Isolation of a Novel Thioflavin S–Derived Compound That Inhibits BAG-1–Mediated Protein Interactions and Targets BRAF Inhibitor–Resistant Cell Lines

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 dosedependently 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.

Choose and Use Your Chemical Probe Wisely to Explore Cancer Biology

“…Thioflavin S is a complex mixture of compounds and, originally, its biological activity was not definitively attributed to any one component; however, a subsequent study isolated and purified Thio-2 (Figure 5A), a component which retained the ability of Thioflavin S to block the BAG-1/HSC70 interaction (Enthammer et al., 2013). Despite this advance, the Thioflavin class of molecules, to which Thio-2 belongs, exhibit multiple sources of off-target activities, including CYP1A1-mediated generation of reactive intermediates and DNA-adduct formation (Chakraborty et al., 2010) as well as PAINS motifs (see next section), casting strong doubt upon the credentials of these compounds as high-quality chemical probes.…”

Targeting BAG-1: A novel strategy to increase drug efficacy in acute myeloid leukemia

“…We decided to impair BAG-1 function, and test whether it might influence leukemia cells sensitivity to drugs. We used Thioflavin S (NSC71948), which has recently been shown to interfere with the ability of BAG-1 to interact with HSP70 in vitro [36,37]. We treated three AML cell lines, HL60 (BAG-1 expression corresponding to >75 th percentile), ML-2 (<50 th percentile) and THP-1 (>75 th percentile; Table 3 To assess if observed increase in the efficiency of drug action occurred due to synergic activity among combined drugs, we calculated the combination index (CI) according to the Chou and Talalay method [29].…”

BAG-1 as a biomarker in early breast cancer prognosis: a systematic review with meta-analyses

“…Bcl-2-associated athanogene-1 exists as three main isoforms, which are produced by alternative translation initiation from a single mRNA (Packham et al , 1997). Bcl-2-associated athanogene-1L is found in the nucleus, whereas BAG-1M and BAG-1S are generally found in the cytoplasm (Packham et al , 1997; Takayama et al , 1998; Brimmell et al , 1999; Schneikert et al , 1999; Yang et al , 1999; Knee et al , 2001), and the possibility of BAG-1 directed therapy has been suggested from laboratory studies (Sharp et al , 2009a, 2009b; Enthammer et al , 2013). In the clinical setting, BAG-1 mRNA has been incorporated as a prognostic biomarker in Oncotype DX (Paik et al , 2004) and PAM50 (Parker et al , 2009) multigene assays, which estimate prognosis following surgery, and can be used to assess the potential benefit of chemotherapy for breast cancer.…”