Karl Welzenbach

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.

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.

Intracellular Phospho‐Flow cytometry reveals novel insights into TCR proximal signaling events. A comparison with Western blot

Phospho‐site specific antibodies become increasingly available, enabling the study of signaling events by Western blotting (WB) or intracellular flow cytometry (Phospho‐Flow). Here we compared data generated by WB or Phospho‐Flow regarding the kinetics and degree of phosphorylation of membrane proximal TCR signaling molecules. Phosphorylation events in Jurkat T cells were triggered by anti‐CD3 stimulation (OKT3) or by oxidative stress (H2O2) and were analyzed by Phospho‐Flow or WB. Both techniques showed that OKT3‐ or H2O2‐induced, transient phosphorylation of ZAP70 or LAT was dependent on functional Lck. Phospho‐Flow data revealed differences in the kinetics and the degree of H2O2‐ or OKT3‐mediated protein phosphorylation compared with WB data. In addition, using Phospho‐Flow we discovered that H2O2‐induced phosphorylation of TCR signaling proteins was inhibited by small molecular weight kinase inhibitors far more potently than OKT3‐triggered protein phosphorylation, despite a superior induction of phosphorylation by H2O2.This finding was confirmed by WB. Interestingly, we identified by Phospho‐Flow that, in P116 Jurkat cells lacking ZAP70 protein expression, H2O2 potently triggered the phosphorylation of ZAP70 residues Y493 and Y292 but not Y319. The phosphorylation of these ZAP70 tyrosine residues cells was blocked by an Lck inhibitor, suggesting the existence of an Lck‐coupled truncated ZAP70 protein or a novel isoform of ZAP70 in P116 cells. Phospho‐Flow is a largely quantitative technology with excellent throughput, highly suited in studying the function or inhibition of TCR signaling pathways and allowing the detection of novel pathway insights. It can serve as a good complement to Western blot analysis.

CD47 fusion protein targets CD172a+ cells in Crohn’s disease and dampens the production of IL-1β and TNF

CD172a+ cells producing IL-1β and TNF are increased in inflamed tissues in Crohn’s disease and can be targeted by CD47 fusion protein.

Targeting SIRP-α protects from type 2-driven allergic airway inflammation.

The interplay between innate and adaptive immune responses is essential for the establishment of allergic diseases. CD47 and its receptor, signal regulatory protein α (SIRP‐α), govern innate cell trafficking. We previously reported that administration of CD47+/+ but not CD47−/− SIRP‐α+ BM‐derived DC (BMDC) induced airway inflammation and Th2 responses in otherwise resistant CD47‐deficient mice. We show here that early administration of a CD47‐Fc fusion molecule suppressed the accumulation of SIRP‐α+ DC in mediastinal LN, the development of systemic and local Th2 responses as well as airway inflammation in sensitized and challenged BALB/c mice. Mechanistic studies highlighted that SIRP‐α ligation by CD47‐Fc on BMDC did not impair Ag uptake, Ag presentation and Ag‐specific DO11.10 Tg Th2 priming and effector function in vitro, whereas in vivo administration of CD47‐Fc or CD47‐Fc‐pretreated BMDC inhibited Tg T‐cell proliferation, pinpointing that altered DC trafficking accounts for defective Th priming. We conclude that the CD47/SIRP‐α axis may be harnessed in vivo to suppress airway SIRP‐α+ DC homing to mediastinal LN, Th2 responses and allergic airway inflammation.

Phosphorylation by Sphingosine Kinase 2 is Essential for in vivo Potency of FTY720 Analogues

active principle (S)-FTY720-phosphate which binds to four of the five known sphingosine-1-phosphate receptors (S1P1–5). It has been convincingly demonstrated that the immunomodulatory effect is primarily mediated by S1P1. Sphingosine kinases (SPHKs) have been identified as the enzymes that catalyze the mono-O-phosphorylation of the amino alcohol head group of FTY720. We and others have found that SPHK2 is much more efficient than SPHK1 in phosphorylation of FTY720 (Table 1). Indeed, in vivo experiments have demonstrated that SPHK2 is essential for the lymphopenia induced by FTY720. Although apoptosis induced by FTY720 at higher concentrations has been shown to be receptor independent, a recent publication suggests that SPHK2 is also essential for this pathway. The finding that only one of the prochiral hydroxy groups in FTY720 is phosphorylated in vivo suggests that the enzymatic phosphorylation reaction can be tuned by offering various stereochemical features in the substrate. For the chiral analogues 1a and 1b (Figure 2) it has already been demonstrated that induction of lymphopenia correlates with SPHK phosphorylation efficiency as well as with the potency as agonists of the corresponding phosphates toward S1P1. For potent S1P1 agonists that do not induce lymphopenia, a difference in SPHK activity has already been suspected as a possible reason for their lack of in vivo efficacy. Herein we report the synthesis and systematic investigation of FTY720-like amino alcohols featuring two stereocenters in the head group in order to establish the relative importance of SPHK phosphorylation compared with potency toward S1P1 for lymphopenia induction in vivo. Primary alcohols 1a and 1b were prepared as reported previously by using a Schcllkopf auxiliary-based approach. For the synthesis of these two compounds as well as the secondary alcohols 2a and 3a, the N-Boc-protected amino alcohol 4 served as the precursor (Scheme 1). Using the protocol described by Ley et al. , this alcohol was oxidized to the aldehyde 5. The addition of methyl magnesium bromide resulted [a] Dr. K. Hcgenauer, Dr. A. Billich, Dr. P. Nussbaumer Novartis Institutes for Biomedical Research Brunner Strasse 59, 1235 Wien (Austria) Fax: (+43)1-80166-354 E-mail : klemens.hoegenauer@novartis.com [b] C. Pally, Dr. M. Streiff, Dr. T. Wagner, Dr. K. Welzenbach Novartis Institutes for Biomedical Research 4002 Basel (Switzerland) Supporting information for this article is available on the WWW under http://www.chemmedchem.org or from the author: all experimental protocols and analytical data. Figure 1. FTY720 is phosphorylated by SPHKs. Table 1. In vivo and in vitro data.

Assembly of binding loops on aromatic templates as VCAM-1 mimetics

The design and synthesis of cyclic mimetics of VCAM‐1 protein that reproduce the integrin‐binding domain are presented. The unprotected peptide precursor 37–43, Thr‐Gln‐Ile‐Asp‐Ser‐Pro‐Leu, was grafted onto functional templates of type naphthalene, biphenyl and benzyl through the chemoselective formation of C‐ and N‐terminal oximes resulting in a mixture of four isomeric forms due to syn–anti isomerism of the oxime bonds. Some isomers could be monitored by HPLC and identified by NMR. The molecule containing a naphthalene‐derived template was found to inhibit the VCAM‐1/VLA‐4 interaction more efficiently than previously reported for sulfur‐bridged cyclic peptides containing similar sequences. The finding confirms the importance of incorporating conformational constraints between the terminal ends of the peptide loop 37–43 in the design of synthetic inhibitors of the VCAM‐1/integrin interaction. Copyright

ADAM17 is the main sheddase for the generation of human triggering receptor expressed in myeloid cells (hTREM2) ectodomain and cleaves TREM2 after Histidine 157

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Here, the question was addressed at which position sheddases cleave TREM2 and what are the proteases involved in this process. Using both pharmacological and genetic approaches we report that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNFα converting enzyme) while ADAM10 plays a minor role. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimers disease. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered.