Cells process an enormous amount of information from their environment and translate this information into phenotypic behaviour that will dictate critical biologic activities. To do this, cells must collect, store, and respond to information both inside and outside the cell. This information is stored in the chemical composition of its cytoplasm, and these conditions will regulate complex biochemical pathways that relay the information from the cell’s exterior to the effector proteins that modulate behaviour. These pathways, known as signal transduction pathways, are largely regulated by phosphorylation. Protein kinases are the class of enzymes responsible catalyzing the phosphorylation of intermediary signaling enzymes (resulting in their activation), and thus, stimulate the transduction of an external stimulus to intracellular machinery. As cellular conditions change, protein kinases sensitive to these conditions will modulate their activity through a change in shape (conformation). Research shows that disregulating signaling pathways through aberrant kinase activity can lead to grievous disease, making them good drug targets for cancer treatments and other conditions. Selective pressure has lead to the 500+ kinases in the proteome to adopt well-conserved active and inactive conformations, making it difficult to understand how nature can distinguish between them. Due to low abundance of kinases, quantitative analysis of kinase conformations is difficult with existing methodology. To this end, we have developed a catch-and-release system that allows us to enrich and quantitatively distinguish between kinases of specific conformations. This project will focus on the development trifunctional probe that serves as a chemical handle to enrich kinases in the novel system. The new catch-and-release system employs improvements over existing biotin-steptavidin systems by taking advantage of bio-orthogonal chemistry and specific elution conditions that increase signal-to-noise ratios. Our probe marks significant progress towards a panel of probes that will profile kinases for diagnostic means and drug target identification.

SRC-Family Kinases (SFKs) are a family of non-receptor tyrosine kinases that play key roles in intracellular signaling transduction.  They are multi-domain proteins containing two regulatory domains, SH2 and SH3, and a catalytic domain.  The two regulatory domains are vital in controlling kinase activity and interactions with other proteins in cell signaling events. Current therapeutic development strategies target the ATP binding site of the kinase domain to inhibit enzymatic activity. Recently, it has been shown that the binding of ligands to the active sites of these kinases can stabilize conformational changes in the SH2/SH3 domains modulating the accessibility of these domains necessary for protein-protein interactions, potentially providing novel kinase regulation through both enzymatic activity and protein-protein interaction. Disregulation of SFK kinases plays an important role in tumor metastasis. A recent unbiased screening of kinase mutations in cancer cells indicated a considerable amount of inactivating mutations, which indicates that there are important non-catalytic functions of kinases which play significant roles in cancer biology.  The long term goal of our study is to better understand these non-catalytic functions in SFK signaling pathways so as to help develop better approaches to treating cancers. Using an array of ATP competitive inhibitors, we have biochemically profiled some of these conformational changes in SFKs which modulate the accessibility of these regulatory domains in particular conformations to help elucidate the role these non-catalytic activities have in intracellular signaling.  So far, it has been shown that alpha C-helix-out inhibitors favor the SH2/SH3 intra-molecularly engaged, inactive conformation, while smaller Type1 inhibitors favor the extended, active conformations of these SFKs.  Currently, the plan is to further profile these conformations, as well as to determine how specific ATP binding site conformations affect SFK function in intracellular signaling. Towards this end, a panel of alpha C-helix-out and Type 1 inhibitors are being synthesized and tested.

Protein kinases are enzymes that take phosphate groups from adenosine triphosphate (ATP) molecules, the energy in your body, and add them to other specific molecules inside the cell. This transfer of the phosphate group can activate or deactivate the target molecule, thus regulating cellular activities such as growth, replication, and respiration. The kinases studied here are hemopoietic cell kinases (Hck kinases) under the class of Scr kinases. Hck kinases are present in bone marrow and are often associated with myeloid leukemias and myeloproliferative disorders. Studies have shown that in the active conformation, the Hck kinase detaches two of its domains, SH2 and SH3, resulting in a more extended conformation. Using a Waters Synapt G2 HDMS, ion mobility mass spectrometry (IM-MS) techniques were used to probe the size of inactive and active Hck kinases by measuring drift times of their gas-phase ions traveling through a neutral background gas under the presence of a weak electric field. Drift times were then converted into collision cross section (CCS) values, which depend on the size and shape of the ion of interest. Eight different small molecules (< 500 Da) were used to inhibit Hck kinases, and changes in conformation were investigated using IM-MS. Preliminary data shows no significant changes in global conformation or size, conflicting with the previous studies. To investigate more subtle changes in conformation, collision induced unfolding (CIU) methods are used. By selecting an ion of interest using a quadrupole mass filter, protein unfolding is monitored using ion mobility as a function of ion activation prior to ion mobility analysis. This process allows an investigation of the subtle conformational changes in the Hck kinase upon the binding of small inhibitor molecules.