Identification of the mechanism of action of 5,6-dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide as STAT3 inhibitor

Signal Transducer and Activator of Transcription 3 (STAT3) is an emerging anticancer target. One of the strategies to achieve STAT3 inhibition could be the disruption of the homodimerization process by small molecules targeting the protein-protein interaction. 5,6-Dimethyl-1H,3H-2,1,3-benzothiadiazole-2,2-dioxide (EL-1) has been identified as promising STAT3 inhibitor by a virtual screening approach. Preliminary structure-activity relationship (SAR) studies were carried out to optimize the hit compound, but any modification of its structure resulted in the decrease of its activity. To rationally guide the design of more potent derivatives, EL-1 mechanism of action was investigated. Most of the known STAT3 inhibitors act as covalent binders interacting with five superficial cysteines. EL-1 in vitro reactivity toward cysteines was tested by means of mass spectrometry detection of thiol adduct, using glutathione as a reactive cysteine surrogate. Preliminary LC-UV data showed a reactivity between EL-1 and GSH in different conditions (i.e. presence/absence of an antioxidant and different pH). At acidic pH, EL-1 is not reactive. However, at neutral and basic pH EL-1 reacts with GSH trough an electrophilic attack to the thiol group, triggered by EL-1 intermediates generated either by resonance or oxidation mechanisms. High-resolution mass spectrometry allowed the identification of EL-1 – GSH adducts and provided an insight into the reactivity of this molecule. The fundamental functional groups for EL-1 reactivity were identified, thus enabling a rational SAR investigation. Derivatives of EL-1 are being synthetized and they will be tested to identify improved candidate compounds for the potent and selective inhibition of STAT3.