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HER3 (or ErbB3) is a member of the human epidermal growth factor receptor (HER/EGFR) family of receptor tyrosine kinases. HER3 carries inactivating mutations in its kinase domain and therefore is denoted as a pseudokinase. Despite lacking catalytic activity, the pseudokinase domain of HER3 plays an important role in activating EGFR and HER2 kinases, resulting in potent downstream signaling. Recent advancements in cancer research identify HER3 as an important therapeutic target in breast, lung, gastric, ovarian and colorectal cancers, but due to HER3's pseudokinase status, its inhibition remains a major challenge. Our past work demonstrated that the pseudokinase domain of HER3 tightly binds ATP. This finding suggested an exciting opportunity - it might be possible to regulate HER3 signaling through modulation of its nucleotide-binding pocket. However, due to lack of available compounds, this idea was left untested. Here we are reporting that the allosteric activator function of HER3 can be modulated through this site via the ATP-competitive inhibitor bosutinib. We present a first structure of the HER3 pseudokinase domain with an inhibitor, bosutinib, bound in the nucleotide-binding site. To our knowledge, this is the first structure of any pseudokinase bound to an inhibitor. We then perform in vitro assays to show for the first time that HER3 function can be modulated through small molecule binding to the nucleotide-binding pocket. Although we show that bosutinib has the opposite effect from what is desired in cancer therapies, our study provides the proof of principle for small molecule regulation of the HER3 function. This finding opens an exciting direction for the generation of a new class of HER3-specific therapeutics that directly target the pseudokinase domain.

DNA-binding small molecules are present in the cellular environment and are ubiquitously used in biochemistry and biotechnology. Here, we use single molecule magnetic tweezers experiments to study the effect of small-molecule ligands on DNA mechanical properties.Using the magnetic tweezers ability to control both the applied stretching force and torque, we have systematically characterized the mechanical properties of DNA in the presence of Ethidium Bromide (EtBr), a well-known intercalator, and Netropsin, an anti-microbial drug and known minor groove binder. In addition, we have characterized the interactions of Topotecan, a clinically used anti-tumor drug, with bare DNA.Our results show a lengthening of DNA upon EtBr intercalation to an extension of ∼1.5 times the initial contour length and a decrease in DNA twist by 29 +- 3 degrees per intercalation event, in agreement with previous estimates from bulk experiments. Further effects of intercalation are a stabilization of the double strand under high forces and negative torques and a decrease in both the bending and twisting persistence lengths. In contrast, minor groove binding by Netropsin does not change the contour or persistence length significantly, but increases the twist per base of the DNA.For Topotecan, both intercalative and minor-groove binding have been postulated. Our magnetic tweezers results indicate that drug binding at > 50 micromolar concentrations has consequences qualitatively similar to the changes observed for EtBr, in support of a intercalative binding mode for Topotecan.These results point to important consequences of intercalative ligand binding for DNA torsional behavior, which we characterized at the single-molecule level using magnetic tweezers. Ultimately, such insights can help elucidate the effects of small molecule drugs in the cellular environment and their interference with DNA transcription and replication.