bp-1-102 and stattic

Signal transducer and activator of transcription (STAT) proteins have important biological functions; however, deregulation of STAT signaling is a driving force behind the onset and progression of inflammatory diseases and cancer. While their biological roles suggest that STAT proteins would be valuable targets for developing therapeutic agents, STAT proteins are notoriously difficult to inhibit using small drug-like molecules, as they do not have a distinct inhibitor binding site. Despite this, a multitude of small-molecule STAT inhibitors have been proposed, primarily focusing on inhibiting STAT3 protein to generate novel cancer therapies. Demonstrating that inhibitors bind to their targets in cells has historically been a very challenging task. With the advent of modern target engagement techniques, such as the cellular thermal shift assay (CETSA), interactions between experimental compounds and their biological targets can be detected with relative ease. To investigate interactions between STAT proteins and inhibitors, we herein developed STAT CETSAs and evaluated known STAT3 inhibitors for their ability to engage STAT proteins in biological settings. While potent binding was detected between STAT proteins and peptidic STAT inhibitors, small-molecule inhibitors elicited variable responses, most of which failed to stabilize STAT3 proteins in cells and cell lysates. The described STAT thermal stability assays represent valuable tools for evaluating proposed STAT inhibitors.

Topics: Aminosalicylic Acids; Cell Line, Tumor; Cyclic S-Oxides; Heating; Humans; Peptides; Protein Binding; Protein Stability; STAT3 Transcription Factor; Sulfonamides

Inhibitors for signal transducer and activator of transcription 3 (STAT3), Stattic, BP-1-102, and LLL12 significantly induce apoptosis in transformed Ba/F3 cells expressing an oncogenic fusion protein, nucleophosmin-anaplastic lymphoma kinase (NPM-ALK) that induces the activation of STAT3. We found that the antioxidant reagent, N-acetyl cysteine (NAC) prevented the abilities of Stattic and BP-1-102, but not LLL12 to induce apoptosis in transformed cells expressing NPM-ALK, providing a novel problem in use of STAT3 inhibitors. We herein investigated the mechanisms how NAC prevented the effects of Sttatic and BP-1-102.. Ba/F3 cells expressing NPM-ALK and SUDHL-1 cells were treated with antioxidants such as NAC, Trolox or edaravone in combination with STAT3 inhibitors. Phosphorylation of STAT3, cell proliferation rate, cell viability, cell cycle, internucleosomal DNA fragmentation and the intracellular accumulation of reactive oxygen species (ROS) was investigated. The binding of STAT3 inhibitors and NAC was analyzed by LC-MS.. NAC but not Trolox and edaravone diminished the abilities of Stattic and BP-1-102 to induce apoptosis in cells expressing NPM-ALK. The ROS levels in cells expressing NPM-ALK were not markedly affected by the treatments with Stattic and BP-1-102 in combination with NAC, suggesting that NAC inhibited the activity of Stattic and BP-1-102 independent of its antioxidant activity. LC-MS analysis revealed that NAC directly bound to Stattic and BP-1-102. Furthermore, these NAC adducts exhibited no cytotoxicity, and failed to affect the activity of STAT3.. NAC antagonizes the activities of Stattic and BP-1-102, which inhibit STAT3 activation by interacting with cysteine residues in STAT3.

Topics: Acetylcysteine; Aminosalicylic Acids; Anthraquinones; Antioxidants; Apoptosis; Cell Line, Tumor; Cell Survival; Cyclic S-Oxides; Humans; Phosphorylation; Reactive Oxygen Species; Signal Transduction; STAT3 Transcription Factor; Sulfonamides