Published OnlineFirst September 18, 2013; DOI: 10.1158/1535-7163.MCT-13-0142 Molecular Cancer Small Molecule Therapeutics Therapeutics Isolation of a Novel Thioflavin S–Derived Compound That Inhibits BAG-1–Mediated Protein Interactions and Targets BRAF Inhibitor–Resistant Cell Lines Marion Enthammer1, Emmanouil S. Papadakis3, Maria Salome Gachet2, Martin Deutsch1, Stefan Schwaiger2, Katarzyna Koziel1, Muhammad Imtiaz Ashraf1, Sana Khalid1, Gerhard Wolber2, Graham Packham3, Ramsey I. Cutress3, Hermann Stuppner2, and Jakob Troppmair1 Abstract Protein–protein interactions mediated through the C-terminal Bcl-2–associated athanogene (BAG) domain of BAG-1 are critical for cell survival and proliferation. Thioflavin S (NSC71948)—a mixture of compounds resulting from the methylation and sulfonation of primulin base—has been shown to dose- dependently inhibit the interaction between BAG-1 and Hsc70 in vitro. In human breast cancer cell lines, with high BAG-1 expression levels, Thioflavin S reduces the binding of BAG-1 to Hsc70, Hsp70, or CRAF and decreases proliferation and viability. Here, we report the development of a protocol for the purification and isolation of biologically active constituents of Thioflavin S and the characterization of the novel compound Thio-2. Thio-2 blocked the growth of several transformed cell lines, but had much weaker effects on untransformed cells. Thio-2 also inhibited the proliferation of melanoma cell lines that had become resistant to treatment with PLX4032, an inhibitor of mutant BRAF. In transformed cells, Thio-2 interfered with intracellular signaling at the level of RAF, but had no effect on the activation of AKT. Thio-2 decreased binding of BAG-1 to Hsc70 and to a lesser extent BRAF in vitro and in vivo, suggesting a possible mechanism of action. Given that tumors frequently develop resistance to kinase inhibitors during treatment, Thio-2 and related compounds may offer promising alternative strategies to currently available therapies. Mol Cancer Ther; 12(11); 2400–14. Ó2013 AACR. Introduction ylation and requires the assembly of signalosomes, which, RAF kinases are part of an evolutionarily conserved along with the core components of the signaling cascade, core-signaling cascade downstream of activated receptor contain accessory molecules required for modulation, tyrosine kinases and the small G-protein RAS (1). The compartmentalization, and specificity (1–3). RAF kinases three RAF isoforms, ARAF, BRAF, and CRAF (also RAF-1), are upstream of a three-tiered mitogen–activated protein are involved in mitogen, survival, and differentiation kinase (MAPK) cascade comprising the dual-specificity signaling. Their activation is a complex process involving Ser/Thr MAPK kinases (MAP2K) MEK1/2 and their interactions with proteins and lipids as well as phosphor- substrates ERK1/2, which are required for most reported RAF effects (4, 5). Several proteins have been described, which may be important for RAF activation and signaling under specific 1 Authors' Affiliations: Daniel Swarovski Research Laboratory, Depart- conditions at distinct cellular sites. These include the Bcl- ment of Visceral, Transplant and Thoracic Surgery, Innsbruck Medical University; 2Institute of Pharmacy/Pharmacognosy, Center of Molecular 2–associated athanogene 1 (BAG-1; refs. 6–10). BAG-1 was Biosciences, University of Innsbruck, Innsbruck, Austria; and 3Cancer originally identified as a Bcl-2–interacting protein with Research UK Centre, Cancer Sciences Division, University of Southampton Faculty of Medicine, Southampton General Hospital, Southampton, United antiapoptotic activity (11). BAG-1 is a member of a family Kingdom of proteins characterized by at least one copy of an Note: Supplementary data for this article are available at Molecular Cancer approximately 100 amino acid–long evolutionarily con- Therapeutics Online (http://mct.aacrjournals.org/). served a-helical BAG domain that allows them to interact with and regulate the Hsp70 family of molecular chaper- Current address for G. Wolber: Freie Universitat€ Berlin, Institute of Phar- macy, Department Pharmaceutical and Medicinal Chemistry, Berlin, ones (12, 13). BAG-1 can bind to the kinase domain of Germany. CRAF (14) or BRAF (15), and in vitro experiments showed Corresponding Author: Jakob Troppmair, Innsbruck Medical University, that BAG-1/CRAF interaction leads to the activation of Innrain 66, Innsbruck 6020, Austria. Phone: 43-512-504-27819; Fax: 43- CRAF independently of RAS (14). RAS-independent RAF 512-504-24624; E-mail: [email protected] activation, which results in the activation of ERK1/2, is also doi: 10.1158/1535-7163.MCT-13-0142 observed in cells overexpressing BAG-1 (16). ERK1/2 acti- Ó2013 American Association for Cancer Research. vation can be terminated by stress-induced upregulation 2400 Mol Cancer Ther; 12(11) November 2013 Downloaded from mct.aacrjournals.org on September 25, 2021. © 2013 American Association for Cancer Research. Published OnlineFirst September 18, 2013; DOI: 10.1158/1535-7163.MCT-13-0142 Thio-2 Inhibitor of BAG-1–Mediated Protein Interactions of Hsp70 (16). Mechanistically, this may be explained by (42). Thioflavin S also reduces viability of wild-type (wt) the competition between CRAF and Hsp70 for a common but not BAG-1–deficient mouse embryonic fibroblasts binding site in helix 2 of the BAG domain. Genetic dis- (42). Until now, hit-to-lead development of drug-like ruption of BAG-1 in mice has no effect on the activation of inhibitors has been precluded by complexity of the Thio- ERK1/2 by mitogens (15). Nevertheless, subcellular local- flavin S mixture. Here, we report the isolation and puri- ization studies have suggested that BAG-1 forms a mito- fication of the compounds Thio-2, Thio-3, Thio-5, and chondrial survival signaling complex with Hsp70/AKT Thio-6 from Thioflavin S and their structural characteri- and BRAF, the absence of which may account for zation. We also demonstrate the ability of Thio-2 to block increased apoptosis in the liver and developing nervous signal propagation via the MAPK pathway, to impede the system of BAG-1–deficient mouse embryos (15). Evidence proliferation of RAF-transformed cells, and inhibit the for BAG-1 involvement in RAF-driven transformation has interaction of BAG-1 with Hsc70 and to a lesser degree been provided by the demonstration that BAG-1 hetero- RAF in HEK293 cells. zygosity in mice expressing a constitutively active form of CRAF in type II pneumocytes significantly reduces onco- gene-induced lung adenoma growth (17). In this model, Materials and Methods RAF-downstream signaling was unaffected, whereas Reagents reduced BAG-1 expression specifically targeted tumor General chemicals were of molecular biology grade and cells to apoptosis (17). were purchased either from Sigma-Aldrich or Fisher Activating mutations in RAF kinases are almost exclu- Scientific, unless otherwise stated. Thioflavin S practical sively restricted to BRAF and are present in approximate- grade (CAS 1326-12-1) was purchased from Sigma- ly 10% of all human tumors with the highest incidence of Aldrich. Calculation of molarity was based on informa- approximately 42% (Catalogue of Somatic Mutations in tion provided by the National Cancer Institute Develop- Cancer Database, Wellcome Sanger Trust) detected in mental Therapeutics Program (NCI-DTP; Rockville, MD; melanoma (1, 5, 18). Most commonly BRAF mutant mel- http://dtp.cancer.gov). Analytical grade reagents (n-hex- anoma are V600E (74%–90%; ref. 19) and 16% to 29% are ane, diethyl ether, ethyl acetate, n-butanol, petroleum V600K mutations (20, 21). Because of the role of RAF ether, acetone, dichloromethane, and methanol) were kinases in promoting cancer cell growth, mainly through supplied by VWR. High-performance liquid chromato- enforcing cell-cycle progression and enhancing cell sur- graphy (HPLC)-grade acetonitrile and methanol were vival (1, 5, 18, 22, 23), the RAS–RAF–MEK module has obtained from Merck. Deionized water (18.2 MO Â cm) become a promising target for therapeutic intervention. was obtained from an Arium 611 UV system (Sartorius This led to the development of small molecular weight Stedim Biotech GmbH). The MEK-inhibitor U0126 was inhibitors of RAF and MAP–ERK kinase (MEK; refs. 5, 24), purchased from Promega Corporation and prepared as a with recent clinical studies reporting that highly specific 10 mmol/L stock solution in dimethyl sulfoxide (DMSO; BRAF inhibitors are effective in the treatment of metastatic Sigma-Aldrich). AZD6244 (selumetinib) was obtained melanoma (25–27). However, initial promise has been from Eubio and prepared as a 10 mmol/L stock in DMSO. hampered by the development of resistance (28–30), Staurosporine (100 mmol/L stock) was obtained from which is characterized by the reactivation of ERK1/2 Sigma-Aldrich. RAF inhibitors sorafenib (BAY43-9006) (31–33) and has been attributed to various mechanisms and PLX4032 were obtained from Axon Medchem BV including activating NRAS mutations (29), CRAF over- and prepared as 50 mmol/L and 10 mmol/L stocks in expression (34), compensatory upregulation of MAP2K DMSO, respectively. Thioflavin S (Sigma-Aldrich) and kinase COT (28), activating MEK1 mutations (35), and purified compounds (Thio-2, Thio-3, and Thio-5; for iso- amplification of mutant BRAF (36). lation and purification see below) were prepared as a 10 Disruption of specific protein–protein interactions (PPI) mmol/L stock solution in DMSO. All inhibitors were within signalosomes may provide an alternative approach, finally diluted in culture medium