Bruton’s Tyrosine Kinase (BTK) is a flexible, multidomain kinase that is predominantly expressed in B-cells. B-cells are a crucial component of adaptive immune response. The growth, development and proliferation of B-cells are controlled by signaling through the B-cell receptor pathway. Aberrant activation of the BCR pathway has been linked to malignancies and autoimmune diseases. BTK is an effector molecule that is activated in response to stimulation of the B-cell receptor. BTK acts as a molecular switch for this pathway. Hence, BTK has been a very sought-after drug target.

Because of the therapeutic importance of BTK, it is important to get a better understanding of the regulatory elements that modulate BTK structure and activity. The conformational organization of the BTK SRC module (SH2-SH3-kinase domains) in its autoinhibitory form has been well characterized. However, there is still ambiguity with regard to the role of BTK pleckstrin homology–Tec homology (PHTH) domain. In addition to its phospholipid targeting function, previous studies have highlighted the autoinhibitory role of the PH domain. In this dissertation, we will explore the role of BTK PHTH domain in regulating kinase activity. We used NMR (Nuclear Magnetic Resonance) spectroscopy to map where and how the PHTH domain affects the kinase domain. For this we assigned backbone amide resonances of the BTK kinase domain and titrated unlabeled BTK PHTH domain into the 15N-BTK kinase domain. In addition, we used hydrogen/deuterium exchange mass spectroscopy (HDX-MS) and evolutionary sequence comparison to further complement the results obtained from NMR titration studies. Our results indicate that the PHTH binding interface on the kinase domain is localized on the activation loop face of the kinase domain. Our NMR results also show interesting allosteric changes on PHTH-kinase binding, these changes are propagated from the activation loop face of the kinase to the active site and possibly to the regulatory domains. Our results provide a more complete picture of the autoinhibitory conformation of full length BTK which is valuable for rational drug design.

The dissertation presents existing literature focused on understanding the interplay between the dynamics of various regulatory elements within SRC module kinases and how regulatory domains influence the conformational ensemble sampled by these proteins. Changes introduced by mutations or ligand binding to the regulatory interface can cause conformational changes within the active site and vice versa. Similarly, this conformational selectivity can occur in the context of drug binding as well.

In addition, the dissertation describes recent advances in drug screening approaches in our laboratory. We used the existing information regarding BTK structure and regulation to identify putative allosteric small molecule modulators of BTK. We built a workflow using virtual screening, two orthogonal in vitro screens (Saturated Transfer Difference NMR and Thermal Shift assays), and further validated hits using Surface Plasmon Resonance (SPR), an activity assay and NMR spectroscopy. The unpublished results yielded a few interesting compounds that require further development.