Geldanamycin Derivatives Research Articles

Synthesis and characterization of 64Cu-labeled Geldanamycin derivative for imaging HSP90 expression in breast cancer

Heat shock protein 90 (HSP90) plays a crucial role in cancer cell growth and metastasis by stabilizing overexpressed signaling proteins. Inhibiting HSP90 has emerged as a promising anti-cancer strategy. In this study, we aimed to develop and characterize a HSP90-targeted molecular imaging probe, [64Cu]Cu-DOTA-BDA-GM, based on a specific HSP90 inhibitor, geldanamycin (GM), for PET imaging of cancers. GM is modified at the C-17 position with 1,4-butane-diamine (BDA) and linked to 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) for 64Cu radiolabeling. We evaluated the probe's specific binding to HSP90-expressing cells using Chinese hamster ovary (CHO) cells and breast cancer cells including MDA-MB-231, MDA-MB-435S, MCF7, and KR-BR-3 cell lines. A competition study with non-radioactive GM-BDA yielded an IC50 value of 1.35 ± 0.14 nM, underscoring the probe's affinity for HSP90. In xenograft models of MDA-MB-231 breast cancer, [64Cu]Cu-DOTA-BDA-GM showcased targeted tumor localization, with significant radioactivity observed up to 18 h post-injection. Blocking studies using unlabeled GM-BDA and treatment with the anticancer drug Vorinostat (SAHA), which can affect the expression and activity of numerous proteins, such as HSPs, confirmed the specificity and sensitivity of the probe in cancer targeting. Additionally, PET/CT imaging in a lung metastasis mouse model revealed increased lung uptake of [64Cu]Cu-DOTA-BDA-GM in metastatic sites, significantly higher than in non-metastatic lungs, illustrating the probe's ability to detect metastatic breast cancer. In conclusion, [64Cu]Cu-DOTA-BDA-GM represents a sensitive and specific approach for identifying HSP90 expression in breast cancer and metastases, offering promising implications for clinical diagnosis and monitoring.

Modifications of geldanamycin via CuAAC altering affinity to chaperone protein Hsp90 and cytotoxicity

Functionalization of alkyne (1) and azide (2) derivatives of geldanamycin (GDM) via dipolar cycloaddition CuAAC yielded 35 new congeners (3–37) with C(17)-triazole arms bearing caps of different nature (basic vs. acidic, hydrophilic vs. hydrophobic). Confrontation of biological data (anticancer activity vs. toxicity in normal cells) with lipophilicity (clogP), dissociation constants (Kd) of complexes with Hsp90 and binding modes to Hsp90 revealed SAR in specific subgroups of GDM derivatives. The most potent GDM congeners 14–16, bearing C(17)-triazole-benzyl-halogen arms exhibited the most optimal clogP values of 2.7–3.1 at favourable binding to Hsp90 (KdHsp90 at μM level). The anticancer activity of 14–16 (IC50 = 0.23–0.41 μM) is higher than those of GDM (IC50 = 0.58–0.64 μM) and actinomycin D (ActD, IC50 = 0.62–0.71 μM) in SKBR-3, SKOV-3 and PC-3 cell lines, with a comparable cytotoxicity in healthy cells. The relationship between structure and attractive anticancer potency (IC50 = 0.53–0.74 μM) is also observed for congeners with C(17)-triazole-saccharide or C(17)-triazole-unsaturated arms. In the former, the absolute configuration at C(4) (ᴅ-glucose vs. ᴅ-galactose) whereas in the latter the length of the unsaturated arm influences the cytotoxic effects due to different binding strength (Kd, ΔE) and modes with Hsp90. Among all triazole congeners of GDM that are biologically attractive and exhibit lower toxicity in normal cells than GDM and ActD, the derivative 22, bearing the C(17)-triazole-cinnamyl arm, shows the lowest Kd (Hsp90), optimal clogP = 2.82, the best pro-apoptotic properties in SKBR-3 and SKOV-3 and the best selectivity indices (SI). For the most potent GDM derivatives with C(17)-triazole arm, the docking studies have suggested the importance of the intermolecular stabilization between the arm and the D57 or Y61 of Hsp90.

LZY3016, a novel geldanamycin derivative, inhibits tumor growth in an MDA-MB-231 xenograft model.

A novel geldanamycin derivative LZY3016 was synthesized as an antitumor agent. Compound LZY3016 exhibited potent anti-proliferation activity toward MDA-MB-231 (IC50 = 0.06 μM), which was more effective than positive drug 17-AAG. In vivo hepatotoxicity assay displayed that serum AST/ALT levels in LZY3016-treated mice were both significantly less than those in the geldanamycin (GA) group. LZY3016 showed potent antitumor activity in an MDA-MB-231 xenograft mouse model, suggesting LZY3016 is an up-and-coming antitumor candidate. The theoretical binding mode between LZY3016 and Hsp90 was obtained by molecular dynamics simulation.

The HSP90 inhibitor 17-AAG induced calcium-mediated apoptosis in filarial parasites.

The available antifilarial medications are effective only against the larval stage of the filarial parasite. As a result, there is a pressing need for an adulticidal drug. The development of drugs requires the identification of molecular targets that are critical for parasite life. In this study, we observed the effect of 17-N-allyl-17-demethoxygeldanamycinon the survival of adult filarial parasites. The 17-N-allyl-17-demethoxygeldanamycin (17-AAG) is a derivative of geldanamycin (GA), which is an inhibitor of heat shock protein(HSP)90. It is less toxic as compared to geldanamycin. The motility and viability of the adult filarial parasite Setaria cervi were decreased on exposure to 17-AAG at 2.5 and 5.0 μM/ml concentrations. The 17-AAG treated parasites showed induction of oxidative stress as evidenced by decreased activity of various antioxidant enzymes like glutathione s-transferase, glutathione reductase, thioredoxin reductase, and an increase in ROS production in comparison to control. Oxidative stress may lead to altered calcium homeostasis. Indeed, in 17-AAG treated worms, there was a rise in calcium in the cytosol and mitochondria, as well as a decrease in the ER. We also observed enhanced activity of phospholipase C in the treated parasite, suggesting the opening of calcium channels located on the ER membrane. ER stress is marked by a reduced level of protein disulfide isomerase. Further, 17-AAG treated worms showed an increase in apoptotic marker enzyme activities like calpain, cyt-c, and caspase-3. The 2D-gel electrophoresis technique showed 142 protein spots in the control and 112 spots in the 17-AAG treated parasite. Thus, 17-AAG induced oxidative stress, and altered calcium, and proteostasis of parasites, which led to apoptosis.

Rational design, synthesis, and biological evaluation of novel C6-modified geldanamycin derivatives as potent Hsp90 inhibitors and anti-tumor agents

Heat shock protein 90 (Hsp90) is an appealing anticancer drug target that provoked a tremendous wave of investigations. Geldanamycin (GA) is the first identified Hsp90 inhibitor that exhibited potent anti-cancer activity, but the off-target toxicity associated with the benzoquinone moiety hampered its clinical application. Until now, structure optimization of GA is still in need to fully exploit the therapeutic value of Hsp90. Due to the structural complexity and synthetic challenge of this compound family, conventional optimization is bound to be costly but high efficiency is expected to be reachable by combining the art of rational design and total synthesis. Described in this paper is our first attempt at this approach aiming at rational modification of the C6-position of GA. The binding affinities towards Hsp90 of compound 1 (C6-ethyl) and 2 (C6-methyl) were designed and predicted by using Discovery Studio. These compounds were synthesized and further subjected to a thorough in vitro biological evaluation. We found that compounds 1 and 2 bind to Hsp90 protein with the IC50 of 34.26 nmol/L and 163.7 nmol/L, respectively. Both compounds showed broad-spectrum antitumor effects. Replacing by ethyl, compound 1 exhibited more potent bioactivity than positive control GA, such as in G2/M cell cycle arrest, cell apoptosis and client proteins degradations. The results firstly indicated that the docking study is able to provide a precise prediction of Hsp90 affinities of GA analogues, and the C6 substituent of GA is not erasable without affecting its biological activity.

Newly Designed Geldanamycin Analogues for Targeted Cancer-Causing Hsp90 Protein Inhibitor: Molecular Docking Study

Cancer is currently a major public health concern worldwide. Previous studies have shown that heat shock protein 90 (Hsp90) is the key common cause of cancer. Thus, Hsp90 is one of the important molecular targets for the development of Hsp90 cancer drug based on geldanamycin (GDM) and alvespimycin (17-DMAG). Herein, novel geldanamycin derivatives, S1-S6 were designed as potential Hsp90 cancer drug by targeting signal transduction pathway, especially against oncogenic client protein from Hsp90. The binding of S1-S6 in the cavity of Hsp90 were investigated by molecular docking using the iGEMDOCK v2.1 software. The results illustrated that S1-S6 bound in the binding site of Hsp90 with similar manner to GDM and 17-DMAG. The binding energies of S1-S6 in Hsp90 (PDB ID:1YET) (-137.49 to -123.24 kcal/mol) were comparable to that of GDM (-133.06 kcal/mol) while the binding energies of S1-S6 in Hsp90 (PDB ID:1OSF) (-137.49 to -131.22 kcal/mol) were slightly higher than that of 17-DMAG (-145.31 kcal/mol). S1-S6 interacted well by hydrogen bonding with key amino acids in the binding site of Hsp90, which could inhibit the cancer cell growth. Therefore, S1-S6 containing novel geldanamycin derivatives could be promising molecules for anti-cancer drug against Hsp90 2 types in the future.

Identification of Predictive Biomarkers of Response to HSP90 Inhibitors in Lung Adenocarcinoma.

Heat shock protein 90 (HSP90) plays an essential role in lung adenocarcinoma, acting as a key chaperone involved in the correct functioning of numerous highly relevant protein drivers of this disease. To this end, HSP90 inhibitors have emerged as promising therapeutic strategies, even though responses to them have been limited to date. Given the need to maximize treatment efficacy, the objective of this study was to use isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomic techniques to identify proteins in human lung adenocarcinoma cell lines whose basal abundances were correlated with response to HSP90 inhibitors (geldanamycin and radicicol derivatives). From the protein profiles identified according to response, the relationship between lactate dehydrogenase B (LDHB) and DNA topoisomerase 1 (TOP1) with respect to sensitivity and resistance, respectively, to geldanamycin derivatives is noteworthy. Likewise, rhotekin (RTKN) and decaprenyl diphosphate synthase subunit 2 (PDSS2) were correlated with sensitivity and resistance to radicicol derivatives. We also identified a relationship between resistance to HSP90 inhibition and the p53 pathway by glucose deprivation. In contrast, arginine biosynthesis was correlated with sensitivity to HSP90 inhibitors. Further study of these outcomes could enable the development of strategies to improve the clinical efficacy of HSP90 inhibition in patients with lung adenocarcinoma.

Inhibition of heat shock protein 90 destabilizes receptor tyrosine kinase ROR1 in lung adenocarcinoma.

We have previously identified receptor tyrosine kinase‐like orphan receptor 1 (ROR1) as a direct transcriptional target of TTF‐1/NKX2‐1, a lineage‐survival oncogene in lung adenocarcinoma. ROR1 sustains prosurvival signaling from multiple receptor tyrosine kinases including epidermal growth factor receptor, MET, and insulin‐like growth factor 1 receptor in part by maintaining the caveolae structure as a scaffold protein of cavin‐1 and caveolin‐1. In this study, a high throughput screening of the natural product library containing 2560 compounds was undertaken using a cell‐based FluoPPI assay detecting ROR1‐cavin‐1 interaction. As a result, geldanamycin (GA), a known inhibitor of heat shock protein 90 (HSP90), was identified as a potential inhibitor of ROR1. Geldanamycin, as well as two GA derivatives tested in the clinic, 17‐allylamino‐17‐demethoxygeldanamycin (17‐AAG) and 17‐dimethylaminoethylamino‐17‐demethoxygeldanamycin (17‐DMAG), decreased ROR1 protein expression. We found that ROR1 physically interacted with HSP90α, but not with other HSP90 paralogs, HSP90β or GRP94. Geldanamycin in turn destabilized and degraded ROR1 protein in a dose‐ and time‐dependent manner through the ubiquitin/proteasome pathway, resulting in a significant suppression of cell proliferation in lung adenocarcinoma cell lines, for which the kinase domain of ROR1, but not its kinase activity or N‐glycosylation, was required. Our findings indicate that HSP90 is required to sustain expression of ROR1 crucial for lung adenosarcoma survival, suggesting that inhibition of HSP90 could be a promising therapeutic strategy in ROR1‐positive lung adenocarcinoma.

Synthesis of novel geldanamycin derivatives

The toxicity associated with the geldanamycin family of benzoquinone ansamycins when used as heat shock protein-90 inhibitor molecular therapeutics is ameliorated by substitution at the 19-position. The resulting 19-substituted derivatives have greater potential for success in oncology clinical trials and for other medicinal purposes such as the treatment of neurodegenerative conditions. Having overcome hurdles associated with the sensitivity and complexity of these molecules, through a variety of synthetic approaches, the synthesis of a series of 19-substituted geldanamycin derivatives is reported herein using optimised Stille and Suzuki coupling reactions. Further compounds were accessible via copper-mediated coupling and nucleophilic addition reactions The new compounds are of significant medicinal interest, in view of their significantly reduced toxicity previously observed for this class of substrate compared to their 19-unsubstituted counterparts that have been evaluated in the clinic.

Anticancer activity and toxicity of new quaternary ammonium geldanamycin derivative salts and their mixtures with potentiators

Geldanamycin (GDM) has been modified by different type neutral/acidic/basic substituents (1–7) and by quinuclidine motif (8), transformed into ammonium salts (9–13) at C(17). These compounds have been characterised by spectroscopic and x-ray methods. Derivative 8 shows better potency than GDM in MCF-7, MDA-MB-231, A549 and HeLa (IC50s = 0.09–1.06 µM). Transformation of 8 into salts 9–13 reduces toxicity (by 11-fold) at attractive potency, e.g. MCF-7 cell line (IC50∼2 µM). Our studies show that higher water solubility contributes to lower toxicity of salts than GDM in healthy CCD39Lu and HDF cells. The use of 13 mixtures with potentiators PEI and DOX enhanced anticancer effects from IC50∼2 µM to IC50∼0.5 µM in SKBR-3, SKOV-3, and PC-3 cancer cells, relative to 13. Docking studies showed that complexes between quinuclidine-bearing 8–13 and Hsp90 are stabilised by extra hydrophobic interactions between the C(17)-arms and K58 or Y61 of Hsp90.

Abstract A20: 17-Aminogeldanamycin inhibits cytoprotective UPR pathways and cooperates with inhibitors of the MAPK signaling cascade in apoptosis induction

Abstract Geldanamycin is an inhibitor of Heat Shock Protein 90 (HSP90). Attenuation of HSP90 activity results in accumulation of unfolded proteins, induction of endoplasmic reticulum (ER) stress, and activation of unfolded protein response (UPR). HSP90 is a chaperone protein participating in appropriate folding of more than 100 proteins, including IRE1α and BRAFV600E. In melanoma cells, BRAFV600E was correlated with chronically induced ER stress and involved in UPR-induced resistance to apoptosis. As geldanamycin is highly hepatotoxic, geldanamycin derivatives exerting similar proapoptotic properties but higher selectivity towards cancer cells are needed. In the present study, 17-aminogeldanamycin exerted higher proapoptotic activity than geldanamycin in patient-derived melanoma cell populations as demonstrated by using fluorescence time-lapse imaging (IncuCyte ZOOM). We have also shown that 17-aminogeldanamycin inhibits ERK1/2 activity and cell proliferation,as well as induces apoptosis transiently, enhancing expression of HSP70 and GRP78. GRP78 regulates activity of the UPR pathway, and we have demonstrated that 17-aminogeldanamycin inhibits IRE1α-dependent pathway of UPR responsible for degradation of unfolded proteins and reduces levels of 36 kDa product of ATF6 cleavage in selected melanoma cell populations. Moreover, we have shown that combination of 17-aminogeldanamycin and trametinib induces apoptosis earlier and more efficiently. Our study suggests that cooperation of 17-aminogeldanamycin and trametinib might result from concurrent inhibition of the MAPK and protective UPR pathways but can be also associated with the basal UPR activity, which is diverse among drug-naïve melanoma cell populations. Further studies are necessary to fully explore antimelanoma capabilities of 17-aminogeldanamycin and its combination with targeted therapy. This work was financially supported by Grant 2014/15/B/NZ7/00947 from National Science Centre (Poland), and 502-03/1-156-01/502-14-318 from Medical University of Lodz. Citation Format: Aleksandra H. Mielczarek-Lewandowska, Mariusz L. Hartman, Malgorzata M. Sztiller-Sikorska, Marta Osrodek, Anna E. Gajos-Michniewicz, Malgorzata E. Czyz. 17-Aminogeldanamycin inhibits cytoprotective UPR pathways and cooperates with inhibitors of the MAPK signaling cascade in apoptosis induction [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr A20.

Abstract LB-234: Development of HER2 targeted HSP90 inhibitors delivery system

Abstract Heat shock protein 90 (HSP90) plays a crucial role in the survival and growth of cancer cells by improvement of the stability and activity of many signaling proteins such as Akt, Raf-1 and HER2/neu. HSP90 inhibitors (HSP90i) such as geldamanycin (GM), AUY922 and STA-9090 have been developed for inhibition of multiple signaling pathways that are initiated by HSP90. Although geldanamycin derivatives (GM) which is the first class of HSP90 inhibitors were disappointing in the initial clinical trials, these studies have shown promise in patients with HER2 positive cancers. To improve anticancer efficacy of HSP90i, HER2 targeted HSP90i delivery system (HER2-HBD) was constructed because of difficulty in chemical modification of HSP90i. HER2-HBD consist of HER2 scFv and HSP90i binding domain, was expressed from 293 cells. This system could incorporate HSP90i into recombinant antibody fusion protein based on biological binding affinity. HER2-HBD and HSP90i was spontaneously formed HER2-HBD/HSP90i complex. HER2-HBD/HSP90i complex specifically delivered HSP90i into HER2 positive cancer cell and reduced cancer cell growth in vitro and in vivo test. HER2-HBD recombinant system based on biological affinity between HSP90i and HBD was not required chemical reaction to conjugate antibody and drug. This system might be useful targeted delivery systems for HSP90i without tailored conjugation method to develop antibody-drug conjugate (ADC) and also applied to develop targeted delivery systems for biological inhibitor drugs. Citation Format: Kwangsuk Lim, Yudong Hong, Young-Wook Won. Development of HER2 targeted HSP90 inhibitors delivery system [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr LB-234.

Effect of geldanamycin on the expression of the matrix molecules and angiogenetic factors in a gastric cancer cell line

ABSTRACT Angiogenesis is the formation of new blood vessels. Angiogenesis affects cancer growth and is a useful target for cancer therapeutics. The effects of geldanamycin on angiogenesis in cases of gastric cancer are poorly understood. We investigated the effects of different doses of 17-allylamino-17-demethoxygeldanamycin (17-AGG), a semi-synthetic derivative of geldanamycin, on the interactions between cellular matrix proteins and angiogenesis factors in a gastric cancer cell line. We examined cancer cells on laminin and collagen I coated surfaces to determine their response to the angiogenic effect of these matrix molecules. We also evaluated the expression levels of VEGF, MMP-9, ES and TSP-1 using ELISA. We found that application of 17-AAG to the gastric cancer cell line on culture dish plastic decreased VEGF, TSP-1, ES and MMP-9 expression, whereas of all of these proteins were increased by laminin and collagen coating. 17-AAG currently is in clinical trial phase 2 and may be a promising drug for treatment of gastric cancer.

Cytotoxicity activity of geldanamycin derivatives against various cancer cell lines

Cytotoxicity activity of geldanamycin derivatives against various cancer cell lines

The molecular chaperone Hsp90α deficiency causes retinal degeneration by disrupting Golgi organization and vesicle transportation in photoreceptors.

Heat shock protein 90 (Hsp90) is an abundant molecular chaperone with two isoforms, Hsp90α and Hsp90β. Hsp90β deficiency causes embryonic lethality, whereas Hsp90α deficiency causes few abnormities except male sterility. In this paper, we reported that Hsp90α was exclusively expressed in the retina, testis, and brain. Its deficiency caused retinitis pigmentosa (RP), a disease leading to blindness. In Hsp90α-deficient mice, the retina was deteriorated and the outer segment of photoreceptor was deformed. Immunofluorescence staining and electron microscopic analysis revealed disintegrated Golgi and aberrant intersegmental vesicle transportation in Hsp90α-deficient photoreceptors. Proteomic analysis identified microtubule-associated protein 1B (MAP1B) as an Hsp90α-associated protein in photoreceptors. Hspα deficiency increased degradation of MAP1B by inducing its ubiquitination, causing α-tubulin deacetylation and microtubule destabilization. Furthermore, the treatment of wild-type mice with 17-DMAG, an Hsp90 inhibitor of geldanamycin derivative, induced the same retinal degeneration as Hsp90α deficiency. Taken together, the microtubule destabilization could be the underlying reason for Hsp90α deficiency-induced RP.

Impact of Heat Shock Protein 90 Inhibition on the Proteomic Profile of Lung Adenocarcinoma as Measured by Two-Dimensional Electrophoresis Coupled with Mass Spectrometry.

Heat shock protein 90 (HSP90) is an important chaperone in lung adenocarcinoma, with relevant protein drivers such as EGFR (epidermal growth factor receptor) and EML4-ALK (echinoderm microtubule-associated protein-like protein4 fused to anaplastic lymphoma kinase) depending on it for their correct function, therefore HSP90 inhibitors show promise as potential treatments for lung adenocarcinoma. To study responses to its inhibition, HSP90 was pharmacologically interrupted by geldanamycin and resorcinol derivatives or with combined inhibition of HSP90 plus HSP70 in lung adenocarcinoma cell lines. Two-dimensional electrophoresis was performed to identify proteomic profiles associated with inhibition which will help to understand the biological basis for the responses. HSP90 inhibition resulted in altered protein profiles that differed according the treatment condition studied. Results revealed 254 differentially expressed proteins after treatments, among which, eukaryotic translation initiation factor3 subunit I (eIF3i) and citrate synthase demonstrated their potential role as response biomarkers. The differentially expressed proteins also enabled signalling pathways involved in responses to be identified; these included apoptosis, serine-glycine biosynthesis and tricarboxylic acid cycle. The proteomic profiles identified here contribute to an improved understanding of HSP90 inhibition and open possibilities for the detection of potential response biomarkers which will be essential to maximize treatment efficacy in lung adenocarcinoma.

Anti-Inflammatory Activity of Geldanamycin and Its Derivatives in LPS-Induced RAW 264.7 Cells

Geldanamycin (1) had been isolated as a major compound from Streptomyces zerumbet W14; an endophyte of Zingiber zerumbet (L.) Smith. Two new geldanamycin derivatives; 17-(tryptamine)-17-demethoxygeldanamycin (2) and 17-(5’-methoxytryptamine)-17-demethoxygeldanamycin (3) were synthe- sized and their anti-inflammatory activity was evaluated in LPS-induced macrophage RAW 264.7 cells by investigating their effects on the inhibition of production of NO, PGE2, TNF-α, IL-1β, IL-6 and IL-10. The data obtained were consistent with the modulation of TNF-α, IL-1β, IL-6, IL-10 production by these derivatives at concentration of 1 to 5 μg/ml. A similar effect was also observed when LPS-induced NO release and PGE2 production were tested. The inhibitory effects were shown in concentration-dependent manners. From the obtained results, it was concluded that two new gelda- namycin derivatives possess anti-inflammatory activity on LPS-induced RAW 264.7 cells. They could be useful for the management of inflammatory diseases.

New geldanamycin derivatives with anti Hsp properties by mutasynthesis.

Mutasynthetic supplementation of the AHBA blocked mutant strain of S. hygroscopicus, the geldanamycin producer, with 21 aromatic and heteroaromatic amino acids provided new nonquinoid geldanamycin derivatives. Large scale (5 L) fermentation provided four new derivatives in sufficient quantity for full structural characterisation. Among these, the first thiophene derivative of reblastatin showed strong antiproliferative activity towards several human cancer cell lines. Additionally, inhibitory effects on human heat shock protein Hsp90α and bacterial heat shock protein from H. pylori HpHtpG were observed, revealing strong displacement properties for labelled ATP and demonstrating that the ATP-binding site of Hsps is the target site for the new geldanamycin derivatives.

Correction: In vitro Biological Characterization of a Novel, Synthetic Diaryl Pyrazole Resorcinol Class of Heat Shock Protein 90 Inhibitors.

The molecular chaperone heat shock protein 90 (HSP90) has emerged as an exciting molecular target. Derivatives of the natural product geldanamycin, such as 17-allylamino-17-demethoxy-geldanamycin (17-AAG), were the first HSP90 ATPase inhibitors to enter clinical trial. Synthetic small-molecule HSP90 inhibitors have potential advantages. Here, we describe the biological properties of the lead compound of a new class of 3,4-diaryl pyrazole resorcinol HSP90 inhibitor (CCT018159), which we identified by high-throughput screening. CCT018159 inhibited human HSP90beta with comparable potency to 17-AAG and with similar ATP-competitive kinetics. X-ray crystallographic structures of the NH(2)-terminal domain of yeast Hsp90 complexed with CCT018159 or its analogues showed binding properties similar to radicicol. The mean cellular GI(50) value of CCT018159 across a panel of human cancer cell lines, including melanoma, was 5.3 mumol/L. Unlike 17-AAG, the in vitro antitumor activity of the pyrazole resorcinol analogues is independent of NQO1/DT-diaphorase and P-glycoprotein expression. The molecular signature of HSP90 inhibition, comprising increased expression of HSP72 protein and depletion of ERBB2, CDK4, C-RAF, and mutant B-RAF, was shown by Western blotting and quantified by time-resolved fluorescent-Cellisa in human cancer cell lines treated with CCT018159. CCT018159 caused cell cytostasis associated with a G(1) arrest and induced apoptosis. CCT018159 also inhibited key endothelial and tumor cell functions implicated in invasion and angiogenesis. Overall, we have shown that diaryl pyrazole resorcinols exhibited similar cellular properties to 17-AAG with potential advantages (e.g., aqueous solubility, independence from NQO1 and P-glycoprotein). These compounds form the basis for further structure-based optimization to identify more potent inhibitors suitable for clinical development.

In vitro investigation of the effect of matrix molecules on the behavior of colon cancer cells under the effect of geldanamycin derivative.

The chaperone-binding drug, 17-allylamino-17-demethoxygeldanamycin, has recently come into clinical use. It is a derivative of geldanamycin, an ansamycin benzoquinone antibiotic with anti-carcinogenic effect. Understanding the effect of this drug on the cancer cells and their niche is important for treatment. We applied 17-allylamino-17-demethoxygeldanamycin to colon cancer cell line (Colo 205) on matrix molecules to investigate the relationship of apoptosis with terminal deoxynucleotidyl transferase dUTP nick end labeling immunocytochemistry and related gene expression. We used laminin and collagen I for matrix molecules and vascular endothelial growth factor for angiogenic structure. We also examined apoptosis-related signaling pathway including mitochondrial proteins, cytochrome c, Bcl-2, caspase-9, Apaf-1 expression using real-time polymerase chain reaction. There was clear effect of 17-allylamino-17-demethoxygeldanamycin that killed more cells on tissue culture plastic compared to matrix molecules. The IC50 value was 0.58 µg/mL for tissue culture plastic compared with 0.64 µg/mL for laminin and 0.75 µg/mL for collagen I. The analyses showed that more cells on matrix molecules underwent apoptosis compared to that on tissue culture plastic. Apoptosis-related gene expression was similar in which Bcl-2 expression decreased and proapoptotic gene expression of the cells on matrix molecules increased compared to that on tissue culture plastic. However, the application of 17-allylamino-17-demethoxygeldanamycin was more effective for the cells on collagen I compared to the cells on laminin. There was also a decrease in angiogenesis as shown by the vascular endothelial growth factor staining. This was more pronounced by coating of the tissue culture plastic with matrix molecules. Our results supported the anti-cancer effect of 17-allylamino-17-demethoxygeldanamycin, and this effect depended on matrix molecules. This effect occurs through apoptosis, and related genes were also altered. All these genes may serve for novel target under the effect of matrix substrate. However, correct interpretation of the results requires further studies.