Class Of Hsp90 Inhibitors Research Articles

Abstract 2977: Novel HSP90 inhibitors with senolytic activity for a targeted therapy in a hormone-induced breast cancer senescence model

Abstract Background: The research into senescent cells, particularly their accumulation and contribution to aging and age-related diseases, has gained significant momentum in recent years. This interest is primarily driven by findings in mouse models, where the excessive buildup of senescent cells is linked to various age-related pathologies. Consequently, there has been a concerted effort in the scientific community to identify and develop senolytics. A notable development in this field is the identification of senolytics within the class of HSP90 inhibitors. These inhibitors, initially investigated for their potential in cancer therapy, have been repurposed due to their high selectivity for senescent cells. Our research has led to new compounds with mild inhibitory activity against HSP90α in response to these challenges. These compounds stand out for their lack of toxic side effects and their demonstrated senolytic activity across multiple cellular models. Objective: The primary objective of this research is to identify and characterize innovative HSP90 inhibitors that exhibit senolytic properties, specifically in the context of hormone-driven cancers. Methods and Results: Utilizing a structure-based virtual screening technique, we identified several compounds displaying strong affinity and predicted inhibitory activity against the target protein, HSP90α. Among these, two inhibitors designated K4 and K5, demonstrated notable senolytic activity in vitro without inducing cytotoxic effects in non-senescent cells. These compounds were tested for their efficacy in senolytic activity and displayed half-maximal inhibitory concentrations (IC50) of 155 nM and 111 nM, respectively. The experimental model for this study was the MCF7 cell line, which was treated with 4-Hydroxytamoxifen (Tam, 10uM for 96 hours) to induce a state of senescence. Subsequent growth curve analyses and beta-galactosidase assays established that Tam treatment effectively halted cell proliferation and induced a senescence state in about 40% of the cell population compared to the ethanol control. When treated with K4 and K5 (10 uM for an additional 96 hours), a significant reduction in the senescent cell population was observed, with K5 showing a particularly pronounced increase in the mortality rate of these cells (up to 2.5-fold compared to the DMSO control). Western blot analysis supported these results, suggesting the effectiveness of K5 combined with Tam in inhibiting proliferation and inducing cell death in MCF7 cells. Conclusion: Our findings propose a novel therapeutic approach for hormone-dependent breast cancer, combining the dual roles of tamoxifen and HSP90 inhibitors to target senescent cancer cells. This strategy could reduce tumor recurrence and contribute to the advancement of cancer treatment methods. Citation Format: Luca Cis, Michela Gottardi Zamperla, Veronica Barbi, Pietro Cigala Fulgosi, Sara De Martino, Aurora Aiello, Simona Nanni, Chiara Cencioni, Davide Pirolli, Paola Tabarelli De Fatis, Giovanni Battista Ivaldi, Marco Malavolta, Christian Steinkühler, Maria Cristina De Rosa, Antonella Farsetti, Sandra Atlante, Carlo Gaetano. Novel HSP90 inhibitors with senolytic activity for a targeted therapy in a hormone-induced breast cancer senescence model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2977.

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.

Discovery of 5-aryl-3-thiophen-2-yl-1H-pyrazoles as a new class of Hsp90 inhibitors in hepatocellular carcinoma

Although hepatocellular carcinoma (HCC)-related mortality has increased over the past decades, treatment options are still very limited, underlining the need for developing new therapeutic strategies. The molecular chaperone heat shock protein 90 (Hsp90) plays a key role in post-translational maturation of many oncogenic client proteins that are important for survival and proliferation of cancer cells. Thus, inhibitors of Hsp90 are promising targets for many cancer types. In this study, 15 diarylpyrazole compounds were screened against MCF7 and HepG2 cell lines. Compound 8, which contained a thiophene group, demonstrated the highest antiproliferative activity against HepG2 cells having an IC50 of 0.083 μM. Four additional diarylpyrazoles, each containing a thiophene group, were prepared and screened for antiproliferative activity. None of these four compounds exhibited superior activity to compound 8 on HepG2 cells. Therefore, compound 8 was selected for further in vitro assays. Cell cycle arrest was observed at the G2 phase in compound 8-treated cells. Compound 8 also caused a 7.7-fold increase in caspase-3. These results confirm the apoptotic effect of compound 8 on HepG2 cells. Moreover, compound 8 inhibited Hsp90 (IC50 = 2.67 ± 0.18 µM) in an in vitro assay and caused a 70.8% reduction in Hsp90 levels in a HepG2 cell-based assay. Additionally, compound 8 caused significant reduction in the levels of Hsp90 client proteins (Akt, c-Met, c-Raf, and EGFR) and a 1.57-fold increase in Hsp70. Molecular docking studies were also performed to predict the binding mode of compound 8 and followed by molecular dynamics simulations to give further insights into the binding mode of 8.

Medium-Throughput Detection of Hsp90/Cdc37 Protein-Protein Interaction Inhibitors Using a Split Renilla Luciferase-Based Assay.

The protein-folding chaperone Hsp90 enables the maturation and stability of various oncogenic signaling proteins and is thus pursued as a cancer drug target. Folding in particular of protein kinases is assisted by the co-chaperone Cdc37. Several inhibitors against the Hsp90 ATP-binding site have been developed. However, they displayed significant toxicity in clinical trials. By contrast, the natural product conglobatin A has an exceptionally low toxicity in mice. It targets the protein-protein interface (PPI) of Hsp90 and Cdc37, suggesting that interface inhibitors have an interesting drug development potential. In order to identify inhibitors of the Hsp90/Cdc37 PPI, we have established a mammalian cell lysate-based, medium-throughput amenable split Renilla luciferase assay. This assay employs N-terminal and C-terminal fragments of Renilla luciferase fused to full-length human Hsp90 and Cdc37, respectively. We expect that our assay will allow for the identification of novel Hsp90/Cdc37 interaction inhibitors. Such tool compounds will help to evaluate whether the toxicity profile of Hsp90/Cdc37 PPI inhibitors is in general more favorable than that of ATP-competitive Hsp90 inhibitors. Further development of such tool compounds may lead to new classes of Hsp90 inhibitors with applications in cancer and other diseases.

A novel chalcone-based molecule, BDP inhibits MDA‑MB‑231 triple-negative breast cancer cell growth by suppressing Hsp90 function.

Triple-negative breast cancer (TNBC) is a molecularly diverse and heterogeneous disease and the molecular heterogeneity of TNBC increases the difficulty in improving survival rates. To date, therapeutic approaches for the treatment of TNBC such as hormonal chemotherapy and trastuzumab-based therapy have been limited by the lack of target receptors such as estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor2 (Her2), emphasizing the urgent need for identifying new therapeutic options. In this regard, heat shock protein90 (Hsp90) has emerged as an attractive therapeutic target for TNBC. Hsp90 plays a central role in regulating correct folding, stability, and function of numerous oncogenic proteins. In the present study, we evaluated the invitro effect of a small molecule Hsp90 inhibitor, (E)-3-(2-bromo-3,4,5-trimethoxyphenyl)-1-(2,4-dihydroxyphenyl)prop-2-en-1-one (BDP) on TNBC cell line, MDA‑MB‑231. This study indicated that BDP efficiently inhibited the growth of MDA‑MB‑231 cells in a dose- and time-dependent manner. BDP induced overall degradation of multiple oncogenic proteins including EGFR, Her2, Met, Akt, c‑Raf, and Cdk4, consequently leading to apoptotic cell death. The flow cytometric analysis revealed that BDP promoted cell cycle arrest at G2/M phases. Moreover, BDP treatment attenuated the migration of MDA‑MB‑231 cells and impaired MMP9 activity, which are essential processes for tumor metastasis. Collectively, BDP represents a new class of Hsp90 inhibitor and shows therapeutic potential for TNBC treatment.

Chalcone-templated Hsp90 inhibitors and their effects on gefitinib resistance in non-small cell lung cancer (NSCLC).

The molecular chaperone Hsp90 has emerged as an attractive cancer therapeutic target due to its role in cellular homeostasis by modulating the stabilization and maturation of many oncogenic proteins. In this study, we designed and synthesized a series of Hsp90 inhibitors that hybridized NVP-AUY992 (2) and PU3 (3) in the chalcone scaffold using a structure-based approach. Our results indicate that compound 1g inhibited the proliferation of gefitinib-resistant non-small cell lung cancer (H1975) cells, downregulated the expression of client proteins of Hsp90 including EGFR, MET, Her2 and Akt, and up-regulated the expression of Hsp70. The compound 1g represents a new class of Hsp90 inhibitors with a chalcone structure. The design, synthesis, and evaluation of 1g are described herein.

Targeting the entry region of Hsp90's ATP binding pocket with a novel 6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl amide

The molecular chaperone Hsp90 plays an important role in cancer cell survival and proliferation by regulating the maturation and stabilization of numerous oncogenic proteins. Due to its potential to simultaneously disable multiple signaling pathways, Hsp90 has emerged as an attractive therapeutic target for cancer treatment. In this study, the design, synthesis, and biological evaluation of a series of Hsp90 inhibitors are described. Among the synthetic compounds, 6,7-dihydrothieno [3,2-c]pyridin-5(4H)-yl amide 19 exhibits a remarkable binding affinity to the N-terminus of Hsp90 in a fluorescence polarization (FP) binding assay (IC50 = 50.3 nM). Furthermore, it effectively inhibits the proliferation of H1975 non-small cell lung cancer (NSCLC) and Skbr3 breast cancer cell lines with GI50 values of 0.31 μM and 0.11 μM, respectively. Compound 19 induces the degradation of the Hsp90 client proteins including EGFR, Her2, Met, c-Raf, and Akt, and consequently promotes apoptotic cancer cell death. Compound 19 also inhibits the growth of H1975 xenografts in NOD-scid IL2R gammanull mice without any apparent body-weight loss. The immunohistologic evaluation indicates that compound 19 decreases the expression of Akt in xenograft tumor tissue via an inhibition of the Hsp90 chaperon function. Additionally, the cytochrome P450 assay indicates that compound 19 has no effect on the activities of five major P450 isoforms (IC50 > 50 μM for 1A2, 2C9, 2C19, 2D6, and 3A), suggesting that clinical interactions between compound 19 and the substrate drugs of the five major P450 isoforms are not expected. Overall, compound 19 represents a new class of Hsp90 inhibitor with its 6,7-dihydrothieno[3,2-c]pyridin-5(4H)-yl-amide structure, and it has the therapeutic potential to overcome drug resistance in cancer chemotherapy.

Abstract 1732: Effect of 19-substituted benzoquinone ansamycin Hsp90 inhibitors on Hsp90/Cdc37/co-chaperone complexes and casein kinase 2 (CK2) activity

Abstract Benzoquinone ansamycin (BQA) Hsp90 inhibitors such as 17-DMAG and 17-AAG have off target toxicities including hepatotoxicity. Mechanisms underlying the toxicity of BQAs are a function of their ability to redox cycle and/or arylate cellular nucleophiles at the unsubstituted 19-position of the molecule. Therefore, we designed 19-substituted BQAs (19BQAs) to prevent conjugation with glutathione and protein thiols as an approach to reduce the hepatotoxicity and minimize off target effects of the BQA class of Hsp90 inhibitors. Our data demonstrated that 19BQAs had reduced toxicity to liver cells relative to their parent quinones while maintaining Hsp90 inhibitory activity, validating the overall approach. 19BQAs depend on tumor cell levels of NQO1 (NAD(P)H: quinone oxidoreductase 1) for optimal activity. We examined 19-phenyl-17-DMAG as a model compound in human isogenic breast cancer cell lines MDA468 (NQO1 null) and MDA468/NQ16 (NQO1 overexpressing) and in NQO1expressing BT474 breast cancer cells. 19-phenyl-17-DMAG induced growth inhibition, apoptosis and the molecular signature of Hsp90 inhibition including decreases in Hsp90 client protein levels and compensatory induction of Hsp70. In addition, treatment of MD468/NQ16 and BT474 cell lines with 19-phenyl-17-DMAG or 17-DMAG also caused the dissociation of Hsp90 from the co-chaperone Cdc37. In these studies we also observed that treatment with19-phenyl-17-DMAG or 17-DMAG resulted in inhibition of Cdc37 phosphorylation. A major kinase responsible for phosphorylation of Cdc37 is casein kinase 2 (CK2) and inhibitors of CK2 are currently under development as anticancer drugs. CK2 activity assays, immunoprecipitation and western blot analysis demonstrated that both 19-phenyl-17-DMAG and 17-DMAG inhibited CK2 kinase activity and reduced CK2α protein subunit expression in NQO1-expressing MD468/NQ16 and BT474 breast cancer cells. Decreased phosphorylation of Cdc37 led to dissociation of the Hsp90/Cdc37/client complex and resulted in the degradation of multiple kinase clients including Raf-1, Cdk4, Akt and HER2. These data suggest that inhibition of both Hsp90 and CK2 by Hsp90 inhibitors may play a role in their antitumor effects. (Supported by CA51210). Citation Format: Chuan-Hsin Chang, David Ross, David Siegel, Christopher J Moody, Russell Kitson. Effect of 19-substituted benzoquinone ansamycin Hsp90 inhibitors on Hsp90/Cdc37/co-chaperone complexes and casein kinase 2 (CK2) activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1732. doi:10.1158/1538-7445.AM2015-1732

Synthesis and biological evaluation of 3,5-disubstituted-4-alkynylisoxozales as a novel class of HSP90 inhibitors.

A series of 3,5-disubstitute-4-alkynylisoxazole derivatives were designed and synthesized through palladium(II)-copper(I) catalyzed Sonogashira cross-coupling reaction of an alkynyl moiety and an isoxazole scaffold as novel HSP90 inhibitors. The resultant compounds displayed moderate to potent binding affinity to HSP90 proteins, and also demonstrated good cell growth inhibitory activity against various cancer cell lines (A549, K562, MCF-7, DU145 and Hela). Some compounds (18d, 18e, 19a, 19d, 20c and 20q) show similar or better binding affinity towards HSP90α and HSP90β comparing to NVP-AUY922. In addition, compounds 18e, 19a and 20q exhibited potent inhibitory activity against various human cancer cell lines.

Computational predictions of corroles as a class of Hsp90 inhibitors.

Corroles have been shown experimentally to cause cell cycle arrest, and there is some evidence that this might be attributed to an inhibitory effect of corroles on Heat shock protein 90 (Hsp90), which is known to play a vital role in cancer cell proliferation. In this study, we used molecular dynamics to examine the interaction of gallium corroles with Hsp90, and found that they can bind preferentially to the ATP-binding N-terminal site. We also found that structural variations of the corrole ring can influence the binding energies and affinities of the corrole to Hsp90. We predict that both the bis-carboxylated corrole (4-Ga) and a proposed 3,17-bis-sulfonated corrole (7-Ga) are promising alternatives to Ga(III) 5,10,15-tris(pentafluorophenyl)-2,17-bis(sulfonic acid)-corrole (1-Ga) as anti-cancer agents.

Abstract 1791: 19-Substituted benzoquinone ansamycin Hsp90 inhibitors: Effects on Hsp90 co-chaperones and Hsp90-Hsf1 complexes in cellular systems

Abstract The 19-substituted benzoquinone ansamycin (BQA) class of Hsp90 inhibitors were developed to decrease the off-target toxicity of their parent unsubstituted BQAs, geldanamcyin, 17-AAG, and 17-DMAG. We have shown that 19-BQAs do not react with thiols and show decreased toxicity to liver cell systems relative to parent BQAs. As a class, Hsp90 inhibitors exhibit anti-cancer activity by decreasing the levels of Hsp90 client proteins critical to cell growth and survival. Some Hsp90 inhibitors also disrupt interaction with Hsp90 co-chaperones, including cdc37, contributing to inhibited growth. Upon inhibition, the transcription factor heat shock factor 1 (Hsf1) dissociates from Hsp90 leading to a compensatory induction of other heat shock proteins, including Hsp70 which is commonly used as a molecular biomarker of cellular Hsp90 inhibition. We undertook a detailed study of the mechanism of action of 19-BQAs in HER2-positive breast cancer cells (BT474) using 19-phenyl- and 19-methyl-DMAG as model compounds. Their ability to disrupt association of Hsp90 with the co-chaperones p23 and cdc37, the kinetics of decreased Hsp90-Hsf1 levels, nuclear Hsf1 accumulation and increases in Hsp70 levels were defined. Both 19-phenyl- and 19-methyl -DMAG disrupted Hsp90-p23 association, confirming binding of the compounds at the N-terminal ATPase site of Hsp90. 19-Phenyl-DMAG and 19-methyl-DMAG also disrupted association of Hsp90 with cdc37, although 19-phenyl-DMAG was more potent. A detailed time course of decreased levels of Hsp90 client proteins and Hsp induction showed that increased Hsp70 was a more sensitive cellular marker as it could be detected at lower drug concentrations. Increases in Hsp70 did not appear to be a generalized stress response, since 19-substituted-BQAs caused dissociation of the Hsp90-Hsf1 dimer, nuclear translocation of Hsf-1, and subsequent increases in Hsp70 levels in a temporal manner. These data indicate that 19-substituted BQAs may exhibit their growth inhibitory effects in breast cancer cells through disruption of Hsp90 and its co-chaperones, particularly cdc37. The N-terminal ATPase site binding and Hsp90 inhibitory activity of these compounds was confirmed through disruption of Hsp90-p23 and Hsp90-Hsf1 associations, respectively (Supported by NCI grant CA51210). Citation Format: Derek A. Drechsel, Chuan-Hsin Chang, Russell Kitson, David Siegel, Christopher J. Moody, David Ross. 19-Substituted benzoquinone ansamycin Hsp90 inhibitors: Effects on Hsp90 co-chaperones and Hsp90-Hsf1 complexes in cellular systems. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1791. doi:10.1158/1538-7445.AM2014-1791

Abstract 1619: Hsp90 inhibitor drug conjugates (HDCs): Construct design and preliminary evaluation

Abstract Background: Most cytotoxic agents are often broadly active but non-selective, and have the disadvantage of high toxicity due to collateral damage to normal tissues. Drugs that target specific protein drivers of cancer cell growth are more tumor selective, yet often lead to tumor resistance via point mutations in their target or activation of alternative signaling pathways. Targeted delivery strategies, such as Antibody-Drug Conjugates (ADCs), offer a solution to these limitations by delivering potent anti-cancer payloads more directly to tumors. Hsp90 is a chaperone protein required by many cancer cells to maintain the stability and function of numerous proteins that drive cancer cell growth, survival, and metastasis. Small molecule inhibitors of Hsp90 such as ganetespib, PU-H71 and 17-AAG are found to be retained in tumors with half- time up to 65 hours in mouse xenografts. These properties are believed to be due to overexpression of an active form of Hsp90 in cancer cells as compared to normal tissues, and have recently been applied for tumor imaging in patients. Results: We have developed a small molecule drug conjugate platform technology using the unique properties of Hsp90 proteins and Hsp90 inhibitors. We have synthesized HDCs with various Hsp90 inhibitor scaffolds including resorcinol, purine, geldanamycin, etc. A cell-based client protein degradation assay is carried out to confirm the intracellular uptake and the Hsp90 binding of the conjugates. While different Hsp90 inhibitor scaffolds offer different DMPK and toxicology profiles, cellular uptake and Hsp90 binding are not greatly affected by the different chemical classes of Hsp90 inhibitors. Over 30 payloads have been conjugated with Hsp90 inhibitors so far. We have selected the payloads based on the hypothesis that HDC can improve the safety profile of a cytotoxic drug, expand the application of chemotherapeutic agents to different tumor types, combat drug resistance, and enable novel anticancer approach. Examples of payloads include topoisomerase inhibitors (camptothecin), microtubule modulators (taxanes), proteasome inhibitors (carfilzomib), CDK inhibitors (flavopiridol) and others. Unlike most other conjugate technologies, HDC does not require lengthy spacing between the anchor Hsp90 inhibitor and the payload. Linker cleavage mechanism is considered a key feature in the HDC design. We have been able to incorporate several linkers such as disulfide, hydrazone, peptide, carbamate, carboxylate, etc. into our HDC designs. Conclusion: In this HDC platform, we provide a method which can be applied to many well-studied mechanisms for modulating cancer pathways and stopping tumor cell growth. We have created a promising platform technology which can result in many anticancer agents in the near future. Citation Format: Weiwen Ying, Dinesh Chimmanamada, Junyi Zhang, Teresa Przewloka, Jun Jiang, Genliang Lu, Sami Osman, James Loch, Dharma Vutukuri, Shoujun Chen, Robert Stein, John Chu, David Proia, Pat Rao, Takayo Inoue, Luisa Shin Ogawa, Ritu Singh, Noriaki Tatsuta. Hsp90 inhibitor drug conjugates (HDCs): Construct design and preliminary evaluation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1619. doi:10.1158/1538-7445.AM2014-1619

Abstract 1788: 19-Substituted benzoquinone ansamycins. Hsp90 inhibitors with decreased off-target toxicity

Abstract Benzoquinone ansamycin (BQA) Hsp90 inhibitors such as 17-DMAG and 17-AAG have off-target toxicities in clinical trials including hepatotoxicity. Mechanisms underlying the toxicity of quinones are a function of their ability to redox cycle and/or arylate cellular nucleophiles at the unsubstituted 19-position of the molecule. Therefore, we designed 19-substituted BQAs to prevent glutathione conjugation and non-specific interactions with protein thiols as an approach to reduce the hepatotoxicity and minimize off-target effects of the BQA class of Hsp90 inhibitors. In this study, the results showed that 19-substituted BQAs did not react with glutathione at the 19-position, while marked reactivity was observed using parent BQAs. Importantly, while parent 17-DMAG induced cell death in primary and cultured mouse hepatocytes, 19-phenyl and 19-methyl 17-DMAG showed reduced toxicity, validating the overall approach. There was no significant difference between the redox cycling ability of either 19-phenyl or 19-methyl 17-DMAG with their parental BQAs in both mouse and human liver microsomes. Accordingly, this suggests that arylation reactions at the unsubstituted 19-position are predominantly responsible for hepatotoxicity. 19-substituted17-DMAG inhibited purified Hsp90 ATPase activity in an NQO1-dependent manner that demonstrated increased inhibitory efficacy of the hydroquinone ansamycin relative to its parent quinone. In human breast cancer cells, 19-phenyl BQAs induced growth inhibition in an NQO1-dependent manner with molecular signatures of Hsp90 inhibition, including decreases in client proteins and compensatory induction of Hsp70. These data indicate that 19-substituted BQAs may be useful Hsp90 inhibitors with decreased off target toxicity (Supported by NCI grant CA51210) Citation Format: Chuan-Hsin Chang, Derek A. Drechsel, Russell R.A. Kitson, David Siegel, Qiang You, Donald S. Backos, Cynthia Ju, Christopher J. Moody, David Ross. 19-Substituted benzoquinone ansamycins. Hsp90 inhibitors with decreased off-target toxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1788. doi:10.1158/1538-7445.AM2014-1788

Fragment-based hit discovery and structure-based optimization of aminotriazoloquinazolines as novel Hsp90 inhibitors

In the last decade the heat shock protein 90 (Hsp90) has emerged as a major therapeutic target and many efforts have been dedicated to the discovery of Hsp90 inhibitors as new potent anticancer agents. Here we report the identification of a novel class of Hsp90 inhibitors by means of a biophysical FAXS-NMR based screening of a library of fragments. The use of X-ray structure information combined with modeling studies enabled the fragment evolution of the initial triazoloquinazoline hit to a class of compounds with nanomolar potency and drug-like properties suited for further lead optimization.

Multivariate analysis in the identification of biological targets for designed molecular structures: The BIOTA protocol

In this work the new protocol BIOlogical Target Assignation (BIOTA) for the prediction of the biological target from molecular structures is proposed. BIOTA is based on the Principal Components Analysis (PCA) application on a matrix of ligands versus molecular descriptors. The application of BIOTA could allow to hypothesize the mechanism of action of a candidate drug prior to its biological evaluation or to repurpose old drugs. The protocol can be fine-tuned by choosing opportune targets (biological or not) and molecular descriptors, and it can be useful in every fields in with it is possible to collect set of compounds with known properties.The robustness of the protocol depends from different factors: the correctness of biological data, the optimization of the molecular structures and their molecular descriptors calculation, the selection of the biological targets. The application of BIOTA to a new class of Hsp90 inhibitors was able to predict quite correctly their affinity for Hsp90.

Targeting the hydrophobic region of Hsp90’s ATP binding pocket with novel 1,3,5-triazines

Heat shock protein 90 (Hsp90) is a molecular chaperone that plays an important role in regulating the maturation and stabilization of many oncogenic proteins. In an attempt to discover a new class of Hsp90 inhibitors, a series of 1,3,5-triazine compounds were rationally designed, synthesized, and their biological activities were evaluated. Compound 3b was found to degrade Hsp90’s client proteins of Her2, Met and Akt and to induce the expression level of Hsp70. The binding mode of 3b in the ATP-binding site of Hsp90 was predicted by the molecular docking.

A Chemical–Biological Study Reveals C9-type Iridoids as Novel Heat Shock Protein 90 (Hsp90) Inhibitors

The potential of heat shock protein 90 (Hsp90) as a therapeutic target for numerous diseases has made the identification and optimization of novel Hsp90 inhibitors an emerging therapeutic strategy. A surface plasmon resonance (SPR) approach was adopted to screen some iridoids for their Hsp90 α binding capability. Twenty-four iridoid derivatives, including 13 new natural compounds, were isolated from the leaves of Tabebuia argentea and petioles of Catalpa bignonioides. Their structures were elucidated by NMR, electrospray ionization mass spectrometry, and chemical methods. By means of a panel of chemical and biological approaches, four iridoids were demonstrated to bind Hsp90 α. In particular, the dimeric iridoid argenteoside A was shown to efficiently inhibit the chaperone in biochemical and cellular assays. Our results disclose C9-type iridoids as a novel class of Hsp90 inhibitors.

1679P - Heat Shock Protein 90 Inhibition Downregulates C-Kit Expression in Gist Cells Through Diminishing Transcription and Enhancing Protein Degradation via both Autophagy and Proteasome Pathways

ABSTRACT Background Gastrointestinal stromal tumor (GIST) is a mutant oncoprotein c-Kit droved cancer. Patients suffered drug resistance after prolonged standard treatment of tyrosine kinase inhibitor (TKI), imatinib mesylate (IM). Inhibition of heat-shock protein 90 (Hsp90), a chaperone responsible for protein maturation and stability, causing its client proteins degradation, as c-Kit, is a developing therapy for cancer. However, the mechanisms of Hsp90 inhibition-induced c-Kit downregulation in post-translational and transcriptional levels have rarely been investigated. Methods and results Our data showed that NVP-AUY922 (AUY922), a new class of Hsp90 inhibitor, inhibited the growth of GIST882 and GIST48 cells with mutant c-Kit protein expression that was accompanied with reduction of both total and phosphor-c-Kit protein and induction of apoptosis. Pharmacological inhibition on either autophagy or proteasome degradation pathway could partially reverse endogenous c-Kit turnover and AUY922-induced c-Kit reduction. These findings were further confirmed by the co-localization of c-Kit with either LC-3B or acridine orange-labeled autophagosome in confocal microscopy, and delay of c-Kit degradation by silencing autophagosome essential Beclin-1 protein. In addition, AUY922 could also reduce c-Kit mRNA without affecting its degradation. Our preliminary data showed that activities and nuclear levels of several transcription factors in GIST cells were diminished after AUY922 treatment. DNA affinity precipitation assay and chromatin immunoprecipitation (ChIP) will be performed to validate the binding of the candidate transcript factors on the c-Kit promoter. Conclusions This is the first report showing the involvement of autophagy in endogenous as well as Hsp90 inhibitor-induced c-Kit degradation. Moreover, AUY922 treatment resulted in downregulation of c-Kit through protein degradation and transcriptional mRNA regulation in GIST cells. These findings address the mechanisms of AUY922 on c-Kit protein downregulation and highlight the potential use of AUY922 in TKI-refractory, c-Kit-expressing GIST. Disclosure All authors have declared no conflicts of interest.

Natural Iminosugar (+)-Lentiginosine Inhibits ATPase and Chaperone Activity of Hsp90

Heat shock protein 90 (Hsp90) is a significant target in the development of rational cancer therapy due to its role at the crossroads of multiple signaling pathways associated with cell proliferation and cell viability. The relevance of Hsp90 as a therapeutic target for numerous diseases states has prompted the identification and optimization of novel Hsp90 inhibitors as an emerging therapeutic strategy. We performed a screening aimed to identify novel Hsp90 inhibitors among several natural compounds and we focused on the iminosugar (+)-lentiginosine, a natural amyloglucosidases inhibitor, for its peculiar bioactivity profile. Characterization of Hsp90 inhibition was performed using a panel of chemical and biological approaches, including limited proteolysis, biochemical and cellular assays. Our result suggested that the middle domain of Hsp90, as opposed to its ATP-binding pocket, is a promising binding site for new classes of Hsp90 inhibitors with multi-target anti-cancer potential.

Heat Shock Protein 90 and Role of Its Chemical Inhibitors in Treatment of Hematologic Malignancies

Heat shock protein 90 (Hsp90) is a conserved and constitutively expressed molecular chaperone and it has been shown to stabilize oncoproteins and facilitate cancer development. Hsp90 has been considered as a therapeutic target for cancers and three classes of Hsp90 inhibitors have been developed: (1) benzoquinone ansamycin and its derivatives, (2) radicicol and its derivates, and (3) small synthetic inhibitors. The roles of these inhibitors in cancer treatment have been studied in laboratories and clinical trials, and some encouraging results have been obtained. Interestingly, targeting of Hsp90 has been shown to be effective in inhibition of cancer stem cells responsible for leukemia initiation and progression, providing a strategy for finding a cure. Because cancer stem cells are well defined in some human leukemias, we will focus on hematologic malignancies in this review.