Sarah C. Bodary

Regulation of Integrin Function by the Urokinase Receptor

Integrin function is central to inflammation, immunity, and tumor progression. The urokinase-type plasminogen activator receptor (uPAR) and integrins formed stable complexes that both inhibited native integrin adhesive function and promoted adhesion to vitronectin via a ligand binding site on uPAR. Interaction of soluble uPAR with the active conformer of integrins mimicked the inhibitory effects of membrane uPAR. Both uPAR-mediated adhesion and altered integrin function were blocked by a peptide that bound to uPAR and disrupted complexes. These data provide a paradigm for regulation of integrins in which a nonintegrin membrane receptor interacts with and modifies the function of activated integrins.

ANGPTL3 Stimulates Endothelial Cell Adhesion and Migration via Integrin αvβ3 and Induces Blood Vessel Formation in Vivo

The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to αvβ3 and induced integrin αvβ3-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin αvβ3 and suggest a possible role in the regulation of angiogenesis.

Characterization of a candidate Borrelia burgdorferi beta3-chain integrin ligand identified using a phage display library.

The spirochaetal agents of Lyme disease, Borrelia burgdorferi (sensu lato) bind to integrins αIIbβ3, αvβ3 and α5β1 in purified form and on the surfaces of human cells. Using a phage display library of B. burgdorferi (sensu stricto) DNA, a candidate ligand for β3‐chain integrins was identified. The native B. burgdorferi protein, termed p66, is known to be recognized by human Lyme disease patient sera and to be expressed on the surface of the spirochaete. We show here that recombinant p66 binds specifically to β3‐chain integrins and inhibits attachment of intact B. burgdorferi to the same integrins. When expressed on the surface of Escherichia coli, this protein increases the attachment of E. coli to a transfected cell line that expresses αvβ3, but not to the parental cell line, which expresses no β3‐chain integrins. Localization of p66 on the surface of B. burgdorferi, the ability of recombinant forms of the protein to bind to β3‐chain integrins and the fact that p66 and B. burgdorferi bind to β3‐chain integrins in a mutually exclusive manner make p66 an attractive candidate bacterial ligand for integrins αIIbβ3 and αvβ3.

Mapping the intercellular adhesion molecule-1 and -2 binding site on the inserted domain of leukocyte function-associated antigen-1.

By extensive mutagenic analysis of the inserted domain (I-domain) of the α-chain (CD11a) of the leukocyte function-associated antigen-1 (LFA-1), we have defined a putative binding surface for intercellular adhesion molecules 1 and 2 (ICAM-1 and -2). This analysis showed that individually mutating Leu-205 or Glu-241 to alanine completely abolished LFA-1 binding to ICAM-1 or -2 without affecting I-domain structure, as assayed by antibody binding. Mutating Thr-243 to alanine also had a profound effect on LFA-1 binding to ICAM-1 and -2, as seen by complete loss of binding to ICAM-1 and a significant reduction (70% decrease) in binding to ICAM-2. Mutating Glu-146 to alanine reduced LFA-1 binding to ICAM-1 or -2 by 70%, and mutating His-264 or Glu-293 to alanine reduced binding to ICAM-1 or -2 by about 30–40%. Mutating Thr-175 to alanine reduced binding to ICAM-1 by about 30% and binding to ICAM-2 by about 70%. Interestingly, mutating Lys-263 to alanine preferentially abolished LFA-1 binding to ICAM-2. Using these data, we have generated a model of the interface between the LFA-1 I-domain and residues in the first domain of ICAM-1 that have been shown to be critical for this interaction. In addition, this model, together with the ICAM-2 crystal structure, has been used to map residues that are likely to mediate LFA-1 I-domain binding to ICAM-2.

Adhesion molecules as therapeutic targets for autoimmune diseases and transplant rejection

Inflammatory disorders such as autoimmune diseases and graft rejection are mediated by activated leukocytes, particularly T lymphocytes, which penetrate the inflamed tissue and perpetuate or amplify the immune reaction. In an unstimulated state, leukocytes do not readily adhere to the vascular endothelium. However, inflammatory signals induce the expression of proteins on the endothelial cell surface that promote the adhesion and extravasation of activated immune cells from the circulation into the underlying tissues. Key among these molecules are P- and E-selectin, intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) on the endothelial cells, and their respective counter receptors , P-selectin glycoprotein ligand-1 (PSGL-1), leukocyte function-associated antigen-1 (LFA-1) and very late antigen-4 (VLA-4), on the leukocytes. In vitro blockade of these molecules inhibits the adhesion of leukocytes. In many cases there is attenuation of leukocyte activation as well. Adhesion blockade in animal models prevents or ameliorates graft rejection and disease severity in autoimmune models. Clinical studies with humanised monoclonal antibodies which interfere with LFA-1/ICAM-1 or VLA-4/VCAM-1 interactions have shown significant efficacy and good safety profiles in autoimmune disease, including psoriasis, multiple sclerosis and inflammatory bowel disease. Thus, adhesion blockade is emerging as a useful therapeutic strategy in several inflammatory settings.

Blockade of CD11a by Efalizumab in Psoriasis Patients Induces a Unique State of T-Cell Hyporesponsiveness

Efalizumab (anti-CD11a) interferes with LFA-1/ICAM-1 binding and inhibits several key steps in psoriasis pathogenesis. This study characterizes the effects of efalizumab on T-cell activation responses and expression of surface markers on human circulating psoriatic T cells during a therapeutic trial. Our data suggest that efalizumab may induce a unique type of T-cell hyporesponsiveness, directly induced by LFA-1 binding, which is distinct from conventional anergy described in animal models. Direct activation of T cells through different activating receptors (CD2, CD3, CD3/28) is reduced, despite T cells being fully viable. This hyporesponsiveness was spontaneously reversible after withdrawal of the drug, and by IL-2 in vitro. In contrast to the state of anergy, Ca(+2) release is intact during efalizumab binding. Furthermore, lymphocyte function-associated antigen-1 (LFA-1) blockade resulted in an unexpected downregulation of a broad range of surface molecules, including the T-cell receptor complex, co-stimulatory molecules, and integrins unrelated to LFA-1, both in the peripheral circulation and in diseased skin tissue. These observations provide evidence for the mechanism of action of efalizumab. The nature of this T-cell hyporesponsiveness suggests that T-cell responses may be reduced during efalizumab therapy, but are reversible after ceasing efalizumab treatment.

Competition between intercellular adhesion molecule-1 and a small-molecule antagonist for a common binding site on the αl subunit of lymphocyte function-associated antigen-1

The lymphocyte function‐associated antigen‐1 (LFA‐1) binding of a unique class of small‐molecule antagonists as represented by compound 3 was analyzed in comparison to that of soluble intercellular adhesion molecule‐1 (sICAM‐1) and A‐286982, which respectively define direct and allosteric competitive binding sites within LFA‐1s inserted (I) domain. All three molecules antagonized LFA‐1 binding to ICAM‐1‐Immunoglobulin G fusion (ICAM‐1‐Ig) in a competition ELISA, but only compound 3 and sICAM‐1 inhibited the binding of a fluorescein‐labeled analog of compound 3 to LFA‐1. Compound 3 and sICAM‐1 displayed classical direct competitive binding behavior with ICAM‐1. Their antagonism of LFA‐1 was surmountable by both ICAM‐1‐Ig and a fluorescein‐labeled compound 3 analog. The competition of both sICAM‐1 and compound 3 with ICAM‐1‐Ig for LFA‐1 resulted in equivalent and linear Schild plots with slopes of 1.24 and 1.26, respectively. Cross‐linking studies with a photoactivated analog of compound 3 localized the high‐affinity small‐molecule binding site to the N‐terminal 507 amino acid segment of the α chain of LFA‐1, a region that includes the I domain. In addition, cells transfected with a variant of LFA‐1 lacking this I domain showed no significant binding of a fluorescein‐labeled analog of compound 3 or ICAM‐1‐Ig. These results demonstrate that compound 3 inhibits the LFA‐1/ICAM‐1 binding interaction in a directly competitive manner by binding to a high‐affinity site on LFA‐1. This binding site overlaps with the ICAM‐1 binding site on the α subunit of LFA‐1, which has previously been localized to the I domain.

RGD Mimetics containing a central hydantoin scaffold: αVβ3 vs αIIbβ3 selectivity requirements

Abstract The synthesis of a series of RGD mimetic α V β 3 antagonists containing a hydantoin scaffold is shown. The results demonstrate some of the structural requirements for the design of selective α V β 3 antagonists (vs α IIb β 3 ) in terms of the Arg-mimetic, the distance between N- and C-terminus and the lipophilic side chain.

Putting the pieces together: contribution of fluorescence polarization assays to small-molecule lead optimization

Fluorescence polarization assays with both purified receptor and intact cells have been developed to assess potency and selectivity of antagonists of the interaction of the lymphocyte receptor, LFA-1, and its endothelial ligand, ICAM-1. Fluorescein isothiocyanate conjugated small molecule probes were optimized for use in binding assay with LFA-1 and a closely related receptor, MAC-1. In the assays, the antagonists compete with the fluorescent probe for binding to the receptor. This enables the determination of IC50 and consequently Ki values of the antagonists for each of the receptors. Routine use of polarization assay with tranfected cells, in addition to purified receptors, has become feasible with the availability of sensitive plate readers that are able to detect 1 nM fluorescent probe in 15 (mu) l sample volumes with good signal to noise. These measurements aid in the iterative synthesis of more potent and selective compounds.

αvβ3 Antagonists Based on a Central Thiophene Scaffold

Abstract A series of novel, highly potent αvβ3 antagonists based on a thiophene scaffold and containing an acylguanidine as an Arg-mimetic is described. A number of structural features, such as cyclic versus open guanidine and a variety of lipophilic side chains, carbamates, sulfonamides and β-amino acids were explored with respect to inhibition of αvβ3 mediated cell adhesion and selectivity versus αIIbβ3 binding. In addition, compound 19 was found to be active in the TPTX model of osteoporosis.