Full-length Bruton's tyrosine kinase (BTK) has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3-SH2-kinase core. Precisely ...how the BTK N-terminal domains (the Pleckstrin homology/Tec homology PHTH domain and proline-rich regions PRR contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveal only the SH3-SH2-kinase core with no electron density visible for the PHTH-PRR segment. Cryo-electron microscopy (cryoEM) data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH-PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3-SH2-kinase core. On the way to activation, disassembly of the SH3-SH2-kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with phosphatidylinositol (3,4,5)-trisphosphate. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.
Bruton’s Tyrosine Kinase (BTK) is a nonreceptor tyrosine kinase that belongs to the TEC family. Mutations in the BTK gene cause X-linked agammaglobulinemia (XLA) leading to an arrest in B-cell ...development. BTK is also a drug target for B-cell lymphomas that rely on an intact B-cell receptor signaling cascade for survival. All FDA approved drugs for BTK target the ATP binding site of the catalytic kinase domain, leading to potential adverse events due to off-target inhibition. In addition, acquired resistance mutations occur in a subset of patients, rendering available BTK inhibitors ineffective. Therefore, allosteric sites on BTK should be explored for drug development to target BTK more specifically and in combination with active site inhibitors. Virtual screening against nonactive site pockets and in vitro experiments resulted in a series of small molecules that bind to BTK outside of the active site. We characterized these compounds using biochemical and biophysical techniques and narrowed our focus to compound “C2”. C2 activates full-length BTK and smaller multidomain BTK fragments but not the isolated kinase domain, consistent with an allosteric mode of action. Kinetic experiments reveal a C2-mediated decrease in K m and an increase in k cat leading to an overall increase in the catalytic efficiency of BTK. C2 is also capable of activating the BTK XLA mutants. These proof-of-principle data reveal that BTK can be targeted allosterically with small molecules, providing an alternative to active site BTK inhibitors.
Since Dr. Ogden Bruton’s 1952 paper describing the first human primary immunodeficiency disease, the peripheral membrane binding signaling protein, aptly named Bruton’s tyrosine kinase (BTK), has ...been the target of intense study. Dr. Bruton’s description of agammaglobulinemia set the stage for ultimately understanding key signaling steps emanating from the B cell receptor. BTK is a multidomain tyrosine kinase and in the decades since Dr. Bruton’s discovery it has become clear that genetic defects in the regulatory domains or the catalytic domain can lead to immunodeficiency. This finding underscores the intricate regulatory mechanisms within the BTK protein that maintain appropriate levels of signaling both in the resting B cell and during an immune challenge. In recent decades, BTK has become a target for clinical intervention in treating B cell malignancies. The survival reliance of B cell malignancies on B cell receptor signaling has allowed small molecules that target BTK to become essential tools in treating patients with hematological malignancies. The first-in-class Ibrutinib and more selective second-generation inhibitors all target the active site of the multidomain BTK protein. Therapeutic interventions targeting BTK have been successful but are plagued by resistance mutations that render drug treatment ineffective for some patients. This review will examine the molecular mechanisms that drive drug resistance, the long-range conformational effects of active site inhibitors on the BTK regulatory apparatus, and emerging opportunities to allosterically target the BTK kinase to improve therapeutic interventions using combination therapies.
Full-length Bruton’s tyrosine kinase (BTK) has been refractory to structural analysis. The nearest full-length structure of BTK to date consists of the autoinhibited SH3–SH2–kinase core. Precisely ...how the BTK N-terminal domains (the Pleckstrin homology/Tec homology PHTH domain and proline-rich regions PRR contain linker) contribute to BTK regulation remains unclear. We have produced crystals of full-length BTK for the first time but despite efforts to stabilize the autoinhibited state, the diffraction data still reveal only the SH3–SH2–kinase core with no electron density visible for the PHTH–PRR segment. Cryo-electron microscopy (cryoEM) data of full-length BTK, on the other hand, provide the first view of the PHTH domain within full-length BTK. CryoEM reconstructions support conformational heterogeneity in the PHTH–PRR region wherein the globular PHTH domain adopts a range of states arrayed around the autoinhibited SH3–SH2–kinase core. On the way to activation, disassembly of the SH3–SH2–kinase core opens a new autoinhibitory site on the kinase domain for PHTH domain binding that is ultimately released upon interaction of PHTH with phosphatidylinositol (3,4,5)-trisphosphate. Membrane-induced dimerization activates BTK and we present here a crystal structure of an activation loop swapped BTK kinase domain dimer that likely represents the conformational state leading to trans-autophosphorylation. Together, these data provide the first structural elucidation of full-length BTK and allow a deeper understanding of allosteric control over the BTK kinase domain during distinct stages of activation.
Bruton’s Tyrosine Kinase (BTK) is a non-receptor tyrosine kinase and is a member of the TEC family of kinases. BTK plays an integral role in many immunological cellular signaling pathways and has ...become a target for therapeutic development. BTK is composed of, from N- to C- terminus, the regulatory Pleckstrin Homology – Tec Homology (PHTH), Src-homology 3 (SH3), Src-homology 2 (SH2) domains followed by the catalytic Kinase domain. All domains N- terminal to the Kinase domain play a role in regulating the activity of BTK. The nearest full- length structure of BTK is that of the SH3-SH2-Kinase fragment in the autoinhibitory conformation whereby the SH3 and SH2 domains dock onto the backside of the Kinase domain, maintaining autoinhibition. The PHTH domain plays two roles: autoinhibition by binding to the Kinase domain and localizing BTK to the membrane by dimerization at PIP3 lipids. Existing drugs targeting BTK all bind to the active site of Kinase domain and patients taking these drugs may acquire resistance mutations rendering drug treatment ineffective. Therefore, the allosteric regulatory mechanisms in BTK should be exploited for therapeutic development.This dissertation explores harnessing the allosteric regulatory interactions of BTK for the development of a small molecule screen and the characterization of small molecules allosterically targeting BTK. Since the allosteric regulatory mechanisms are being explored in this dissertation, the first chapter provides a historical perspective of allostery, kinases, and targeting kinases in instances of disease. The second chapter is a modified manuscript published in the journal Frontiers in Cell and Developmental Biology that details the current understanding of existing active site inhibitors, mechanisms of drug resistance, and the allosteric regulatory mechanisms of BTK. The final two chapters provide the ways in which we have capitalized onthe allosteric regulatory mechanisms of BTK for small molecule screening. The third chapter is a preliminary manuscript to be submitted for publication and details the screening approach taken against the SH3-SH2-Kinase fragment of BTK and the characterization of the hits received from the screen. The fourth chapter outlines the progress for developing a cellular based screen targeting the regulatory mechanisms of the BTK PHTH domain. Overall, this dissertation provides the strategies we have taken for targeting BTK allosterically with small molecules. This work highlights the importance of the allosteric regulation of BTK and why targeting BTK allosterically is promising in a clinical setting.
The reality confronting ecosystem managers today is one of heterogeneous, rapidly transforming landscapes, particularly in the areas more affected by urban and agricultural development. A landscape ...management framework that incorporates all systems, across the spectrum of degrees of alteration, provides a fuller set of options for how and when to intervene, uses limited resources more effectively, and increases the chances of achieving management goals. That many ecosystems have departed so substantially from their historical trajectory that they defy conventional restoration is not in dispute. Acknowledging novel ecosystems need not constitute a threat to existing policy and management approaches. Rather, the development of an integrated approach to management interventions can provide options that are in tune with the current reality of rapid ecosystem change.
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