Molecular determinants regulating Bruton’s tyrosine kinase activity and their mechanism: a combined computational and experimental approach

Understanding allostery in proteins is critical in understanding their unique regulatory mechanisms and this knowledge can be exploited to develop highly specific, targeted therapies. In this dissertation, we have investigated the unique sequence elements that regulate the activity of a protein tyrosine kinase called Bruton’s tyrosine kinase or Btk. Btk is a member of the immune signaling pathway in B-cells and is required for B-cells maturation and function. Lack of a three-dimensional structure of full-length Btk kinase has proved a roadblock in understanding how the domains in Btk interact to shift its conformational equilibrium between active and inactive states. Moreover, in-spite of high homology between the catalytic centers of Btk and other well-studied protein tyrosine kinases such as Src, the regulatory mechanisms of these kinases appear to differ significantly creating an impediment to gaining a complete understanding of the mode of Btk regulation. In pursuit of the aim to identify key sequence and structure motifs that regulate Btk activity, we made use of a range of computational tools to better understand the Btk kinase domain and, when possible, the resulting hypotheses were tested using experimental methods. First, we have identified a specific isoleucine residue, conserved in Btk and related kinases, which functions to stabilize the inactive Btk conformation. We showed that substitution of the conserved isoleucine to leucine shifts the conformational equilibrium of the Btk kinase domain to the active state. Next, we showed how a highly conserved tryptophan, located in a linker region adjacent to the Btk kinase domain, stabilizes the active Btk kinase domain conformation through correlated dynamic motions within the kinase domain itself. Finally, sequence-structure information, combined with information theory and molecular dynamics, was used to identify a specific site in the Btk kinase domain that can be targeted to rescue the kinase activity of Btk in the presence of an inactivating disease causing mutation. The work presented here provides new insights into the regulatory mechanisms in Btk as well as potential allosteric sites in the protein, for which modulators of Btk activity could be developed. There is a growing need for the discovery of such allosteric modulators as Btk has been implicated in immunodeficiency disorders such as X-linked agammaglobulinemia as well as B-cells malignancies and breast and colon cancers. Ultimately, increased knowledge about the molecular mechanisms controlling Btk function should lead to the development of novel Btk activity modulators

Targeting Allosteric Pockets in Protein Kinases Using Molecular Modeling and Simulations

The deregulation of protein kinases is often related with the development of several malignancies such as cancer. Therefore, inhibition of protein kinases is an established and often effective pharmacological strategy. However, point mutations in kinases are frequently the cause of drug resistance. To overcome this issue, many efforts are directed towards the design of allosteric drugs, with the goal to inhibit the mutated forms of kinases. In this regard, the comprehension of the molecular basis of the allosteric control of protein kinases is essential for the design of novel allosteric drugs. In this thesis, we studied the molecular basis that allosterically regulates the function of Abelson (Abl) kinase, a relevant pharmaceutical target for the treatment of several malignancies, as chronic myelogenous leukemia. This study proposes a novel mechanism according to which conserved structural motifs dynamically cooperate to regulate allosterically the function of Abl. The information retrieved from this study can be employed for the rational design of new Abl allosteric inhibitors. In addition, we also developed a new algorithm for the detection of protein pockets in MD simulations. This algorithm has been conceived to identify and analyze all the pockets of a given protein without any user a priori information of their localization. It also enables the detection of pockets’ network, characterizing possible allosteric signaling pathways that connect the functional with the allosteric sites. Overall, this tool allows the study of the dynamic properties of pockets and might be employed in the early stages of the drug discovery process to design both orthosteric and allosteric binders

PROGRESS TOWARDS THE STRUCTURAL BASIS OF TEC-FAMILY KINASE ACTIVATION BY HIV-1 NEF

HIV-1 Nef is a viral accessory factor that is essential for virus infectivity, host immune evasion and AIDS progression. Nef lacks intrinsic catalytic activity and functions instead via interactions with multiple classes of host cell proteins involved in signal transduction and endocytic trafficking. Nef interacts with the Src-family tyrosine kinases Hck and Lyn through their SH3 domains, resulting in constitutive kinase activity. Nef also binds to select members of the Tec-family of tyrosine kinases, including Btk, Bmx, and Itk, all of which are expressed in HIV-1 target cells. Of particular interest is Itk, which is expressed in CD4+ T cells and is activated by Nef. Selective Itk inhibitors block Nef-dependent enhancement of HIV-1 infectivity and replication, suggesting an important role in the viral life cycle. While the interaction between Itk and Nef has been demonstrated at the plasma membrane in cell-based fluorescence complementation assays, the structural basis of this interaction has not been reported. Like Src-family kinases, Itk has a core region consisting of sequential SH3, SH2 and kinase domains. In addition, Itk has an N-terminal pleckstrin homology (PH) domain important for membrane targeting as well as a Tec homology(TH) region involved in kinase regulation. To explore the structure of the Nef:Tec-family kinase (TFK) complexes, I have created a panel of bacterial expression constructs for the Itk and Btk regulatory region. These include the entire PH-TH-SH3-SH2 region, the SH3-SH2 region, and the isolated SH3 domain, all of which have yielded mg amounts of soluble protein. I have also produced recombinant, N-terminally myristoylated (Myr) Nef in bacteria, a post-translational modification essential for Nef membrane localization in cells. Preliminary Surface Plasmon Resonance (SPR) studies show that Myr-Nef binds membrane bilayers with low µM affinity in a Myr-dependent manner. These proteins will provide the foundation for future structural determination of Nef-TFK complexes by X-ray crystallography as well as the nature of this interaction in lipid bilayers, the physiological site of interaction in HIV-infected cells

Using evolutionary covariance to infer protein sequence-structure relationships

During the last half century, a deep knowledge of the actions of proteins has emerged from a broad range of experimental and computational methods. This means that there are now many opportunities for understanding how the varieties of proteins affect larger scale behaviors of organisms, in terms of phenotypes and diseases. It is broadly acknowledged that sequence, structure and dynamics are the three essential components for understanding proteins. Learning about the relationships among protein sequence, structure and dynamics becomes one of the most important steps for understanding the mechanisms of proteins. Together with the rapid growth in the efficiency of computers, there has been a commensurate growth in the sizes of the public databases for proteins. The field of computational biology has undergone a paradigm shift from investigating single proteins to looking collectively at sets of related proteins and broadly across all proteins. we develop a novel approach that combines the structure knowledge from the PDB, the CATH database with sequence information from the Pfam database by using co-evolution in sequences to achieve the following goals: (a) Collection of co-evolution information on the large scale by using protein domain family data; (b) Development of novel amino acid substitution matrices based on the structural information incorporated; (c) Higher order co-evolution correlation detection. The results presented here show that important gains can come from improvements to the sequence matching. What has been done here is simple and the pair correlations in sequence have been decomposed into singlet terms, which amounts to discarding much of the correlation information itself. The gains shown here are encouraging, and we would like to develop a sequence matching method that retains the pair (or higher order) correlation information, and even higher order correlations directly, and this should be possible by developing the sequence matching separately for different domain structures. The many body correlations in particular have the potential to transform the common perceptions in biology from pairs that are not actually so very informative to higher-order interactions. Fully understanding cellular processes will require a large body of higher-order correlation information such as has been initiated here for single proteins

In vitro and in vivo studies of Bruton tyrosine kinase (BTK) mutations & inhibition

Bruton tyrosine kinase (BTK) is a non-receptor protein kinase that belongs to the TEC family kinases. It plays an important role in the B-cell receptor signaling pathway (BCR) and its pharmacological inhibition has been demonstrated as an effective strategy for the treatment of B-cell malignancies. Ibrutinib, acalabrutinib and zanubrutinib are small molecules and irreversible BTK binders that have been approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) for the treatment of several B-cell malignancies. Irreversible inhibitors block BTK catalytic activity by covalently binding to the cysteine (C) 481 located in the kinase domain. Mutations at this residue abrogate the possibility of forming a covalent bond, thereby decreasing the efficacy of the inhibitor. The most common mutation found in treated patients is the cysteine-481 to serine substitution (C481S). However, other less frequent substitutions have also been identified, such as, T474I and T474S substitutions in the BTK gatekeeper residue or PLCg2 gain-of-function substitutions e.g. S707Y and R665W. In paper I we studied a novel C481S knock-in mouse model. Our analysis of these mice reveled no phenotype alterations, as compared to wild-type mice, and demonstrated that C481S substitution has no detectable effect on BTK´s function or on the development of hematopoietic cells. We demonstrated that isolated B-lymphocytes carrying C481S were resistant to irreversible but sensitive to reversible BTK inhibitors (BTKis). This was achieved by analyzing BTK catalytic activity, cell-viability and expression of cell activation markers. Additionally, we confirmed that irreversible BTKis impaired T-lymphocyte activation in a BTK independent manner. This demonstrates the potential of this mouse model to be used in the study of BTKindependent, both therapeutic and adverse, effects caused by irreversible BTKis. Resistance to BTKis has become one of the most critical concerns in long term ibrutinib treated patients. The cause of the resistance to irreversible BTKis is less frequently associated to the gatekeeper residue, in contrast what is observed for other kinase inhibitors such as the fusionprotein BCR-ABL inhibitor imatininb or the EGFR inhibitor gefitinib. In paper II we aimed to understand the role of gatekeeper and combined gatekeeper/C481 BTK variants in the resistance to reversible and irreversible BTKis. We evaluated protein expression, catalytic activity and susceptibility to BTKis of 16 BTK single and double variants. We found that double T474I/C481S, T474M/C481S and T474M/C481T variants were insensitive to ³16 fold irreversible inhibitor pharmacological serum concentration. On the other hand, reversible BTKis showed a variable inhibition pattern. RN486 seemed to have highest therapeutic potential for patients that develop resistance to combined gatekeeper/C481 BTK variants

THE CONCISE GUIDE TO PHARMACOLOGY 2017/18:Enzymes

The Concise Guide to PHARMACOLOGY 2017/18 provides concise overviews of the key properties of nearly 1800 human drug targets with an emphasis on selective pharmacology (where available), plus links to an open access knowledgebase of drug targets and their ligands (www.guidetopharmacology.org), which provides more detailed views of target and ligand properties. Although the Concise Guide represents approximately 400 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.13877/full. Enzymes are one of the eight major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, ligand-gated ion channels, voltage-gated ion channels, other ion channels, nuclear hormone receptors, catalytic receptors and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2017, and supersedes data presented in the 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature Committee of the Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate

Evolutionary plasticity of SH3 domain binding by Nef proteins of the HIV-1/SIVcpz lentiviral lineage

The accessory protein Nef of human and simian immunodeficiency viruses (HIV and SIV) is an important pathogenicity factor known to interact with cellular protein kinases and other signaling proteins. A canonical SH3 domain binding motif in Nef is required for most of these interactions. For example, HIV-1 Nef activates the tyrosine kinase Hck by tightly binding to its SH3 domain. An archetypal contact between a negatively charged SH3 residue and a highly conserved arginine in Nef (Arg77) plays a key role here. Combining structural analyses with functional assays, we here show that Nef proteins have also developed a distinct structural strategy-termed the "R-clamp"-that favors the formation of this salt bridge via buttressing Arg77. Comparison of evolutionarily diverse Nef proteins revealed that several distinct R-clamps have evolved that are functionally equivalent but differ in the side chain compositions of Nef residues 83 and 120. Whereas a similar R-clamp design is shared by Nef proteins of HIV-1 groups M, O, and P, as well as SIVgor, the Nef proteins of SIV from the Eastern chimpanzee subspecies (SIVcpzP.t.s.) exclusively utilize another type of Rclamp. By contrast, SIV of Central chimpanzees (SIVcpzP.t.t.) and HIV-1 group N strains show more heterogenous R-clamp design principles, including a non-functional evolutionary intermediate of the aforementioned two classes. These data add to our understanding of the structural basis of SH3 binding and kinase deregulation by Nef, and provide an interesting example of primate lentiviral protein evolution.Peer reviewe

Types and effects of protein variations.

Variations in proteins have very large number of diverse effects affecting sequence, structure, stability, interactions, activity, abundance and other properties. Although protein-coding exons cover just over 1 % of the human genome they harbor an disproportionately large portion of disease-causing variants. Variation ontology (VariO) has been developed for annotation and description of variation effects, mechanisms and consequences. A holistic view for variations in proteins is made available along with examples of real cases. Protein variants can be of genetic origin or emerge at protein level. Systematic names are provided for all variation types, a more detailed description can be made by explaining changes to protein function, structure and properties. Examples are provided for the effects and mechanisms, usually in relation to human diseases. In addition, the examples are selected so that protein 3D structural changes, when relevant, are included and visualized. Here, systematics is described for protein variants based on VariO. It will benefit the unequivocal description of variations and their effects and further reuse and integration of data from different sources