′-dimethoxyacetophenone (compound 1) through cyclization. The hydroxyl group was converted to a bromo group by treatment with PBr3 to give compound 3. Brominated compound 3 was coupled with an aldehyde (compound 4) to obtain the desired hydroxyl compound 5. The aldehyde compound 4 was synthesized from 2,4-dihydroxybenzaldehyde (compound 7) and 3-methylbut-2-enal through regioselective cyclization and continuous methylation by Jacob’s procedure. Finally, compound 6 (L80) (Fig. 1A) was formed by the acetylation of compound 5.

L80 Exhibited Markedly Reduced Toxicity to Normal Cells Compared with Deguelin.

Previous research reported that high concentrations of deguelin can induce behavioral disorder similar to Parkinson’s disease–like syndrome in rats treated with deguelin at high doses (Caboni et al., 2004). We first assessed the possible permeation of L80 and deguelin through the blood-brain barrier (BBB) by performing the parallel artificial membrane permeability assay (Kansy et al., 1998). The parallel artificial membrane permeability assay permits the fast determination of artificial membrane permeation properties of small molecules by passive diffusion (Kansy et al., 1998) Each compound can be classified as either permeable [central nervous system (CNS)+; −log Pe < 5.4] or impermeable (CNS−; −log Pe > 5.7) (Di et al., 2003). The −log Pe for deguelin and L80 were 4.78 ± 0.20 and 4.82 ± 0.25, respectively (Fig. 1B; Supplemental Fig. S1A; Supplemental Table S1), suggesting that the two drugs may be BBB permeable. We then tested the effects of L80 and deguelin on viability of hippocampal cell line, HT-22. Treatment with deguelin showed significant cytototoxicity in HT-22 cells in a concentration-dependent manner (Fig. 1C; Supplemental Fig. S1B). In contrast, L80 displayed no obvious inhibitory effects on the viability of HT-22 cells even at the highest concentration of 10 μM. These data suggest that, although both deguelin and L80 could be permeable to BBB, L80 may be less toxic to the CNS compared with its mother compound deguelin. We further tested the potential cytotoxicity of L80 and deguelin on normal lung epithelial, retinal pigment epithelial, and vascular endothelial cells. To this end, immortalized human bronchial epithelial cell lines (HBE and BEAS-2B), retinal pigment epithelial cells, and HUVECs were incubated with L80 or deguelin. More than 80% of HBECs were viable after treatment with 10 μM L80 (Fig. 1D; Supplemental Fig. S1C). In contrast, deguelin treatment even at 1 μM showed significant decrease in lung epithelial cell lines. Similarly, cytotoxic effects of L80 on vascular endothelial and retinal epithelial cells were less compared with deguelin (Fig. 1E; Supplemental Fig. S1, D and E). These overall data indicate markedly reduced cytotoxicity of L80 to normal cells compared with deguelin.

L80 Functions as a Potent Inhibitor of NSCLC Cell Proliferation and Anchorage-Dependent and Anchorage-Independent Colony Formation.

In a previous study, we demonstrated that deguelin inhibits the growth of and induces apoptosis in NSCLC cell lines, with minimum effects on normal HBE cells at in vitro concentrations that are attainable in vivo (Lee et al., 2004). Hence, we first examined the effects of L80 on viability of NSCLC cell lines (i.e., H1299, A549, H460, and H226B). Because resistance to traditional chemotherapeutic agents is an obstacle to anticancer therapies, we also included NSCLC cell sublines (i.e., H226B/R, H460/R) with acquired resistance against paclitaxel after prolonged exposure. These cell lines were treated with 10−7 to 10−5 M L80 for 3 days; then cell viability was measured by the MTT assay. As shown in Fig. 2A and Supplemental Fig. S2A, L80 significantly inhibited viability of human NSCLC cells. The inhibitory effect of L80 on the drug-resistant sublines (H460/R and H226B/R) was comparable with that on the corresponding parental cells (H460 and H226B), suggesting that L80 might be applicable to both naive and chemo-resistant lung cancer cells (Fig. 2, A and B; Supplemental Fig. S1A). We next examined the effects of L80 on the anchorage-dependent colony forming abilities of the NSCLC cells. As illustrated in Fig. 2C and Supplemental Fig. S2B, all tested NSCLC cells showed significantly decreased colony formation after 0.1 to 10 μM of L80 treatment. Because tumorigenic cells can often grow in semisolid media, such as soft agar (Hanahan and Weinberg, 2000), we further tested the effects of L80 on colony formation of NSCLC cells using a semisolid culture condition. Consistent with the cells grown in monolayer culture conditions, L80 strongly suppressed the indicated NSCLC cell growth in soft agar (Fig. 2D; Supplemental Fig. S2C). Notably, H460/R and H226B/R cells formed significantly fewer colonies in soft agar than their corresponding parental cells after treatment with 10 μM L80, suggesting that L80 might deregulate cellular events that regulate anchorage-independent colony forming abilities of NSCLC cells carrying acquired paclitaxel resistance. These results suggest that L80 effectively inhibits survival, and the colony-forming abilities of both naive and chemo-resistant NSCLC cells with minimal toxicity to normal HBE cells.

Fig. 2.
Fig. 2.

Inhibitory effects of L80 on the proliferation and colony formation of human lung cancer cells. (A–B) H1299, A549, H460, and H226B cells and paclitaxel-resistant lung cancer cells (H460/R and H226B/R) were treated with increasing concentrations of L80 or deguelin (Deg) for 3 days. Cell viability was determined by the MTT assay. (C) H1299, A549, H460, H460/R, H226B, and H226B/R cells were seeded onto six-well plates at a density of 300 cells/well and then treated with L80 (0, 0.1, 1, and 10 μM). After two weeks, colonies were stained with crystal violet and counted. (D) The anchorage-independent growth of cells treated with increasing concentrations of L80 was determined by soft agar colony formation assay. All values represent mean ± S.D. of experiments conducted in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 by one-way analysis of variance compared with control group.

L80 Induces Apoptotic Cell Death in NSCLC Cells.

To investigate the mechanisms by which L80 inhibited cancer cell proliferation and colony formation, the effect of L80 on cell cycle progression was examined by flow cytometry. Treatment with 10 μM L80 induced an accumulation of 20.8% of H1299 cells, 18.6% of H460 cells, 9% of H226 cells, 11.3% of H226B/R cells, and 20.8% of H460/R cells in the sub-G0/G1 phase, suggesting that it has proapoptotic activity in NSCLC cells (Fig. 3A; Supplemental Fig. S3A). L80-induced apoptosis was further supported by Western blot analysis showing the concentration-dependent increase in poly-(ADP-ribose) polymerase cleavage (Fig. 3B). These results indicate that the antiproliferative activities of L80 in NSCLC cells are at least in part due to increased apoptosis.

Fig. 3.
Fig. 3.

Induction of apoptosis by treatment with L80. (A) Cells were treated with L80 for 24 hours. The distribution of cells in each phase of the cell cycle was analyzed by flow cytometry. (B) The expression level of cleaved poly-(ADP-ribose) polymerase (PARP) after treatment with L80 in H1299 and H460/R cells was analyzed by Western blot analysis.

L80 Suppresses HIF-1α Expression and Angiogenic Activities in NSCLC Cells.

Based on the previous findings regarding the antiangiogenic activity of deguelin through suppression of HIF-1α (Oh et al., 2007, 2008), we tested the effect of L80 on HIF-1α expression and its target genes, including VEGF and IGF2. Treatment with L80 under hypoxic conditions suppressed the expression of HIF-1α in a concentration-dependent manner (Fig. 4A; Supplemental Fig. S4A) and significantly downregulated hypoxia response element–dependent reporter activity (Fig. 4B; Supplemental Fig. S4B). Consistent with these results, the VEGF and IGF2 mRNA and protein expression levels were markedly decreased in L80-treated NSCLC cells (Fig. 4C; Supplemental S4C), indicating that the L80-mediated decrease in HIF-1α transcriptional activity led to a downregulation of VEGF expression. The inhibitory effect of L80 on VEGF expression was comparable with that of deguelin (Fig. 4D; Supplemental Fig. S4D). Because tumor angiogenesis is stimulated by angiogenic factors secreted from cancer cells, we analyzed whether L80 could inhibit the angiogenesis-stimulating activities of NSCLC cells. Thus, we assessed the effects of conditioned medium (CM) collected from L80-treated H1299 cells on HUVECs. We observed that treatment with CM from L80-treated H1299 cells induced significantly decreased HUVEC tube formation compared with CM from vehicle-treated cells (Fig. 4E; Supplemental Fig. S4E). These findings suggest that L80 possesses the ability to suppress tumor angiogenesis by inhibiting HIF-1α expression.

Fig. 4.
Fig. 4.

L80 exerts its antiangiogenic potential through inhibiting the stability and transcriptional activity of HIF-1α. (A) The HIF-1α expression level in the indicated NSCLC cells treated with increasing concentrations of L80 under hypoxic conditions was analyzed by Western blot analysis. (B) The indicated NSCLC cells were cotransfected with pGL3-basic or pGL3-HRE-Luc and pRL-TK-Luc. After treatment with L80 under hypoxic conditions, luciferase activity was monitored as described in Materials and Methods. Values represent mean ± S.D. of experiments conducted in triplicate. *P < 0.05 and **P < 0.01, by Student’s t test compared with control group. The indicated NSCLC cells treated with increasing concentrations of L80 (C) or H1299 treated with 1 μM of deguelin (Deg) or L80 (D) were subjected to RT-PCR and Western blot analysis for the mRNA and protein expression of VEGF and IGF2. (E) HUVECs were treated with CM obtained from H1299 cells treated with increasing concentrations of L80 and incubated under hypoxic conditions. The morphologic changes of HUVECs were imaged and scored. Values represent mean ± S.D. of experiments conducted in triplicate. *P < 0.05, ***P < 0.001 by one-way analysis of variance compared with control group.

L80 Decreases Migration and Invasion of NSCLC Cells.

Deguelin suppresses the migration and invasion of various types of cancer cells, including pancreatic and prostate cancer cells (Thamilselvan et al., 2011; Boreddy and Srivastava, 2013). Hence, we examined the effects of L80 on NSCLC cell migration and the invasion of NSCLC cells by performing in vitro migration and invasion assays. We observed that pretreatment with 10 μM L80 for 24 hours, which showed no detectable effect on cell proliferation (Fig. 5A; Supplemental Fig. S5A), had consistent inhibitory effects on the migration (Fig. 5B; Supplemental Fig. S5B) and invasion (Fig. 5C; Supplemental Fig. S5C) of H1299, H226B, and H226B/R cells. These findings suggest that L80 might regulate cellular events involved in the migration and invasion of NSCLC cells and their sublines with acquired paclitaxel resistance.

Fig. 5.
Fig. 5.

Inhibition of migration and invasion of lung cancer by treatment with L80. H1299, H226B, and H226B/R cells were seeded onto 96-well plates and incubated for 12 hours to evaluate proliferation effects by MTT assay (A). These cells also were seeded onto Transwells coated with either gelatin only (B) or gelatin and matrigel (C). After incubation for 18–19 hours, the migratory or invaded cells on the bottom of the membrane were stained and counted. All values represent mean ± S.D. of experiments conducted in triplicate. *P < 0.05, **P < 0.01, and ***P < 0.001 by Student’s t test compared with control group.

Effects of L80 on Tumor Growth and CD31 Expression.

We further evaluated the antitumor activities of L80 in H1299 xenograft tumors established in NOD/SCID mice. Mice bearing tumors (with volumes of 50–150 mm3) were randomly grouped and received L80 (20 mg/kg body weight) for 3 weeks. L80 significantly suppressed the growth of H1299 xenograft tumors (Fig. 6A). During the treatment, the body weight of mice did not significantly change between the control and L80-treated groups, suggesting minimal toxicity of L80 to the mice (Supplemental Fig. S6). Consistent with notable antitumor effects, the tumoral expression of cleaved caspase-3, an indicator of apoptotic cell death, was markedly increased in L80-treated tumors (Fig. 6B). The expression of CD31 was decreased in tumors treated with L80, suggesting antiangiogenic effects of L80 (Fig. 6C). These results suggest that L80 displays effective antitumor activities in NSCLC xenograft tumors by inhibiting tumor angiogenesis and by inducing apoptosis.

Fig. 6.
Fig. 6.

The antitumor effect of L80 in a tumor xenograft model. (A) H1299 cells were inoculated into the right flank of NOD/SCID mice. Mice harboring H1299 xenograft tumors were treated with vehicle or L80 for 3 weeks. The tumor sizes were monitored every day and were analyzed by one-way analysis of variance compared with control group (0 mg/kg). (B and C) The expression levels of cleaved caspase-3 and CD31 in xenograft tumors were analyzed by immunohistochemistry. Con, control; DAPI, 4′,6-diamidino-2-phenylindole.

L80 Inhibits Hsp90 Function by Binding to the ATP-Binding Pocket in the C-Terminal Domain of Hsp90.

We investigated the mechanism by which L80 suppressed the antitumor activities of NSCLC cells. Based on our previous results showing that deguelin blocks Hsp90 function by directly binding to the ATP-binding pocket of Hsp90, resulting in the degradation of various client proteins, including HIF-1α (Isaacs et al., 2002; Oh et al., 2007), we assessed whether Hsp90 was involved in L80-mediated anticancer activity. We first performed coimmunoprecipitation assays to examine the effect of L80 on the Hsp90 and HIF-1α interaction in H1299 cells. We observed a strong physical interaction between HIF-1α and Hsp90 in H1299 cells. However, this interaction was strongly diminished in the cells pretreated with L80 for 1 hour, when HIF-1α and Hsp90 protein expression levels were not affected (Fig. 7A; Supplemental Fig. S7A). We further confirmed this finding by performing a pull-down assay. H1299 lysates were incubated with recombinant proteins containing the HLH-PAS-ODD domain of HIF-1α, which is essential for complex formation with Hsp90 and has a sensitivity to Hsp90 inhibitors (Minet et al., 1999; Katschinski et al., 2004), in the presence or absence of L80. The level of Hsp90 bound to the recombinant HIF-1α was determined by Western blot analysis. As shown in Fig. 7B and Supplemental Fig. S7B, the interaction between Hsp90 and HIF-1α was markedly diminished by L80 treatment. The suppression of Hsp90 function was further confirmed by assessing the effect of L80 on the expression of other Hsp90 client proteins. As shown in Fig. 7C and Supplemental Fig. S7C, treatment with L80 effectively inhibited the expression of Hsp90 client proteins, including ErbB2 and Akt, in a concentration-dependent manner. Thus, these results suggest that L80 can directly modulate the function of Hsp90.

Fig. 7.
Fig. 7.

Downregulation of Hsp90 function by treatment with L80. (A) H1299 cells were treated with L80 under hypoxic conditions. Total cell lysates were prepared and immunoprecipitated (IP) with anti–HIF-1α antibodies. The interaction between HIF-1α and Hsp90 was analyzed by Western blot analysis. (B) H1299 lysates interacted with histidine-tagged recombinant HIF-1α in the presence or absence of L80. After pull-down with Ni-NTA agarose beads, the amount of Hsp90 bound to the recombinant HIF-1α was determined by Western blot analysis. (C) The expression levels of ErbB2 and Akt in the indicated NSCLC cells treated with increasing concentrations of L80 were analyzed by Western blot analysis. (D) Recombinant Hsp90 proteins containing the full length (FL) protein or the N-terminal and middle domain (NM), middle (M), and C-terminal (C) domains interacted with ATP-agarose in the presence or absence of L80. The protein level bound to the ATP-agarose beads was determined by Western blot analysis. (E–G) Structural analysis of the binding of L80 to the C-terminal ATP binding pocket of Hsp90. (E) Docking study of ATP and L80 in the C-terminal ATP-binding pocket of hHsp90 homodimer. (F and G) Docking pose of L80 with the C-terminal hHsp90. Con, control.

In general, Hsp90 inhibitors interfere with the binding of ATP to the nucleotide-binding pocket of Hsp90 in the N- or C-terminal domains (Whitesell and Lindquist, 2005). The nucleotide-binding pocket in the N-terminal domain was first identified and is the predominant ATP-binding site in Hsp90. ATP can bind to an additional binding pocket in the C-terminal domain of Hsp90 when the N-terminal binding sites are occupied (Soti et al., 2002; Whitesell and Lindquist, 2005). We investigated whether L80 binds to the ATP-binding pocket of Hsp90, and if so, whether it was the N- or the C-terminal pocket. Thus, we prepared recombinant Hsp90 proteins containing full length protein, the N-terminal and middle domain, the middle domain, and the C-terminal domain. The binding of each Hsp90 domain to ATP-agarose was examined in the presence of L80. We observed a complete disruption of the interaction between ATP-agarose and the C-terminal domain of Hsp90 in the presence of L80 (1 μM), whereas the same concentration of L80 showed a partial or no effect on ATP binding to the full length protein or the N-terminal and middle domain, respectively (Fig. 7D; Supplemental Fig. S7D). These results suggest that L80 is able to bind to the C-terminal ATP-binding pocket of Hsp90 and thus hamper Hsp90 function.

We further conducted a computational molecular modeling to ensure the binding of L80 to the C-terminal ATP-binding pocket of Hsp90. We carried out a docking study of ATP and L80 in the C-terminal ATP-binding pocket of hHsp90 homodimer. The active site was determined based on reported hHsp90 C-terminal ATP-binding site (Sgobba et al., 2008). As shown in Fig. 7E, the active site is located at the dimerization interface. After docking, we compared docking pose and score of ATP with those of L80. As illustrated in Fig. 7F, ATP (white carbon) fits well into the ATP-binding pocket of chain A. The adenine ring occupies deep into the hydrophobic pocket. The phosphate moiety interacts with multiple hydrogen bonds with Glu611, and 3′-hydroxy of sugar moiety forms an additional hydrogen bond with Ser677 in chain A. Although L80 is partially overlaid over the sugar-phosphate moiety of ATP, L80 binds at the center of interface between chain A and B with higher docking score (−logKd = 7.13) than ATP (−logKd = 6.29) (Fig. 7G). Two oxygen atoms of the acetoxy group form the a hydrogen bond with an amide at Lys 615 in chain A, and the oxygen atom of the methoxy group also forms a hydrogen bond with a hydrogen bond at Ser677, which is a part of the ATP-binding pocket in chain B. A full image of the Hsp90:L80 complex (Fig. 7E) reveals that L80 occupies the middle of the active site located at the interface of C-terminal homodimer. Taken together, our docking results suggests that L80 can bind to the C-terminal ATP binding site competitively with ATP, leading to stabilization of the open state of the Hsp90 homodimer by interacting with both A and B chains.

Discussion

The aim of the current study was to determine the toxicity, efficacy, and action mechanisms of a deguelin-derived analog, designated L80, for potential clinical use. The data presented show that the proliferative, angiogenic, and metastatic activities of NSCLC cells and their sublines with acquired chemoresistance were significantly inhibited by L80. L80 also exhibited significant antitumor activity in vivo in H1299 NSCLC xenograft tumors, which was accompanied by increased apoptosis and decreased vascular formation. Mechanistically, L80 effectively disrupted Hsp90 function by directly binding to the C-terminal ATP-binding pocket of Hsp90, leading to the reduced expression of Hsp90 client proteins, including HIF-1α. These data suggest that L80 may have potential as an anticancer drug targeting Hsp90 through mechanisms that are different than those targeted by the well known N-terminal Hsp90 inhibitors.

Hsp90, which belongs to a family of ATPase-containing molecular chaperones, functions to prevent its client proteins from misfolding upon heat shock or other stresses, thereby promoting their stabilization (Whitesell and Lindquist, 2005; Trepel et al., 2010). Cancer cells are normally in a state of proteotoxic pressure due, in part, to the accumulation of mutated molecules that could lead to cell lethality (Bagatell and Whitesell, 2004). Numerous Hsp90 client proteins, such as those in the epidermal growth factor receptor family, c-Met, Akt, Src, and B-Raf, are often mutated or overexpressed in several types of human cancer, including NSCLC (Whitesell and Lindquist, 2005; Shimamura and Shapiro, 2008), most likely to compensate for the insult. These oncogenic kinases mediate cancer cell proliferation, survival, angiogenesis, invasion, and metastasis, all of which play critical roles in cancer progression and resistance to conventional or targeted anticancer agents, including paclitaxel, cisplatin, erlotinib, and trastuzumab (Whitesell and Lindquist, 2005; Solar et al., 2007; Sawai et al., 2008; Bao et al., 2009; Trepel et al., 2010; Wainberg et al., 2013). Therefore, targeting Hsp90 would be an effective way for treating cancer and overcoming anticancer drug resistance, and it is a promising target for anticancer drug design. Consistent with this hypothesis, a growing body of evidence has shown the importance of an Hsp90 inhibitor for the treatment of cancer. Inhibition of Hsp90 induces the simultaneous degradation of numerous oncogenic Hsp90 client proteins, leading to an effective antitumor outcome (Whitesell and Lindquist, 2005; Trepel et al., 2010). The Hsp90 inhibitor ganetespib, alone or in combination with other anticancer drugs, displays effective antitumor effects in lung cancer cells, especially lung cancer harboring mutant K-Ras (Acquaviva et al., 2012). Another Hsp90 inhibitor, NVP-AUY922 [(5Z)-N-ethyl-5-(4-hydroxy-6-oxo-3-propan-2-ylcyclohexa-2,4-dien-1-ylidene)-4-[4-(morpholin-4-ylmethyl)phenyl]-2H-1,2-oxazole-3-carboxamide], has also shown potent antiproliferative and antitumor effects in various lung cancer cells (Garon et al., 2013).

Although several clinical trials to evaluate the effectiveness of Hsp90 inhibitors in lung cancer treatment are ongoing (Jhaveri et al., 2012), the main drawbacks of Hsp90 inhibitors are undesirable side effects, poor water solubility, and toxicity (Trepel et al., 2010). Therefore, it is necessary to develop novel strategies to target Hsp90. Zhang et al. (2008) suggested a novel strategy for Hsp90 inhibition through the disruption of Hsp90-cochaperone interactions. Another strategy targeting the C-terminal ATP-binding pocket of Hsp90 has been proposed because the antibiotic novobiocin binds with low affinity to a C-terminal ATP-binding pocket (Marcu et al., 2000a,b). Recently, more potent analogs of novobiocin have been developed (Burlison et al., 2006, 2008). Although the antitumor activities of these analogs need to be evaluated in relevant in vivo models of human cancer, such new approaches could provide a novel opportunity for Hsp90 inhibition.

We previously reported that deguelin, a natural product that competes with ATP on the Hsp90 ATP-binding pocket, leading to decreases in many Hsp90 client oncoproteins without detectable toxicities to normal lung epithelial cells in vitro and to major organs in mouse tissues in vivo, has potential as an anticancer drug (Oh et al., 2007). However, the potential neuronal toxicity of deguelin, which most likely occurs through the inhibition of NADH dehydrogenase (Caboni et al., 2004), raises concern for its use in human cancer patients. Hence, we attempted to develop deguelin derivatives as lead compounds for the development of novel Hsp90 inhibitors (Oh et al., 2007; Kim et al., 2008; Chang et al., 2012). In our structure-activity relationship studies, the 2,2-dimethyl-2H-chromene moiety of deguelin seemed to be responsible for occupying the ATP-binding pocket of Hsp90 via hydrophobic interactions. In addition, two dimethyoxy groups at the C9 and C10 positions, the terminal methoxy group in the 2,2-dimethyl-2H-chromene moiety, and a hydrogen bond donor group at the C7 position seemed to be important for the antiproliferative activity (Oh et al., 2007; Chang et al., 2012). In recent studies, we and others reported inhibitory effects of a compound containing 2,2-dimethyl-2H-chromene moiety on HIF-1α expression (Tan et al., 2005; Chang et al., 2012). In the current study, we show that L80, which shares these critical moieties of the deguelin, has antiangiogenic and antitumor activities.

We found several features of L80 that make the analog a lead compound for the development of novel anticancer drugs. First, L80 appeared to retain the potent antiproliferative and apoptotic activities of its mother compound in vitro and in vivo with minimal toxicity to normal lung epithelial, hippocampal, retinal pigment epithelial, and vascular endothelial cells. Tissue samples taken from several organs, including liver, of L80-treated mice revealed no pathohistological changes. This finding is important because the main hurdle for the currently available Hsp90 inhibitors in clinical trials is liver and ocular toxicities (Jhaveri et al., 2012). Second, L80 effectively suppressed NSCLC cells’ activities for angiogenesis stimulation, migration, and invasion. This finding is significant because the majority of cancer patients die from metastatic disease; effective therapeutic drugs would be expected not only to lower primary tumor burden but also to interrupt the metastatic cascade. Third, L80 seemed to inhibit Hsp90 function through mechanisms that are different than those regulated by the currently available Hsp90 inhibitors. We found that the blockade of Hsp90 interaction with HIF-1α and the destabilizing of Hsp90 client proteins including HIF-1α by L80 treatment were associated with decreased binding of ATP-agarose to the C-terminal domain of the Hsp90 protein. Considering the role of C-terminal dimerization during Hsp90 conformational cycle and the inverse correlation between N- and C-terminal dimerization (Soti et al., 2002; Ratzke et al., 2010), binding of L80 to the C-terminal Hsp90 homodimer may cause stabilization of open conformation of Hsp90, resulting in prohibiting global conformational changes of Hsp90 such as N-terminal dimerization and the formation of the ATP binding pocket. The computational analysis using molecular docking modeling suggests that L80 can bind to the C-terminal ATP binding site competitively with ATP L80 and lead the stabilization of the open form of Hsp90 homodimer, further confirming the inhibitory effect of L80 on Hsp90 function by binding to the C-terminal ATP binding pocket of Hsp90. These findings suggest that L80 can serve as a functional inhibitor of Hsp90 that blocks ATP binding to the C-terminal binding pocket of Hsp90. Because the dimerization of Hsp90 mediated by the C terminus is critical for its chaperone function and because the ATP-binding pocket at the C terminus is necessary for the close association between two N terminals in the ATP-bound state (Wainberg et al., 2013), targeting the C-terminal domain of Hsp90 could be an alternative and effective allosteric approach to functionally inhibiting Hsp90. Although additional studies are required to explore the detailed mechanisms by which L80 interacts with the ATP-binding pocket in the C terminus of Hsp90, the mode of action of L80 makes it a leading chemical for the development of novel Hsp90 inhibitors. Fourth, L80 did not discriminate naive NSCLC cells from those with acquired resistance to paclitaxel. Previous studies suggested that Hsp90 is involved in anticancer drug resistance and that treatment with an Hsp90 inhibitor overcomes the resistance to conventional or molecular-targeted anticancer drugs (Solar et al., 2007; Sawai et al., 2008; Bao et al., 2009; Wainberg et al., 2013), emphasizing the importance of Hsp90 inhibition to overcome anticancer drug resistance. Although additional in vitro and in vivo studies are required to evaluate the effectiveness of L80, either alone or in combination with paclitaxel, our results suggest that the suppression of Hsp90 function may overcome resistance to paclitaxel, which is frequently used for the treatment of patients with NSCLC (Ramalingam and Belani, 2004). Although the mechanisms mediating the different affinities of L80 to the C- and N-terminal ATP-binding pockets of Hsp90 have not been fully elucidated, these potent in vitro and in vivo antitumor activities of L80 in NSCLC cells support the potential of L80 as a candidate for the development of anticancer agents.

In summary, the present study suggests that L80 displays significant antitumor activities in NSCLC by binding to the ATP-binding pocket in the C terminus of Hsp90, thus disrupting Hsp90 function. Overall, the data presented provide compelling evidence for the continued development of L80-based C-terminal Hsp90 inhibitors as promising alternatives to the currently available N-terminal inhibitors for the treatment of human malignancies, including NSCLC. These results suggest that L80 may be a novel Hsp90 inhibitor with effective antitumor effect on NSCLC. Further studies are warranted to investigate the effectiveness of L80 in additional preclinical and clinical settings.

Authorship Contributions

Participated in research design: S. C. Lee, H. Y. Lee.

Conducted experiments: S. C. Lee, Choi, Seong, S. J. Park, Seo, Hong, H. Kim, M.-Y. Lee.

Contributed new reagents or analytic tools: H. S. Kim, K. C. Kim, J. Lee, Suh, H. J. Park, K. W. Kim.

Performed data analysis: S. C. Lee, H. Y. Lee.

Wrote or contributed to the writing of the manuscript: Min, H. Y. Lee.

Footnotes

    • Received November 16, 2014.
    • Accepted May 5, 2015.

  • This work was supported by grants from the National Research Foundation of Korea (NRF), the Ministry of Science, ICT & Future Planning, Republic of Korea [NRF-2011-0017639, NRF-2011-0019400, and NRF-2011-0030001].

  • dx.doi.org/10.1124/mol.114.096883.

  • ↵Embedded ImageThis article has supplemental material available at molpharm.aspetjournals.org.

Abbreviations

BBB
blood-brain barrier
CM
conditioned medium
CNS
central nervous system
HBE
human bronchial epithelial
HUVEC
human umbilical endothelial cell
L80
(6,7-dimethoxyquinolin-4-yl)(5-methoxy-2,2-dimethyl-2H-chromen-6-yl)methyl acetate
MTT
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
NSCLC
non–small cell lung cancer
NVP-AUY922
(5Z)-N-ethyl-5-(4-hydroxy-6-oxo-3-propan-2-ylcyclohexa-2,4-dien-1-ylidene)-4-[4-(morpholin-4-ylmethyl)phenyl]-2H-1,2-oxazole-3-carboxamide

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Blockade of the C-terminal Hsp90 ATP Binding Domain by L80

Su-Chan Lee, Hye-Young Min, Hoon Choi, Ho Shin Kim, Kyong-Cheol Kim, So-Jung Park, Myeong A Seong, Ji Hae Seo, Hyun-Ju Park, Young-Ger Suh, Kyu-Won Kim, Hyun-Seok Hong, Hee Kim, Min-Young Lee, Jeewoo Lee and Ho-Young Lee
Molecular Pharmacology August 1, 2015, 88 (2) 245-255; DOI: https://doi.org/10.1124/mol.114.096883
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