-
Julie A Di Paolo,
Tao Huang,
Mercedesz Balazs,
James Barbosa,
Kai H Barck,
Brandon J Bravo,
Richard A D Carano,
James Darrow,
Douglas R Davies,
Laura E DeForge, [......],
Scott A Mitchell,
Hong Rong,
Bart L Staker,
J Andrew Whitney, Sherry Yeh,
Wendy B Young,
Christine Yu,
Juan Zhang,
Karin Reif,
Kevin S Currie
[show abstract]
[hide abstract]
ABSTRACT: Bruton's tyrosine kinase (Btk) is a therapeutic target for rheumatoid arthritis, but the cellular and molecular mechanisms by which Btk mediates inflammation are poorly understood. Here we describe the discovery of CGI1746, a small-molecule Btk inhibitor chemotype with a new binding mode that stabilizes an inactive nonphosphorylated enzyme conformation. CGI1746 has exquisite selectivity for Btk and inhibits both auto- and transphosphorylation steps necessary for enzyme activation. Using CGI1746, we demonstrate that Btk regulates inflammatory arthritis by two distinct mechanisms. CGI1746 blocks B cell receptor-dependent B cell proliferation and in prophylactic regimens reduces autoantibody levels in collagen-induced arthritis. In macrophages, Btk inhibition abolishes FcγRIII-induced TNFα, IL-1β and IL-6 production. Accordingly, in myeloid- and FcγR-dependent autoantibody-induced arthritis, CGI1746 decreases cytokine levels within joints and ameliorates disease. These results provide new understanding of the function of Btk in both B cell- or myeloid cell-driven disease processes and provide a compelling rationale for targeting Btk in rheumatoid arthritis.
Nature Chemical Biology 01/2011; 7(1):41-50. · 14.69 Impact Factor
-
Hans D Brightbill,
Surinder Jeet,
Zhonghua Lin,
Donghong Yan,
Meijuan Zhou,
Martha Tan,
Allen Nguyen, Sherry Yeh,
Donnie Delarosa,
Steven R Leong, [......],
Mark S Dennis,
Anan Chuntharapai,
Laura DeForge,
Y Gloria Meng,
Min Xu,
Charles Eigenbrot,
Wyne P Lee,
Canio J Refino,
Mercedesz Balazs,
Lawren C Wu
[show abstract]
[hide abstract]
ABSTRACT: IgE-mediated hypersensitivity is central to the pathogenesis of asthma and other allergic diseases. Although neutralization of serum IgE with IgE-specific antibodies is in general an efficacious treatment for allergic asthma, one limitation of this approach is its lack of effect on IgE production. Here, we have developed a strategy to disrupt IgE production by generating monoclonal antibodies that target a segment of membrane IgE on human IgE-switched B cells that is not present in serum IgE. This segment is known as the M1' domain, and using genetically modified mice that contain the human M1' domain inserted into the mouse IgE locus, we demonstrated that M1'-specific antibodies reduced serum IgE and IgE-producing plasma cells in vivo, without affecting other immunoglobulin isotypes. M1'-specific antibodies were effective when delivered prophylactically and therapeutically in mouse models of immunization, allergic asthma, and Nippostrongylus brasiliensis infection, likely by inducing apoptosis of IgE-producing B cells. In addition, we generated a humanized M1'-specific antibody that was active on primary human cells in vivo, as determined by its reduction of serum IgE levels and IgE plasma cell numbers in a human PBMC-SCID mouse model. Thus, targeting of human IgE-producing B cells with apoptosis-inducing M1'-specific antibodies may be a novel treatment for asthma and allergy.
The Journal of clinical investigation 06/2010; 120(6):2218-29. · 15.39 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Tim-4 is a phosphatidylserine (PS) receptor that is expressed on various macrophage subsets. It mediates phagocytosis of apoptotic cells by peritoneal macrophages. The in vivo functions of Tim-4 in phagocytosis and immune responses, however, are still unclear. In this study, we show that Tim-4 quickly forms punctate caps on contact with apoptotic cells, in contrast to its normal diffused expression on the surface of phagocytes. Despite its expression in marginal zone and tingible body macrophages, Tim-4 deficiency only minimally affects outcomes of several acute immune challenges, including the trapping of apoptotic cells in the marginal zone, the clearance apoptotic cells by tingible body macrophages, and the formation of germinal centers and elicitation of antibody responses against sheep red blood cells (SRBCs). In addition, Tim-4(-/-) resident peritoneal macrophages (rPMs) phagocytose necrotic cells and other opsonized targets normally. However, their ability to bind and engulf apoptotic cells is significantly compromised both in vitro and in vivo. Most importantly, Tim-4 deficiency results in increased cellularity in the peritoneum. Resting rPMs produce higher TNF-alpha in culture. Their response to LPS, on the contrary, is dampened. Our data support an indispensible role of Tim-4 in maintaining the homeostasis of rPMs.
Proceedings of the National Academy of Sciences 05/2010; 107(19):8712-7. · 9.68 Impact Factor
-
Wei Yu Lin,
Qian Gong,
Dhaya Seshasayee,
Zhonghua Lin,
Qinglin Ou,
Shiming Ye,
Eric Suto,
Jean Shu,
Wyne Pun Lee,
Ching-Wei V Lee, [......],
Joseph Beyer,
Dimitry Danilenko, Sherry Yeh,
Laura E DeForge,
Allen Ebens,
Jeffrey S Thompson,
Christine Ambrose,
Mercedesz Balazs,
Melissa A Starovasnik,
Flavius Martin
[show abstract]
[hide abstract]
ABSTRACT: Removal of pathogenic B lymphocytes by depletion of monoclonal antibodies (mAbs) or deprivation of B-cell survival factors has demonstrated clinical benefit in both oncologic and immunologic diseases. Partial clinical responses and emerging data demonstrating incomplete B-cell depletion after immunotherapy fuels the need for improved therapeutic modalities. Lessons from the first generation of therapeutics directed against B-cell-specific antigens (CD20, CD22) are being applied to develop novel antibodies with additional functional attributes. We describe the generation of a novel class of B-cell-directed therapy (anti-BR3 mAbs) that combines the depleting capacity of a therapeutic mAb and blockade of B-cell-activating factor (BAFF)-BR3 B-cell survival. In mice, treatment with antagonistic anti-BR3 antibodies results in quantitatively greater reduction in some B-cell subsets and qualitatively different effects on bone marrow plasma cells compared with BR3-Fc BAFF blockade or with anti-CD20 treatment. Comparative analysis of BR3-Fc and anti-BR3 mAb reveals a lower B-cell dependence for BAFF-mediated survival in nonhuman primates than in mice. This novel class of B-cell-targeted therapies shows species characteristics in mice and primates that will guide translation to treatment of human disease.
Blood 01/2008; 110(12):3959-67. · 9.90 Impact Factor
-
Yingnan Zhang, Sherry Yeh,
Brent A. Appleton,
Heike A. Held,
P. Jaya Kausalya,
Dominic C. Y. Phua,
Wai Lee Wong,
Laurence A. Lasky,
Christian Wiesmann,
Walter Hunziker,
Sachdev S. Sidhu
[show abstract]
[hide abstract]
ABSTRACT: We present a detailed comparative analysis of the PDZ domains of the human LAP proteins Erbin, Densin-180, and Scribble and
the MAGUK ZO-1. Phage-displayed peptide libraries and in vitro affinity assays were used to define ligand binding profiles for each domain. The analysis reveals the importance of interactions
with all four C-terminal residues of the ligand, which constitute a core recognition motif, and also the role of interactions
with more upstream ligand residues that support and modulate the core binding interaction. In particular, the results highlight
the importance of site-1, which interacts with the penultimate residue of ligand C termini. Site-1 was found to be monospecific in the Erbin PDZ domain (accepts tryptophan only), bispecific in the first PDZ domain of ZO-1
(accepts tryptophan or tyrosine), and promiscuous in the Scribble PDZ domains. Furthermore, it appears that the level of promiscuity
within site-1 greatly influences the range of potential biological partners and functions that can be associated with each protein. These
findings show that subtle changes in binding specificity can significantly alter the range of biological partners for PDZ
domains, and the insights enhance our understanding of this diverse family of peptide-binding modules.
Journal of Biological Chemistry 08/2006; 281(31):22299-22311. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The LAP (leucine-rich repeat and PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW(-4)ETW(-1)V; IC(50) = approximately 0.15 microm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable interactions with Trp(-1) are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the beta2-beta3 loop provides a binding site for the Trp(-4) side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.
Journal of Biological Chemistry 03/2003; 278(9):7645-54. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: The LAP (leucine-rich repeatand PDZ-containing) family of proteins play a role in maintaining epithelial and neuronal cell size, and mutation of these proteins
can have oncogenic consequences. The LAP protein Erbin has been implicated previously in a number of cellular activities by
virtue of its PDZ domain-dependent association with the C termini of both ERB-B2 and the p120-catenins. The present work describes
the NMR structure of Erbin PDZ in complex with a high affinity peptide ligand and includes a comprehensive energetic analysis
of both the ligand and PDZ domain side chains responsible for binding. C-terminal phage display has been used to identify
preferred ligands, whereas binding affinity measurements provide precise details of the energetic importance of each ligand
side chain to binding. Alanine and homolog scanning mutagenesis (in a combinatorial phage display format) identifies Erbin
side chains that make energetically important contacts with the ligand. The structure of a phage-optimized peptide (Ac-TGW−4ETW−1V; IC50 = ∼0.15 μm) in complex with Erbin PDZ provides a structural context to understand the binding energetics. In particular, the very favorable
interactions with Trp−1 are not Erbin side chain-mediated (and therefore may be generally applicable to many PDZ domains), whereas the β2-β3 loop
provides a binding site for the Trp−4 side chain (specific to Erbin because it has an unusually long loop). These results contribute to a growing appreciation
for the importance of at least five ligand C-terminal side chains in determining PDZ domain binding energy and highlight the
mechanisms of ligand discrimination among the several hundred PDZ domains present in the human genome.
Journal of Biological Chemistry 02/2003; 278(9):7645-7654. · 4.77 Impact Factor
