Unfortunately, detailed structural biology studies on STAT3 as well as target-based drug discovery attempts have been hampered by difficulties in the expression and purification of the full-length STAT3 and a lack of ligand-bound crystal structures. Considering these, molecular modeling and simulations offer a stylish strategy for the assessment of the druggability of STAT3 dimers and allow investigations of reported activating and inhibiting STAT3 mutants in the atomistic level of fine detail. atomistic level of fine detail. In the present study, we focused on the effects exerted by reported STAT3 mutations within AT-1001 the protein structure, dynamics, DNA-binding, and dimerization, thus linking structure, dynamics, energetics, and the biological function. By employing atomistic molecular dynamics and umbrella-sampling simulations to a series of human being STAT3 dimers, which comprised wild-type protein and four mutations, we explained the modulation of STAT3 activity by these mutations. Counter-intuitively, our results show the D570K inhibitory mutation exerts its effect by enhancing rather than weakening STAT3CDNA relationships, which interfere with the DNA launch by the protein dimer and thus inhibit STAT3 function as a transcription element. We mapped the binding site and characterized the binding mode of a medical candidate napabucasin/BBI-608 at STAT3, which resembles the effect of a D570K mutation. Our results contribute to understanding the activation/inhibition mechanism of STAT3, to explain the molecular mechanism of STAT3 inhibition by BBI-608. Alongside AT-1001 the characterization of the BBI-608 binding mode, we also found out a novel binding site amenable to bind small-molecule ligands, which may pave the AT-1001 way to design novel STAT3 inhibitors and to suggest new strategies for pharmacological interventions to combat cancers associated with poor prognosis. Intro Transmission transducer activator of transcription 3 (STAT3) protein has emerged like a prominent target in tumor progression due to its pivotal part in cell signaling.1 The activation of the STAT3 protein has been also related to drug resistance,2 to the expression of additional anti-apoptotic proteins,3 and to the inflammatory processes in tumor development, among others.4?6 Despite its importance in malignancy progression, the pharmacological drugging of STAT3 is still challenging that demands clarification. Its inclination to aggregate is definitely a major hurdle that helps prevent the determination of the structure in both monomeric and dimeric forms as well as bound to small-molecule inhibitors.7?9 Although many strategies have been explained in the literature to inhibit STAT3, only a few inhibitors are still going through clinical trials (e.g., TTI-101 [ClinicalTrials.gov Identifier: “type”:”clinical-trial”,”attrs”:”text”:”NCT03195699″,”term_id”:”NCT03195699″NCT03195699] or napabucasin (BBI-608)10?12 [ClinicalTrials.gov Identifier: “type”:”clinical-trial”,”attrs”:”text”:”NCT03647839″,”term_id”:”NCT03647839″NCT03647839]), and STAT3 has become probably one of the most challenging cancer-related proteins to target by small molecules. Gaining insights into the atomistic-level characteristics of STAT3 would permit the Has2 recognition of novel strategies to interact with this protein by small molecules and target oncogenic pathways indirectly. The human STAT3 monomer AT-1001 is composed of six highly specialized domains (i.e., N-terminal, coiled-coil domain name, DNA-binding domain name, linker domain name, SH2 domain name, and C-terminal transactivation domain name). Particularly, the DNA-binding domain name (residues: 320C494) is responsible for the DNA-binding when STAT3 is in the dimeric form. An -helix linker domain name joins the latter with the SH2 domain name, which is essential for the binding of STAT3 to phosphorylated receptors and for its dimerization (residues: 493C583). This process is mainly facilitated by the SH2 domain name, as each monomer interacts after the phosphorylation of a specific tyrosine residue (Y705) located in the transactivation domain name.13,14 Therefore, in an attempt to avoid the dimerization, the SH2 domain name has traditionally been the main target for drug design, mostly accompanied by computational studies relying on molecular docking calculations or similar structure-based approaches,15?23 despite no crystallographic data being available up to date to support them. These ligands attempt to compete with phosphorylated p-Y705 at the site known to recognize phosphorylated residues24,25 with limited success. An alternative, represented by OPB-3112126 and OPB-51602,4 is to target an allosteric site at the SH2 domain name: these compounds bind to a pocket different from the one binding p-Y705.4,26 Furthermore, STAT3 can undergo other post-translational modifications besides Y705 phosphorylation, such as S727 phosphorylation,13,27 and it has been experimentally demonstrated that unphosphorylated STAT3 can dimerize and bind to DNA. This provides an alternative strategy to targeting the SH2 domain name directly. Due to the challenges coupled with the effective targeting of STAT3 SH2 domains, other STAT3 domains have thus been explored in the development of potent and selective STAT3 inhibitors. Recently, several inhibitors have AT-1001 been identified to bind to the DNA-binding domain name (DBD), which has been initially overlooked due to its potential specificity problems. Experimental evidence shows that its binding by small molecules permits the dimer formation, whereas disrupts the DNACprotein binding.28?30 The exploration through alternatives to the SH2 domain might be the key to unlock the STAT3 druggability problem. The conservation of the three-dimensional structure of the whole protein is crucial for the activity of.