Gabriele Weitz-Schmidt

Statins as anti-inflammatory agents

The beneficial effects of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) in cardiovascular disease have generally been attributed to their cholesterol-lowering property. However, an increasing number of in vitro and in vivo studies indicate that statins have direct anti-inflammatory effects that are not mediated by their hypocholesterolemic activity. In this article, the HMG-CoA-reductase-dependent and -independent mechanisms by which statins might affect leukocyte adhesion and migration to sites of inflammation are reviewed and the implications for the design of new statin-derived drugs are discussed.

Small Molecule Integrin Antagonists that Bind to the β2 Subunit I-like Domain and Activate Signals in One Direction and Block Them in the Other

Leukocyte integrins contain an inserted (I) domain in their alpha subunits and an I-like domain in their beta(2) subunit, which directly bind ligand and regulate ligand binding, respectively. We describe a novel mechanistic class of integrin inhibitors that bind to the metal ion-dependent adhesion site of the beta(2) I-like domain and prevent its interaction with and activation of the alpha(L) I domain. The inhibitors do not bind to the alpha(L) I domain but stabilize alpha/beta subunit association and can show selectivity for alpha(L)beta(2) compared to alpha(M)beta(2). The inhibitors reveal a crucial intersection for relaying conformational signals within integrin extracellular domains. While blocking signals in one direction to the I domain, the antagonists induce the active conformation of the I-like domain and stalk domains, and thus transmit conformational signals in the other direction toward the transmembrane domains.

The Potent Protein Kinase C-Selective Inhibitor AEB071 (Sotrastaurin) Represents a New Class of Immunosuppressive Agents Affecting Early T-Cell Activation

There is a pressing need for immunosuppressants with an improved safety profile. The search for novel approaches to blocking T-cell activation led to the development of the selective protein kinase C (PKC) inhibitor AEB071 (sotrastaurin). In cell-free kinase assays AEB071 inhibited PKC, with Ki values in the subnanomolar to low nanomolar range. Upon T-cell stimulation, AEB071 markedly inhibited in situ PKCθ catalytic activity and selectively affected both the canonical nuclear factor-κB and nuclear factor of activated T cells (but not activator protein-1) transactivation pathways. In primary human and mouse T cells, AEB071 treatment effectively abrogated at low nanomolar concentration markers of early T-cell activation, such as interleukin-2 secretion and CD25 expression. Accordingly, the CD3/CD28 antibody- and alloantigen-induced T-cell proliferation responses were potently inhibited by AEB071 in the absence of nonspecific antiproliferative effects. Unlike former PKC inhibitors, AEB071 did not enhance apoptosis of murine T-cell blasts in a model of activation-induced cell death. Furthermore, AEB071 markedly inhibited lymphocyte function-associated antigen-1-mediated T-cell adhesion at nanomolar concentrations. The mode of action of AEB071 is different from that of calcineurin inhibitors, and AEB071 and cyclosporine A seem to have complementary effects on T-cell signaling pathways.

Small molecule inhibitors induce conformational changes in the I domain and the I-like domain of lymphocyte function-associated antigen-1. Molecular insights into integrin inhibition.

The β2 integrin lymphocyte function-associated antigen-1 (LFA-1) is a conformationally flexible α/β heterodimeric receptor, which is expressed on the surface of all leukocytes. LFA-1 mediates cell adhesion crucial for normal immune and inflammatory responses. Intracellular signals or cations are required to convert LFA-1 from a nonligand binding to a ligand binding state. Here we investigated the effect of small molecule inhibitors on LFA-1 by monitoring the binding of monoclonal antibodies mapped to different receptor domains. The inhibitors were found to not only induce epitope changes in the I domain of the αLchain but also in the I-like domain of the β2 chain depending on the individual chemical structure of the inhibitor and its binding site. For the first time, we provide strong evidence that the I-like domain represents a target for allosteric LFA-1 inhibition similar to the well established regulatory L-site on the I domain of LFA-1. Moreover, the antibody binding patterns observed in the presence of the various inhibitors establish a conformational interaction between the LFA-1 I domain and the I-like domain in the native receptor that is formed upon activation. Differentially targeting the binding sites of the inhibitors, the L-site and the I-like domain, may open new avenues for highly specific therapeutic intervention in diseases where integrins play a pathophysiological role.

Lymphocyte function antigen-1 mediates leukocyte adhesion and subsequent liver damage in endotoxemic mice

Sepsis is associated with leukocyte activation and recruitment in the liver. We investigated the role of lymphocyte function antigen‐1 (LFA‐1) in endotoxin‐induced leukocyte–endothelium interactions, microvascular perfusion failure, hepatocellular injury and apoptosis in the liver by use of gene‐targeted mice, blocking antibodies and a synthetic inhibitor of LFA‐1 (LFA703). For this purpose, mice were challenged with lipopolysaccharide (LPS)+D‐galactosamine (Gal), and intravital microscopy of the liver microcirculation was conducted 6 h later. The number of firmly adherent leukocytes in response to LPS/Gal was reduced by 48% in LFA‐1‐deficient mice. Moreover, endotoxin‐induced increases of apoptosis and enzyme markers of hepatocellular injury were decreased by 64 and 69–90%, respectively, in LFA‐1‐deficient mice. Furthermore, sinusoidal perfusion was improved in endotoxemic mice lacking LFA‐1. A similar protective pattern was observed in endotoxemic mice pretreated with an antibody against LFA‐1. Thus, immunoneutralization of LFA‐1 reduced endotoxin‐induced leukocyte adhesion by 55%, liver enzymes by 64–66% and apoptosis by 42%, in addition to the preservation of microvascular perfusion. Administration of a novel statin‐derived inhibitor of LFA‐1, LFA703, significantly decreased leukocyte adhesion (more than 56%) and the subsequent liver injury in endotoxemic mice. Thus, this study demonstrates a pivotal role of LFA‐1 in supporting leukocyte adhesion in the liver. Moreover, interference with LFA‐1‐mediated leukocyte adhesion protects against endotoxemic liver damage, and may constitute a potential therapeutic strategy in sepsis.

1,4-Diazepane-2,5-diones as novel inhibitors of LFA-1

1,4-Diazepane-2,5-diones (2) are found to be a new class of potent LFA-1 inhibitors. The synthesis, structure, and biological evaluation of these 1,4-diazepine-2,5-diones and related derivatives are described.

A statin‐based inhibitor of lymphocyte function antigen‐1 protects against ischemia/reperfusion‐induced leukocyte adhesion in the colon

Statins are mainly used to control hypercholesterolemia; however, recent studies have also ascribed anti‐inflammatory effects to the statins. LFA703 is a novel statin‐derived compound, which potently inhibits lymphocyte function antigen‐1 (LFA‐1, CD11a/CD18) but does not affect HMG‐CoA reductase activity. The objective of this study was to examine the anti‐inflammatory mechanisms of LFA703 in ischemia/reperfusion (I/R)‐induced leukocyte–endothelium interactions in the colon. For this purpose, the superior mesenteric artery was occluded for 30 min and leukocyte responses were analyzed in colonic venules after 120 min of reperfusion in mice using inverted intravital fluorescence microscopy. First, the inhibitory mechanisms of LFA703 on leukocyte adhesion were investigated in vitro using a mouse CD4+8+ thymocyte cell line. Immunoneutralization of LFA‐1 and ICAM‐1 abolished leukocyte adhesion, whereas inhibition of VLA‐4 had no effect in this in vitro assay. Indeed, it was found that LFA703 dose‐dependently reduced LFA‐1‐dependent leukocyte adhesion to mouse endothelial cells in vitro with an IC50 of 3.2 μM. I/R caused an increase in leukocyte rolling and adhesion in colonic venules. Immunoneutralization of LFA‐1 significantly reduced I/R‐induced leukocyte adhesion by 89% in colonic venules. In contrast, I/R‐provoked leukocyte rolling was insensitive to inhibition of LFA‐1 function. Administration of 30 mg kg−1 of LFA703 decreased reperfusion‐induced leukocyte adhesion by more than 91%, while the level of leukocyte rolling was unchanged, suggesting that LFA703 effectively blocked LFA‐1‐dependent firm adhesion of leukocyte in the colon. However, LFA703 did not decrease the expression of LFA‐1 on circulating leukocytes. This study demonstrates that LFA‐1 is indeed a critical adhesion molecule in mediating postischemic leukocyte adhesion in the colon. Moreover, this is the first study showing that a statin‐based synthetic compound has the capacity to abolish LFA‐1‐dependent leukocyte adhesion in I/R. These novel findings may have great implications in the clinical treatment of conditions associated with I/R‐induced tissue injury, such as organ transplantation, trauma and major surgery.

Lymphocyte Function-Associated Antigen-1 Blockade by Statins: Molecular Basis and Biological Relevance

Lymphocyte function-associated antigen-1 (LFA-1) belongs to the integrin family and plays an important role in leukocyte trafficking and in T-cell activation. Random screening of chemical libraries identified the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor lovastatin as an inhibitor of the LFA-1/intercellular adhesion molecule (ICAM)-1 interaction. The effect of lovastatin on LFA-1 was found to be unrelated to the inhibition of HMG-CoA reductase and to be mediated by lovastatin binding to a novel allosteric site within LFA-1. The biological relevance of LFA-1 inhibition by statins with respect to the overall benefit of this drug class is reviewed. The implications of the statin effect on LFA-1 for future drug design and therapy are discussed.

Differential effect of LFA703, pravastatin, and fluvastatin on production of IL-18 and expression of ICAM-1 and CD40 in human monocytes.

A novel, proinflammatory cytokine, interleukin (IL)‐18 production was detected in the medium of human monocytes treated with 3‐hydroxy‐3‐methylglutaryl coenzyme‐A (HMG‐CoA) reductase inhibitors, pravastatin, and fluvastatin (0.1 and 1 μM) but not with the statin‐derived lymphocyte function‐associated antigen‐1 (LFA‐1) inhibitor LFA703, which did not inhibit HMG‐CoA reductase. Pravastatin and fluvastatin also induced the production of IL‐18, tumor necrosis factor α (TNF‐α) and interferon‐γ (IFN‐γ) in human peripheral blood mononuclear cells (PBMC) in contrast to LFA703. IL‐18 production by PBMC is located upstream of the cytokine cascade activated by these statins. The IL‐18‐induced cytokine production was demonstrated to be dependent on adhesion molecule expression on monocytes. In the absence and presence of lower concentrations (0.1 and 1 ng/ml) of IL‐18, pravastatin and fluvastatin inhibited the expression of intercellular adhesion molecule (ICAM)‐1 and induced the expression of CD40, whereas LFA703 had no effect. In the presence of higher concentrations (5, 10, and 100 ng/ml) of IL‐18, pravastatin, fluvastatin, and LFA703 similarly inhibited the expression of ICAM‐1 and CD40 as well as the production of IL‐12, TNF‐α, and IFN‐γ in PBMC. The effects of pravastatin and fluvastatin but not LFA703 were abolished by the addition of mevalonate, indicating the involvement of HMG‐CoA reductase in the action of pravastatin and fluvastatin. Thus, the effects of LFA703 were distinct from those of pravastatin and fluvastatin in the presence of lower concentrations of IL‐18. It was concluded that LFA703 has the inhibitory effect on an IL‐18‐initiated immune response without any activation on monocytes.

Design, synthesis and biological evaluation of aryl-substituted sialyl Lewis X mimetics prepared via cross-metathesis of C-fucopeptides

Several aryl substituted C-fucopeptides have been developed as sialyl Lewis X mimetics. Although the compounds have a much simpler structure compared to SLe(x), up to 3-times higher binding affinity toward E-selectin and > 1000 times toward P-selectin was observed. Furthermore, a convenient strategy for generating a number of analogues from a SLe(x) mimetic template at a very late stage of the synthesis was introduced, using a ruthenium catalyzed cross olefin metathesis under benchtop conditions.