Structural Biology in Drug Discovery
Structural Biology in Drug Discovery

Author: Jean-Paul Renaud

Publisher: John Wiley & Sons

Published: 2020-01-09

Total Pages: 1367

ISBN-13: 1118900502

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With the most comprehensive and up-to-date overview of structure-based drug discovery covering both experimental and computational approaches, Structural Biology in Drug Discovery: Methods, Techniques, and Practices describes principles, methods, applications, and emerging paradigms of structural biology as a tool for more efficient drug development. Coverage includes successful examples, academic and industry insights, novel concepts, and advances in a rapidly evolving field. The combined chapters, by authors writing from the frontlines of structural biology and drug discovery, give readers a valuable reference and resource that: Presents the benefits, limitations, and potentiality of major techniques in the field such as X-ray crystallography, NMR, neutron crystallography, cryo-EM, mass spectrometry and other biophysical techniques, and computational structural biology Includes detailed chapters on druggability, allostery, complementary use of thermodynamic and kinetic information, and powerful approaches such as structural chemogenomics and fragment-based drug design Emphasizes the need for the in-depth biophysical characterization of protein targets as well as of therapeutic proteins, and for a thorough quality assessment of experimental structures Illustrates advances in the field of established therapeutic targets like kinases, serine proteinases, GPCRs, and epigenetic proteins, and of more challenging ones like protein-protein interactions and intrinsically disordered proteins

Structural and Functional Analysis of Chemokine Interactions with Glycosaminoglycans and Chemokine Receptors
Structural and Functional Analysis of Chemokine Interactions with Glycosaminoglycans and Chemokine Receptors

Author: Catherina L. Salanga

Publisher:

Published: 2011

Total Pages: 288

ISBN-13: 9781124703862

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Chemokines are involved in cell migration and activation during routine immune surveillance, inflammation and even cancer metastasis. The migration of chemokine receptor-bearing cells, including leukocytes and tumor cells, occurs in response to the secretion of chemokines, which accumulate on cell surfaces through interaction with glycosaminoglycans (GAGs) where they effectively serve as traffic signals to guide cell movement. Engagement of chemokines with their receptors subsequently causes the activation of signaling pathways that result in firm adhesion and extravasation of the cell into tissue, and in the case of leukocytes, activation of defense mechanisms. However, in cancer cells, the signaling pathways can be exploited or redirected, resulting in responses like survival, growth and proliferation. Herein, a structural and functional approach was used to address specific questions about the interactions of chemokines (i) with GAGs and (ii) with chemokine receptors in the context of cancer. Technically, the use of mass spectrometry has been a strong theme throughout these studies. In Chapter 2, a novel application of hydroxyl radical footprinting coupled with mass spectrometry was used to characterize the GAG binding specificity of the chemokine, MCP-3/CCL7. Potential GAG binding epitopes, identified by mass spectrometry, were then validated by mutagenesis and functional assays. In Chapter 3 and 4, a phosphoproteomic mass spectrometry strategy was used to elucidate CXCL12-mediated survival signaling through the receptor, CXCR4, in cells from patients with chronic lymphocytic leukemia (CLL). While signaling cascades involved in chemokine-mediated migration are well established, pathways involved in cell survival and proliferation in cancer, are not. Methods developed for phosphopeptide enrichment, and subsequent analysis via mass spectrometry are described in Chapter 3, and interesting/novel phosphoproteins, potentially involved in CXCL12-mediated CLL survival are described in Chapter 4. In Chapter 5, a functional approach was taken to elucidate the roles of receptors CXCR4 and CXCR7 in breast cancer growth and metastasis. The data show that CXCR7 affects the functional activity of CXCR4 in vitro, and decreases the extent of lung metastases in vivo, without inhibiting primary tumor growth. Overall, these studies serve to better understand some of the regulatory mechanisms that control chemokine function in normal physiology and in cancer.

Structural and Functional Analysis of the Chemokine CCL27 and the Expression and Purification of Silent Chemokine Receptors D6 and DARC
Structural and Functional Analysis of the Chemokine CCL27 and the Expression and Purification of Silent Chemokine Receptors D6 and DARC

Author: Ariane L. Jansma

Publisher:

Published: 2009

Total Pages: 206

ISBN-13:

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Chemokines are small chemoattractant proteins that function by binding to G-protein coupled receptors (GPCRs) on a wide variety of cell types, triggering cascades of intracellular signaling pathways. While they are best known for their role in leukocyte migration, both in standard immune surveillance and maintenance, as well as in response to inflammation, chemokines are involved in a variety of physiological and pathophysiological processes. In addition to their complex function, the chemokine network itself is highly complex, with some chemokines being specific to one receptor, while others activate multiple receptors expressed on different cell types and in some cases, resulting in very different cellular responses. This project in part involves the chemokine CCL27, which is expressed in skin and selectively chemoattracts CLA memory T cells expressing the chemokine receptor, CCR10. The first set of aims for this project involved a comprehensive analysis of the structural and functional mechanism contributing to the biological diversity of CCL27. This was accomplished through a biophysical characterization of the oligomerization properties, residues targeting receptor activation, as well as sites of glycosaminoglycan (GAG) interactions for this chemokine. The results suggest that CCL27 exists in multiple oligomeric states, and its unique oligomerization patterns appear to play a role in the diversity of its multiple binding partners. The second major project aim involves optimization of the expression and purification of the silent chemokine receptors D6 and the Duffy Antigen Receptor for Chemokines (DARC). D6 and DARC are termed silent receptors because they bind many chemokines with high affinity and specificity, but unlike other chemokine receptors, they do not signal through G-proteins. Instead, they act as regulators, either by targeting their chemokine ligands for degradation, or by shuttling them from one location to another. One of the rate-limiting steps in the biophysical characterization of 7-transmembrane helical chemokine receptors is obtaining sufficient amounts of purified, functional protein. In this project, a tetracycline-indicuble mammalian cell expression system is applied to both D6 and DARC. Results from the initial test expressions and purifications indicate that this method was successful in generating solubilized receptor protein.

Study of the Structure and Function of CXC Chemokine Receptor 2
Study of the Structure and Function of CXC Chemokine Receptor 2

Author: Hae Ryong Kwon

Publisher:

Published: 2010

Total Pages: 120

ISBN-13:

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It has been shown that the amino terminus and second extracellular loop (EC2) of CXCR2 are crucial for ligand binding and receptor activation. The lack of an ionic lock motif in the third intracellular loop of CXCR2 focuses an investigation of the mechanism by which these two extracellular regions contribute to receptor recognition and activation. The first objective of this investigation was to predict the structure of CXCR2 based on known structures of crystallized GPCRs. Rhodopsin, [beta]2- adrenergic receptor, CXCR4 were used for homology modeling of CXCR2 structure. Highly conserved motifs found in sequence alignments of the template GPCRs were helpful to generate CXCR2 models. We also studied solvent accessibility of residues in the EC2 of CXCR2 in the inactive state. Most of the residues in the EC2 were found to be solvent accessible in the inactive state, suggesting the residues might be involved in ligand recognition. Second, we studied the role of charged residues in the EC2 of CXCR2 in ligand binding and receptor activation using constitutively active mutants (CAM) of CXCR2, D9K and D9R. Combinatorial mutations consisting of the CAM in the amino terminus and single mutations of charged residues in the EC2 were generated to study two concepts including "attraction" and "repulsion" models. The mutant receptors were used to test their effects on cell surface expression, ligand binding, receptor activation through PLC-[beta]3, and cellular transformation. All the mutations in the repulsion model result in CXCR2 receptors that are unable to bind ligand, suggesting that each of the Arg residues in the EC2 are important for ligand recognition. Interestingly, mutations in the attraction model partially inhibited receptor activation by the CAM D9K, suggesting that Glu198 and Asp199 residues in the EC2 are associated with receptor activation. Furthermore, a novel CAM, E198A/D199A, was identified in this study. These negatively charged residues are very close to a conserved disulfide bond linking the EC2 and the third transmembrane. In this sense, these current discoveries concerning the structural basis of CXCR2 and interdisciplinary approaches would provide new insights to investigate unknown mechanisms of interaction with its cognate ligands and receptor activation.

Structural and Functional Analysis of the Constitutively Active C-C Chemokine Receptor Type 1 (CCR1)
Structural and Functional Analysis of the Constitutively Active C-C Chemokine Receptor Type 1 (CCR1)

Author: Christian Taylor Gilliland

Publisher:

Published: 2013

Total Pages: 232

ISBN-13: 9781303616686

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Chemokine receptors belong to the G protein-coupled receptor (GPCR) family of proteins and are critical mediators of the directed migration of leukocytes in innate and adaptive immune responses. Understanding the behavior of chemokine receptors under basal and agonist-stimulated conditions is essential to developing effective therapeutics for inflammatory and autoimmune diseases. For the first time, the constitutive activity of the C-C chemokine receptor type 1 (CCR1) is uncovered through ligand-independent cellular migration, constitutive phosphorylation and association with [beta]-arrestin-2, and continual internalization followed by recycling back to the plasma membrane. Initial data suggests that CCR1 can act as a scavenging receptor to sequester chemokines intracellularly without canonical G protein signaling, thereby providing biological relevance to receptor constitutive activity. A Ser/Thr-rich cluster in the distal carboxy-terminal tail of CCR1 is identified as the major site of basal phosphorylation and fulfills a necessary, but not sufficient, role in pre-coupling to [beta]-arrestin-2. Site-directed mutagenesis of receptor transmembrane domains and conserved DRY motif has identified residues important for stabilizing CCR1 in a constitutively active state. Activation of CCR1 primarily leads to a conformational rearrangement with [beta]-arrestin-2, while endogenous chemokines induce this change with differential potency and efficacy. Lastly, small metal ion chelator molecules are able to activate desensitization and down-modulation of CCR1 with similar efficacy to natural ligands. Taken together, the work presented herein underlies the complexity of CCR1 function in the presence and absence of ligand and provides new avenues for therapeutic targeting.