The discovery of activation state dependent kinase inhibitors, which bind specifically to the inactive conformation of the protein, is considered to be a promising pathway to improved cancer treatments. Identifying such inhibitors is challenging, however, because they can have Kd values similar to molecules known to inhibit kinase function by interacting with the active form. Further, while inhibitor induced changes within the kinase tertiary structure are significant, few technologies are able to correctly assign inhibitor binding modes in a high-throughput fashion based exclusively on protein-inhibitor complex formation and changes in local protein structure. We have developed a new assay, using ion mobility-mass spectrometry, capable of both rapidly detecting inhibitor binding and classifying the resultant kinase binding modes. Here, we demonstrate the ability of our approach to classify a broad set of kinase inhibitors, using micrograms of protein, without the need for protein modification or tagging.

Alzheimer's disease (AD) is a multifactorial neurodegenerative disease related to the deposition of aggregated amyloid-β (Aβ) peptides in the brain. It has been proposed that metal ion dyshomeostasis and miscompartmentalization contribute to AD progression, especially as metal ions (e.g., Cu(II) and Zn(II)) found in Aβ plaques of the diseased brain can bind to Aβ and be linked to aggregation and neurotoxicity. The role of metal ions in AD pathogenesis, however, is uncertain. To accelerate understanding in this area and contribute to therapeutic development, recent efforts to devise suitable chemical reagents that can target metal ions associated with Aβ have been made using rational structure-based design that combines two functions (metal chelation and Aβ interaction) in the same molecule. This paper presents bifunctional compounds developed by two different design strategies (linkage or incorporation) and discusses progress in their applications as chemical tools and/or potential therapeutics.

On the basis of synergism observed between a selective c-Src kinase inhibitor with an HDAC inhibitor, the development of the first chimeric c-Src kinase and HDAC inhibitor is described. The optimized chimeric inhibitor is shown to be a potent c-Src and HDAC inhibitor. Chimeric inhibitor 4 is further shown to be highly efficacious in cancer cell lines and significantly more efficacious than a dual-targeting strategy using discrete c-Src and HDAC inhibitors.

Lon and ClpXP are the only soluble ATP-dependent proteases within the mammalian mitochondria matrix, which function in protein quality control by selectively degrading misfolded, misassembled or damaged proteins. Chemical tools to study these proteases in biological samples have not been identified, thereby hindering a clear understanding of their respective functions in normal and disease states. In this study, we applied a proteolytic site-directed approach to identify a peptide reporter substrate and a peptide inhibitor that are selective for Lon but not ClpXP. These chemical tools permit quantitative measurements that distinguish Lon-mediated proteolysis from that of ClpXP in biochemical assays with purified proteases, as well as in intact mitochondria and mitochondrial lysates. This chemical biology approach provides needed tools to further our understanding of mitochondrial ATP-dependent proteolysis, and contributes to the future development of diagnostic and pharmacological agents for treating diseases associated with defects in mitochondrial protein quality.

Protein kinase pathways are traditionally mapped by monitoring downstream phosphorylation. Meanwhile, the non-catalytic functions of protein kinases remain under-appreciated as critical components of kinase signaling. c-Src is a protein kinase known to have non-catalytic signaling function important in healthy and disease cell signaling. Large conformational changes in the regulatory domains regulate c-Src's non-catalytic functions. Herein, we demonstrate that changes in the global conformation of c-Src can be monitored using a selective proteolysis methodology. Further, we use this methodology to investigate changes in the global conformation of several clinical and non-clinical mutations of c-Src. Significantly, we identify a novel activating mutation observed clinically, W121R, that can escape down-regulation mechanisms. Our methodology can be expanded to monitor the global conformation of other tyrosine kinases, including c-Abl, and represents an important tool toward the study of elucidating the non-catalytic functions of protein kinases.