The Swiss Society of Experimental Pharmacology in Times of Change

Experimental pharmacology is undergoing fundamental changes. This article describes the challenges and opportunities associated with these changes from the perspective of the Swiss Society of Pharmacology (SSEP), the society which aims to advance experimental pharmacology in Switzerland and abroad

Allosteric LFA-1 inhibitors modulate natural killer cell function.

Natural killer (NK) cells are believed to play an important role in a variety of disease pathologies, including transplant rejection and autoimmunity. None of the therapeutic modalities currently available are known to potently interfere with NK cell activity. Here we demonstrate for the first time that low molecular weight inhibitors of the integrin lymphocyte function-associated antigen-1 (LFA-1) readily block NK cell adhesion, activation, and NK cell-mediated cytolysis in vitro, in contrast to other immunosuppressive agents. These effects were independent of the type of allosteric mechanism by which LFA-1 inhibition was achieved. In addition, we describe a simple, nonradioactive whole-blood assay that should be suitable to monitor NK cell activation in clinical practice. Taken together, our study underlines the importance of LFA-1 in NK cell effector functions and indicates that allosteric LFA-1 inhibitors may become important tools to further elucidate the therapeutic potential of NK cell modulation in immunological diseases

A novel multi-parameter whole blood assay to dissect the pharmacologic effects of different modes of LFA-1 receptor occupancy

The integrin lymphocyte function associated antigen-1 (LFA-1) plays central roles in leukocyte adhesion as well as T cell activation and proliferation, rendering LFA-1 an attractive therapeutic target. Different modes of LFA-1 inhibition have been identified and are reported in preclinical and clinical development, respectively. An important question which remains to be answered is whether these different modes of inhibition will translate into different pharmacodynamic effect profiles. Here we describe a multi-parameter flow cytometry-based methodology which allowed, for the first time, to relate different modes and levels of LFA-1 occupancy to distinct pharmacologic effects at the single cell level in human whole blood cultures. Two classes of allosteric small molecule inhibitors, designated αI and α/β I allosteric LFA-1 inhibitors, were investigated. Interestingly, more than 80% LFA-1 occupancy (as measured by distinct compound-induced LFA-1 epitope changes) was required with αI allosteric inhibitors to achieve half-maximal inhibition of T cell activation and proliferation while less than 40 % receptor occupancy by α/β I allosteric inhibitors was sufficient for the same effect size. Further, α/β I allosteric inhibitors induced partial downregulation of LFA-1 from the cell surface revealing a novel property of this inhibitor class distinct from αI allosteric inhibitors. The assay described here opens the possibility to dissect the molecular mechanisms of LFA-1 inhibitors in a physiological environment. This will be essential to identify the best-suited inhibitors for clinical development and to direct them to the most appropriate target indications. The assay may also be used for therapeutic monitoring of LFA-1 inhibitors and possibly other immunosuppressants

A novel multi-parameter assay to dissect the pharmacological effects of different modes of integrin αLβ2 inhibition in whole blood

The integrin αLβ2 plays central roles in leukocyte adhesion and T cell activation, rendering αLβ2 an attractive therapeutic target. Compounds with different modes of αLβ2 inhibition are in development, currently. Consequently, there is a foreseeable need for bedside assays, which allow assessment of the different effects of diverse types of αLβ2 inhibitors in the peripheral blood of treated patients.; Here, we describe a flow cytometry-based technology that simultaneously quantitates αLβ2 conformational change upon inhibitor binding, αLβ2 expression and T cell activation at the single-cell level in human blood. Two classes of allosteric low MW inhibitors, designated α I and α/β I allosteric αLβ2 inhibitors, were investigated. The first application revealed intriguing inhibitor class-specific profiles.; Half-maximal inhibition of T cell activation was associated with 80% epitope loss induced by α I allosteric inhibitors and with 40% epitope gain induced by α/β I allosteric inhibitors. This differential establishes that inhibitor-induced αLβ2 epitope changes do not directly predict the effect on T cell activation. Moreover, we show here for the first time that α/β I allosteric inhibitors, in contrast to α I allosteric inhibitors, provoked partial downmodulation of αLβ2, revealing a novel property of this inhibitor class.; The multi-parameter whole blood αLβ2 assay described here may enable therapeutic monitoring of αLβ2 inhibitors in patients' blood. The assay dissects differential effect profiles of different classes of αLβ2 inhibitors

Anti-αLβ2 antibodies reveal novel endocytotic cross-modulatory functionality

Antibodies targeting cell surface receptors are considered to enable highly selective therapeutic interventions for immune disorders and cancer. Their biological profiles are found, generally, to represent the net effects of antibody-target interactions. The former therapeutic anti-integrin αLβ2 antibody efalizumab seems to defeat this paradigm by eliciting, via mechanisms currently unknown, much broader effects than would be predicted based on its target specificity.; To elucidate the mechanisms behind these broad effects, we investigated in primary human lymphocytes in vitro the effects of anti-αLβ2 antibodies on the expression of αLβ2 as well as unrelated α4 integrins, in comparison to Fab fragments and small-molecule inhibitors.; We demonstrate that anti-αLβ2 mAbs directly induce the internalization of α4 integrins. The endocytotic phenomenon is a direct consequence of their antibody nature. It is inhibited when monovalent Fab fragments or small-molecule inhibitors are used. It is independent of crosslinking via anti-Fc mAbs and of αLβ2 activation. The cross-modulatory effect is unidirectional and not observed in a similar fashion with the α4 integrin antibody natalizumab.; The present study identifies endocytotic cross-modulation as a hitherto unknown non-canonical functionality of anti-αLβ2 antibodies. This cross-modulation has the potential to fundamentally alter an antibody's benefit risk profile, as evident with efalizumab. The newly described phenomenon may be of relevance to other therapeutic antibodies targeting cluster-forming receptors. Thus, pharmacologists should be cognizant of this action when investigating such antibodies

Improved lymphocyte function-associated antigen-1 (LFA-1) inhibition by statin derivatives: molecular basis determined by x-ray analysis and monitoring of LFA-1 conformational changes in vitro and ex vivo.

The integrin lymphocyte function-associated antigen-1 (LFA-1) (alphaLbeta2; CD11a/CD18) plays an important role in leukocyte migration and T cell activation. LFA-1 is inhibited by the cholesterol-lowering drug lovastatin, which binds to an allosteric site of the alphaL I domain termed the lovastatin site (L-site). Here we report for the first time the x-ray structures of the LFA-1 I domain complexed with derivatives of lovastatin optimized for LFA-1 inhibition. This analysis identified two new subpockets within the L-site occupied by chemical groups of the statin derivatives but not by lovastatin itself. Occupancy of these L-site subpockets led to distinct conformational changes in LFA-1, which were detectable by an epitope-monitoring assay. We utilized this assay to demonstrate improved LFA-1 inhibition in human blood in vitro and in blood samples from treated animals ex vivo. Moreover, we demonstrate that the novel lovastatin-derived LFA-1 inhibitor LFA878 exhibits potent anti-inflammatory effects in carrageenan-induced rat paw edema. In summary, the findings reported here extend the understanding of LFA-1 inhibition at the molecular level, allow for the identification and design of LFA-1 inhibitors of further enhanced potency, and support the expectation that LFA-1 inhibitors binding to the L-site will be of therapeutic value in treating inflammatory diseases

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

1. 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. 2. 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. 3. 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. 4. 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. 5. 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

Downstream effect profiles discern different mechanisms of integrin LFA-1 inhibition

The integrin leukocyte function-associated antigen-1 (αLβ2, LFA-1) plays crucial roles in T cell adhesion, migration and immunological synapse (IS) formation. Consequently, αLβ2 is an important therapeutic target in autoimmunity. Three major classes of αLβ2 inhibitors with distinct modes of action have been described to date: Monoclonal antibodies (mAbs), small molecule α/β I allosteric and small molecule α I allosteric inhibitors. The objective of this study was to systematically compare these three modes of αLβ2 inhibition for their αLβ2 inhibitory as well as their potential agonist-like effects. All inhibitors assessed were found to potently block αLβ2-mediated leukocyte adhesion. None of the inhibitors induced ZAP70 phosphorylation, indicating absence of agonistic outside-in signalling. Paradoxically, however, the α/β I allosteric inhibitor XVA143 induced conformational changes within αLβ2 characteristic for an intermediate affinity state. This effect was not observed with the α I allosteric inhibitor LFA878 or the anti- αLβ2 mAb efalizumab. On the other hand, efalizumab triggered the unscheduled internalization of αLβ2 in CD4+ and CD8+ T cells while LFA878 and XVA143 did not affect or only mildly reduced αLβ2 surface expression, respectively. Moreover, efalizumab, in contrast to the small molecule inhibitors, disturbed the fine-tuned internalization/recycling of engaged TCR/CD3, concomitantly decreasing ZAP70 expression levels. In conclusion, different modes of αLβ2 inhibition are associated with fundamentally different biologic effect profiles. The differential established here is expected to provide important translational guidance as novel αLβ2 inhibitors will be advanced from bench to bedside

Ligand recognition by E- and P-selectin : chemoenzymatic synthesis and inhibitory activity of bivalent sialyl Lewis x derivatives and sialyl Lewis x carboxylic acids

Described is the preparation of five sLex dimers and five sLex carboxylic acids by coupling chemoenzymatically synthesized amino-substituted sialyl Lewis x (sLex) derivative 4 to homobifunctional cross-linkers 20-24 of varying chain length. 20-24 were obtained by alkylating low-molecular-weight oligoethylene glycols with tert-butyl bromoacetate and subsequent transformation of the di-tert-butyl esters into disuccinimide esters. The products were assayed for inhibition against binding of a sLea-polymer to immobilized E- and P-selectin. In the E-selectin assay all dimers had lower IC50 values than the sLex monomer. The results show that comparable binding enhancements can be obtained with linkers of completely different length and rigidity. In the P-selectin assay four of the five sLex carboxylic acids displayed significantly improved inhibitory potency. The lowest IC50 value was observed for the compound with the shortest spacer between the sLex moiety and the additional carboxylate, being ca. 20-40 times more potent than unmodified sLex. These findings should be of importance for the design of new multivalent forms of sLex as well as sLex mimetics as high-affinity selectin ligands