Esteban Masuda

Targeting Syk as a treatment for allergic and autoimmune disorders

Recent advances in our understanding of allergic and autoimmune disorders have begun to translate into novel, effective and safe medicines for these common maladies. Examples include an anti-IgE monoclonal antibody recently approved for severe asthmatics and the TNF-α antagonists that have demonstrated their ability to suppress rheumatoid arthritis, Crohn’s disease and other chronic inflammatory processes. However, protein therapies are difficult and expensive to develop, manufacture and administer. Clearly, there is also a need for small-molecule inhibitors of novel targets that have safe and effective characteristics. Syk is an intracellular protein tyrosine kinase that was discovered 15 years ago as a key mediator of immunoreceptor signalling in a host of inflammatory cells including B cells, mast cells, macrophages and neutrophils. These immunoreceptors, including Fc receptors and the B-cell receptor, are important for both allergic diseases and antibody-mediated autoimmune diseases and thus pharmacologically interfering with Syk could conceivably treat these disorders. In addition, as Syk is positioned upstream in the cell signalling pathway, therapies targeting Syk may be more advantageous relative to drugs that inhibit a single downstream event. Syk inhibition during an allergic or asthmatic response will block three mast cell functions: the release of preformed mediators such as histamine, the production of lipid mediators such as leukotrienes and prostaglandins and the secretion of cytokines. In contrast, commonly used antihistamines or leukotriene receptor antagonists target only a single mediator of this complex cascade. Despite its expression in platelets and other non-haematopoietic cells, the role of Syk in regulating vascular homeostasis and other housekeeping functions is minimal or masked by redundant Syk-independent pathways. This suggests that targeting Syk would be an optimal approach to effectively treat a multitude of chronic inflammatory diseases without undue toxicity.

Retrovirally Delivered Random Cyclic Peptide Libraries Yield Inhibitors of Interleukin-4 Signaling in Human B Cells

Inteins are polypeptide sequences found in a small set of primarily bacterial proteins that promote the splicing of flanking pre-protein sequences to generate mature protein products. Inteins can be engineered in a “split and inverted” configuration such that the protein splicing product is a cyclic polypeptide consisting of the sequence linking two intein subdomains. We have engineered a split intein into a retroviral expression system to enable the intracellular delivery of a library of random cyclic peptides in human cells. Cyclization of peptides could be detected in cell lysates using mass spectrometry. A functional genetic screen to identify 5-amino acid-long cyclic peptides that block interleukin-4 mediated IgE class switching in B cells yielded 13 peptides that selectively inhibited germ line ε transcription. These results demonstrate the generation of cyclic peptide libraries in human cells and the power of functional screening to rapidly identify biologically active peptides.

Amisyn, a Novel Syntaxin-binding Protein That May Regulate SNARE Complex Assembly

The regulation of SNARE complex assembly likely plays an important role in governing the specificity as well as the timing of membrane fusion. Here we identify a novel brain-enriched protein, amisyn, with a tomosyn- and VAMP-like coiled-coil-forming domain that binds specifically to syntaxin 1a and syntaxin 4 bothin vitro and in vivo, as assessed by co-immunoprecipitation from rat brain. Amisyn is mostly cytosolic, but a fraction co-sediments with membranes. The amisyn coil domain can form SNARE complexes of greater thermostability than can VAMP2 with syntaxin 1a and SNAP-25 in vitro, but it lacks a transmembrane anchor and so cannot act as a v-SNARE in this complex. The amisyn coil domain prevents the SNAP-25 C-terminally mediated rescue of botulinum neurotoxin E inhibition of norepinephrine exocytosis in permeabilized PC12 cells to a greater extent than it prevents the regular exocytosis of these vesicles. We propose that amisyn forms nonfusogenic complexes with syntaxin 1a and SNAP-25, holding them in a conformation ready for VAMP2 to replace it to mediate the membrane fusion event, thereby contributing to the regulation of SNARE complex formation.

Systematic identification of regulatory proteins critical for T-cell activation

Background The activation of T cells, mediated by the T-cell receptor (TCR), activates a battery of specific membrane-associated, cytosolic and nuclear proteins. Identifying the signaling proteins downstream of TCR activation will help us to understand the regulation of immune responses and will contribute to developing therapeutic agents that target immune regulation. Results In an effort to identify novel signaling molecules specific for T-cell activation we undertook a large-scale dominant effector genetic screen using retroviral technology. We cloned and characterized 33 distinct genes from over 2,800 clones obtained in a screen of 7 × 108 Jurkat T cells on the basis of a reduction in TCR-activation-induced CD69 expression after expressing retrovirally derived cDNA libraries. We identified known signaling molecules such as Lck, ZAP70, Syk, PLCγ1 and SHP-1 (PTP1C) as truncation mutants with dominant-negative or constitutively active functions. We also discovered molecules not previously known to have functions in this pathway, including a novel protein with a RING domain (found in a class of ubiquitin ligases; we call this protein TRAC-1), transmembrane molecules (EDG1, IL-10Rα and integrin α2), cytoplasmic enzymes and adaptors (PAK2, A-Raf-1, TCPTP, Grb7, SH2-B and GG2-1), and cytoskeletal molecules (moesin and vimentin). Furthermore, using truncated Lck, PLCγ1, EDG1 and PAK2 mutants as examples, we showed that these dominant immune-regulatory molecules interfere with IL-2 production in human primary lymphocytes. Conclusions This study identified important signal regulators in T-cell activation. It also demonstrated a highly efficient strategy for discovering many components of signal transduction pathways and validating them in physiological settings.

Rab37 is a novel mast cell specific GTPase localized to secretory granules

GTPases regulate a myriad of cellular functions including signal transduction, cytoskeletal organization and membrane trafficking. Rab GTPases act to coordinate the membrane dynamics of cells by organizing and regulating the activity of effector proteins important in vesicle trafficking. Rab37 is a novel Rab GTPase specifically expressed in the MC‐9 mast cell line and bone marrow mast cells. Rab37 is 74% identical to Rab26 and 47% identical to Rab8, a GTPase important in Golgi to plasma membrane vesicle trafficking in mammalian cells. When green fluorescent protein tagged Rab37 is expressed in bone marrow mast cells, the secretory granules are labeled. These data suggest that Rab37 may play an important role in mast cell degranulation making this protein a potentially important target for therapeutic intervention in the treatment of allergy.

Tomosyn Binds t-SNARE Proteins via a VAMP-like Coiled Coil

Exocytosis requires the fusion of vesicular and plasma membranes and results in the release of vesicle contents into the extracellular space. This membrane fusion event is mediated in large part by specific interactions of proteins on the vesicle membrane (v-SNAREs) with proteins on the target membrane (t-SNAREs) (6xRothman, J.E. Nature. 1994; 372: 55–63CrossRef | PubMed | Scopus (1801)See all References, 1xBajjalieh, S.M. and Scheller, R.H. J. Biol. Chem. 1995; 270: 1971–1974CrossRef | PubMed | Scopus (165)See all References, 7xSudhof, T.C. Nature. 1995; 375: 645–653CrossRef | PubMedSee all References). In neurotransmitter release, for example, the vesicle protein VAMP (also called synaptobrevin) forms a tight SDS-resistant protein complex with two target membrane proteins: syntaxin and SNAP-25. This trimeric protein complex has been proposed to constitute part of the minimal machinery required for membrane fusion (Jahn and Hanson 1998xJahn, R. and Hanson, P.I. Nature. 1998; 393: 14–15CrossRef | PubMed | Scopus (71)See all ReferencesJahn and Hanson 1998).SNARE complexes assemble into a four-helix bundle formed by the 70 membrane-proximal residues of SNARE proteins. The minimal core consists of two helical domains from SNAP-25 and one from syntaxin arranged in parallel with one helical domain from VAMP (Fasshauer et al. 1998xFasshauer, D., Eliason, W.K., Brunger, A.T., and Jahn, R. Biochemistry. 1998; 37: 10354–10362CrossRef | PubMed | Scopus (175)See all ReferencesFasshauer et al. 1998). Importantly, the generation of the SNARE core complexes is highly regulated. Despite their ability to form SDS-resistant complexes, most VAMP, syntaxin, and SNAP-25 proteins are not bound to each other under steady state conditions. Instead, it appears that the availability of syntaxin and VAMP proteins to form SNARE complexes is limited by their interactions with other proteins, including Munc18 (also called rSec1) and synaptophysin, respectively. Munc18 in vitro competitively inhibits the binding of SNAP-25 or VAMP to syntaxin-1a (Pevsner et al. 1994xPevsner, J., Hsu, S.C., Braun, J.E., Calakos, N., Ting, A.E., Bennett, M.K., and Scheller, R.H. Neuron. 1994; 13: 353–361Abstract | Full Text PDF | PubMed | Scopus (473)See all ReferencesPevsner et al. 1994); yet, how these complexes are rearranged during the priming and triggering of exocytosis remains to be determined.Recently, a new syntaxin-1–binding protein, tomosyn, was shown to have the ability to displace Munc18 from syntaxin-1 and subsequently to form a new complex with syntaxin, SNAP-25 and synaptotagmin (a syntaxin-binding protein) (Fujita et al. 1998xFujita, Y., Shirataki, H., Sakisaka, T., Asakura, T., Ohya, T., Kotani, H., Yokoyama, S., Nishioka, H., Matsuura, Y., Mizoguchi, A. et al. Neuron. 1998; 20: 905–915Abstract | Full Text | Full Text PDF | PubMed | Scopus (188)See all ReferencesFujita et al. 1998). Although the exact nature of the interaction was not determined, tomosyn was found to interact with the coiled coil domain of syntaxin-1a. Furthermore, it was also suggested that tomosyn would be later replaced by VAMP to form the SNARE complex required for membrane fusion, since the characterization of the SNARE proteins present in different high molecular complexes showed that the presence of tomosyn and VAMP was mutually exclusive (Fujita et al. 1998xFujita, Y., Shirataki, H., Sakisaka, T., Asakura, T., Ohya, T., Kotani, H., Yokoyama, S., Nishioka, H., Matsuura, Y., Mizoguchi, A. et al. Neuron. 1998; 20: 905–915Abstract | Full Text | Full Text PDF | PubMed | Scopus (188)See all ReferencesFujita et al. 1998). In this way, tomosyn may play a key role in regulating the generation of fusion-competent SNARE complexes.We have started, among several approaches, to search for proteins that interact with SNARE proteins from mast cells using a yeast two-hybrid system in order to identify and map regulatory components of the exocytic process in these cells. Interestingly, using SNAP-23 (the counterpart for SNAP-25 in mast cells) as a bait, we found 3 different interacting clones among 3 × 106 clones screened which encoded the carboxyl terminus of tomosyn. Similarly, 6 different clones encoding the carboxyl terminus of tomosyn were recovered among 3 × 107 clones screened using syntaxin-4 as a bait. These results indicate that tomosyn, like VAMP, has the capacity to bind not only syntaxin-1a but also syntaxin-4 and SNAP-23. The results also show that tomosyn is expressed in mast cells, specifically the murine mast cell line MC-9, and suggest that, as in neurons and PC12 cells, tomosyn plays a role in the regulation of mast cell exocytosis.Altogether, the 9 cDNA clones encoded peptides containing the carboxy-terminal 82–142 amino acid residues of tomosyn. This defines the carboxyl terminus as the domain of tomosyn that interacts with the coiled coil domains of syntaxin and SNAP-23. Analysis of this carboxy-terminal interacting domain of tomosyn with the COILS program (21 amino acid window size) predicted a coiled coil centered on arginine 1188 with a probability of 0.9. In a blast search of the nonredundant database, two VAMP homologs are the fourth and fifth highest scores behind tomosyn itself and previously discussed tumor suppressor–related genes. In addition, the VAMP1 homology is 10 and the VAMP4 homology is 8 standard deviations above the mean randomized homology found between the sequences (Figure 1Figure 1). Comparison to several other known coiled coil sequences results in insignificant scores ranging from 0–4 standard deviations above the mean randomized homology. Interestingly, sensitive profile searches based on the coiled coil domains of the SNARE proteins have shown that all SNARE coiled coils are distantly related (Weimbs et al. 1998xWeimbs, T., Mostov, K., and Hui Low, S. Trends Cell Biol. 1998; 8: 260–262Abstract | Full Text | Full Text PDF | PubMed | Scopus (100)See all ReferencesWeimbs et al. 1998). Moreover, alignment of the coiled coil domains of the SNARE superfamily reveal the conservation of an arginine residue in the d position of a central heptad repeat in all VAMP-like SNAREs. A glutamine residue replaces the arginine in all other SNARE coiled coils (Weimbs et al. 1998xWeimbs, T., Mostov, K., and Hui Low, S. Trends Cell Biol. 1998; 8: 260–262Abstract | Full Text | Full Text PDF | PubMed | Scopus (100)See all ReferencesWeimbs et al. 1998). The tomosyn predicted coiled coil includes the conserved arginine at position d in the central heptad repeat of the α helix (Figure 1Figure 1). This finding thus argues that tomosyn, like VAMP proteins, is capable of forming trimeric complexes with t-SNAREs and is consistent with the proposed replacement of tomosyn by VAMP during the exocytic process. We propose that tomosyn is the first of a class of membrane trafficking regulators that utilizes a SNARE coil domain to substitute for one of the components of the four-helix bundle that makes up the core complex. Further characterization of the tomosyn molecule should shed light on the regulation of dissociation and association of SNARE complexes during the exocytic membrane fusion events.Figure 1Schematic Representation of Tomosyn and Sequence Alignment of Its Carboxy-Terminal Domain Residues with the Coiled Coil Sequences of Representative VAMP Family ProteinsThe amino acid sequence of the 82 residues from the smallest interacting domain of murine tomosyn was identical to the sequence from rat tomosyn (black box). Numbers indicating the position of tomosyn residues correspond to the rat sequence (ra Tomosyn, U92072). GenBank accession numbers for the other sequences are as follows: mu VAMP1, U61751; mu VAMP4, AF061516; and sc Snc1, U12980 (mu, murine; sc, S. cerevisiae). Black boxes indicate identical residues present in all sequences. White boxes indicate positions that show 75% match (3 out of 4) to similar amino acid residues. The black triangle marks the arginine (R) residue conserved in all VAMP family members. The a and d positions in the heptad repeats are marked by black dots below the alignment.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Discovery and Development of Spleen Tyrosine Kinase (SYK) Inhibitors

■ INTRODUCTION Allergic and autoimmune disorders share significant functional overlap in the biologic pathways responsible for the activation of signal transduction events leading to production of numerous proinflammatory factors involved in disease initiation and progression. Given the reciprocal connections in these mechanistic pathways, it would be advantageous to target strategic master regulators with novel therapeutics to treat allergic and autoimmune diseases. One such crucial regulator is spleen tyrosine kinase (SYK), a member of the cytoplasmic protein tyrosine kinase (PTK) family. Geahlen et al. initially reported SYK as a proteolytically derived fragment (p40) from a parent p72 protein extracted from bovine thymus. The isolated p40 protein was found to be catalytically active, possessing an adenosine 5′-triphosphate (ATP) binding site, and capable of intramolecular autophosphorylation of tyrosine residues. Further studies have led to the observation that antibodies to p40 concomitantly showed cross-reactivity with a 72 kDa protein-tyrosine kinase from the spleen and thymus. Yamamura et al. corroborated the earlier work, employing immunoblot analysis using anticytosolic protein-tyrosine kinase40 antibody made for the N-terminal portion of the 40 kDa kinase, and identified a 72 kDa protein, which is now known as the SYK protein. Application of a partial sequence oligonucleotide probe for the 40 kDa kinase led to a clone comprising the complete coding sequence for the 40 kDa kinase; the clone was made up of 628 amino acids with a molecular weight of 71 618 Da and corresponded to acidophilic SYK kinase. Subsequent to its initial discovery, SYK has garnered substantial attention because of its importance as a key signal transduction regulator through antigen and Fc receptors in hematopoietic cells. The validity of SYK as a target for therapeutic intervention has progressed over the past 15 years, and SYK has recently entered the mainstream of potential pharmaceutical targets with a number of inhibitor classes being reported in the literature. The true potential as a validated target for inhibition by small molecules has also been investigated in the past few years, with advanced clinical trial data only emerging during this period of time (vide infra). Expression of SYK is found extensively in hematopoietic cells, such as mast cells, basophils, B-cells, T-cells, neutrophils, dendritic cells (DC), macrophages, monocytes, erythrocytes, and platelets, as well as nonhematopoietic cells, such as osteoclasts, epithelial cells, fibroblasts, hepatocytes, and neuronal and vascular endothelial cells. Another member of the nonreceptor tyrosine family is ζ-chain-associated protein kinase 70 of 70 kDa (ZAP-70), which is present in the cytoplasm and is closely related to SYK in both homology and function. However, ZAP-70 expression is limited to T lymphocytes and natural killer (NK) cells. SYK and ZAP-70 constitute the cytoplasmic PTK family, possessing tandem N-terminal Src homology-2 domains (SH2) referred to as N-SH2 and C-SH2 domains. These are separated by interdomain A, and the C-SH2 domain is followed by interdomain B which precedes the C-terminal kinase domain (Figure 1). The SH2 domains bind phosphorylated tyrosines, and the tandem SH2 domains of the SYK family neatly dock into a conserved peptide motif containing two phosphorylated tyrosines separated by 9−11 amino acids. These motifs are found in signaling chains of multimeric receptors that respond to foreign and self-antigens in immune cells. Such motifs are referred to as immunoreceptor tyrosine-based activation motifs (ITAMs). Importantly, the tandem SH2 domains also help to maintain SYK family kinases in an inactivated state via intramolecular interactions in resting immune cells. Consequently, the binding of the two SH2 domains to ITAMs of receptors plays a critical role in SYK activation and frees the activated kinase domain for substrate interaction. This mode of activation also serves as a guiding principle to recognize the receptors that activate SYK family kinases. Essentially, the activation of these kinases is mediated by ITAM-containing receptors. SYK, for example, transduces signals from antigen receptors such as the B-cell antigen receptor (BCR) and Fc receptors (FcR) that bind the Fc portion of immunoglobulins, which in turn bind antigens (Figure 2). ZAP-70, consistent with its narrow expression profile, transduces signals from the T cell receptor (TCR) and ITAM-bearing NK cell receptors. These multimeric receptors invariably contain one or more subunits possessing ITAM motifs. It has been recognized that ITAM-bearing subunits are shared with other receptors. For example, specific leukocyte integrins, receptor activator of nuclear factor-κB ligand (RANKL), and C-type lectin receptors (CLR), such as Dectin-2 or Mincle, have been shown to co-opt FcRγ and DNAX-activated protein (DAP12), which contain ITAMs, and when engaged they activate SYK. SYK PTK intrinsic activity has been reported to be up to 100fold more active than ZAP-70, and this difference probably reflects that SYK is easier to activate than ZAP-70. SYK, for example, is not as reliant on phosphorylated ITAM architecture for enzymatic activation as ZAP-70. Interestingly, it has become clear in recent years that incomplete ITAMs, referred to as hemITAMs, are capable of interacting with and mediating SYK activation. In this way, a number of C-type lectin receptors, such as Dectin-1, CLEC2, and CLEC9A, possess hemITAMs and transduce signals through SYK. Altogether, SYK is activated through ITAMs present in BCRs

A Novel E3 Ubiquitin Ligase TRAC-1 Positively Regulates T Cell Activation

TRAC-1 (T cell RING (really interesting new gene) protein identified in activation screen) is a novel E3 ubiquitin ligase identified from a retroviral vector-based T cell surface activation marker screen. The C-terminal truncated TRAC-1 specifically inhibited anti-TCR-mediated CD69 up-regulation in Jurkat cells, a human T leukemic cell line. In this study, we show that TRAC-1 is a RING finger ubiquitin E3 ligase with highest expression in lymphoid tissues. Point mutations that disrupt the Zn2+-chelating ability of its amino-terminal RING finger domain abolished TRAC-1’s ligase activity and the dominant inhibitory effect of C-terminal truncated TRAC-1 on TCR stimulation. The results of in vitro biochemical studies indicate that TRAC-1 can stimulate the formation of both K48- and K63-linked polyubiquitin chains and therefore could potentially activate both degradative and regulatory ubiquitin-dependent pathways. Antisense oligonucleotides to TRAC-1 specifically reduced TRAC-1 mRNA levels in Jurkat and primary T cells and inhibited their activation in response to TCR cross-linking. Collectively, these results indicate that the E3 ubiquitin ligase TRAC-1 functions as a positive regulator of T cell activation.

A novel role for p21-activated protein kinase 2 in T cell activation.

To identify novel components of the TCR signaling pathway, a large-scale retroviral-based functional screen was performed using CD69 expression as a marker for T cell activation. In addition to known regulators, two truncated forms of p21-activated kinase 2 (PAK2), PAK2ΔL1–224 and PAK2ΔS1–113, both lacking the kinase domain, were isolated in the T cell screen. The PAK2 truncation, PAK2ΔL, blocked Ag receptor-induced NFAT activation and TCR-mediated calcium flux in Jurkat T cells. However, it had minimal effect on PMA/ionomycin-induced CD69 up-regulation in Jurkat cells, on anti-IgM-mediated CD69 up-regulation in B cells, or on the migratory responses of resting T cells to chemoattractants. We show that PAK2 kinase activity is increased in response to TCR stimulation. Furthermore, a full-length kinase-inactive form of PAK2 blocked both TCR-induced CD69 up-regulation and NFAT activity in Jurkat cells, demonstrating that kinase activity is required for PAK2 function downstream of the TCR. We also generated a GFP-fused PAK2 truncation lacking the Cdc42/Rac interactive binding region domain, GFP-PAK283–149. We show that this construct binds directly to the kinase domain of PAK2 and inhibits anti-TCR-stimulated T cell activation. Finally, we demonstrate that, in primary T cells, dominant-negative PAK2 prevented anti-CD3/CD28-induced IL-2 production, and TCR-induced CD40 ligand expression, both key functions of activated T cells. Taken together, these results suggest a novel role for PAK2 as a positive regulator of T cell activation.

Pharmacologic inhibition of PKCα and PKCθ prevents GVHD while preserving GVL activity in mice

Allogeneic hematopoietic cell transplantation (HCT) is the most effective therapy for hematopoietic malignancies through T-cell-mediated graft-vs-leukemia (GVL) effects but often leads to severe graft-vs-host disease (GVHD). Given that protein kinase Cθ (PKCθ), in cooperation with PKCα, is essential for T-cell signaling and function, we have evaluated PKCθ and PKCα as potential therapeutic targets in allogeneic HCT using genetic and pharmacologic approaches. We found that the ability of PKCα(-/-)/θ(-/-) donor T cells to induce GVHD was further reduced compared with PKCθ(-/-) T cells in relation with the relevance of both isoforms to allogeneic donor T-cell proliferation, cytokine production, and migration to GVHD target organs. Treatment with a specific inhibitor for both PKCθ and PKCα impaired donor T-cell proliferation, migration, and chemokine/cytokine production and significantly decreased GVHD in myeloablative preclinical murine models of allogeneic HCT. Moreover, pharmacologic inhibition of PKCθ and PKCα spared T-cell cytotoxic function and GVL effects. Our findings indicate that PKCα and θ contribute to T-cell activation with overlapping functions essential for GVHD induction while less critical to the GVL effect. Thus, targeting PKCα and PKCθ signaling with pharmacologic inhibitors presents a therapeutic option for GVHD prevention while largely preserving the GVL activity in patients receiving HCT.