Gebhard Thoma

Targeting CLA/E‐selectin interactions prevents CCR4‐mediated recruitment of human Th2 memory cells to human skin in vivo

Naive Th cells, bearing receptors for cutaneous antigens, become activated in skin‐draining lymph nodes and express cutaneous lymphocyte antigen (CLA), which confers to these cells the capacity to migrate into the skin to exert their normal effector functions. In the case of atopic dermatitis (AD), allergen‐specific Th2 cells generate exacerbated responses and induce skin inflammation. In such a situation, interfering with the specific mechanism of skin homing would provide a therapeutic benefit. Here we report that CLA+ Th2 memory cells, derived from skin lesions of AD patients, selectively migrate to human skin grafts transplanted onto SCID mice in response to CCR4 but not CCR3, CCR8 or CXCR3 ligands. Skin homing of human CCR4+ Th2 memory cells was Pertussis toxin sensitive and restricted to the CLA+ subset. Furthermore, treatment of these mice with anti‐E‐selectin monoclonal antibody was sufficient to prevent CCL22‐mediated Th2 cell migration to human skin, which both, validates the model and highlights the importance of CLA/E‐selectin interactions in the homing process of Th2 cells to the skin. Using this mechanistic model we demonstrate that skin homing of human Th2 memory cells can be efficiently suppressed using a low molecular weight E‐selectin antagonist, which is of clinical relevance for the treatment of inflammatory skin diseases, including AD.

Removal of anti-Galα1,3Gal xenoantibodies with an injectable polymer

Preformed and elicited Abs against the Galalpha1,3Gal terminating carbohydrate chains (alphaGal Abs) are the primary cause of hyperacute and acute vascular xenograft rejection in pig-to-primate transplantation. alphaGal Abs are produced by long-lived Ab-producing cells that are not susceptible to pharmacological immunosuppression. We reasoned that antigen-specific elimination of alphaGal Abs might be achieved in vivo by systemic administration of nonimmunogenic polyvalent alphaGal structures with high avidity for alphaGal Abs. We devised GAS914, a soluble trisaccharide-polylysine conjugate of approximately 500 kDa that effectively competes for alphaGal binding by alphaGal IgM (IC(50), 43 nM) and IgG (IC(50), 28 nM) in vitro. Injections of GAS914 in cynomolgus monkeys, at the dose of 1 mg/kg, resulted in the immediate decrease of more than 90% of circulating alphaGal Abs and serum anti-pig cytotoxicity. In baboons, repeated injections of GAS914 effectively reduced both circulating alphaGal Abs and cytotoxicity over several months. Studies with [(14)C]GAS914 in rhesus monkeys and Gal(-/-) mice indicate that GAS914 binds to circulating alphaGal Abs and that the complex is quickly metabolized by the liver and excreted by the kidney. Remarkably, posttreatment alphaGal Ab titers never exceeded pretreatment levels and no sensitization to either alphaGal or the polylysine backbone has been observed. Furthermore there was no apparent acute or chronic toxicity associated with GAS914 treatment in primates. We conclude that GAS914 may be used therapeutically for the specific removal of alphaGal Abs.

Orally Bioavailable Isothioureas Block Function of the Chemokine Receptor CXCR4 In Vitro and In Vivo

The interaction of the chemokine receptor CXCR4 with its ligand CXCL12 is involved in many biological processes such as hematopoesis, migration of immune cells, as well as in cancer metastasis. CXCR4 also mediates the infection of T-cells with X4-tropic HIV functioning as a coreceptor for the viral envelope protein gp120. Here, we describe highly potent, selective CXCR4 inhibitors that block CXCR4/CXCL12 interactions in vitro and in vivo as well as the infection of target cells by X4-tropic HIV.

Identification of a Potent Janus Kinase 3 Inhibitor with High Selectivity within the Janus Kinase Family

We describe a synthetic approach toward the rapid modification of phenyl-indolyl maleimides and the discovery of potent Jak3 inhibitor 1 with high selectivity within the Jak kinase family. We provide a rationale for this unprecedented selectivity based on the X-ray crystal structure of an analogue of 1 bound to the ATP-binding site of Jak3. While equally potent compared to the Pfizer pan Jak inhibitor CP-690,550 (2) in an enzymatic Jak3 assay, compound 1 was found to be 20-fold less potent in cellular assays measuring cytokine-triggered signaling through cytokine receptors containing the common γ chain (γC). Contrary to compound 1, compound 2 inhibited Jak1 in addition to Jak3. Permeability and cellular concentrations of compounds 1 and 2 were similar. As Jak3 always cooperates with Jak1 for signaling, we speculate that specific inhibition of Jak3 is not sufficient to efficiently block γC cytokine signal transduction required for strong immunosuppression.

Targeting the CXCR4–CXCL12 Axis Mobilizes Autologous Hematopoietic Stem Cells and Prolongs Islet Allograft Survival via Programmed Death Ligand 1

Antagonism of CXCR4 disrupts the interaction between the CXCR4 receptor on hematopoietic stem cells (HSCs) and the CXCL12 expressed by stromal cells in the bone marrow, which subsequently results in the shedding of HSCs to the periphery. Because of their profound immunomodulatory effects, HSCs have emerged as a promising therapeutic strategy for autoimmune disorders. We sought to investigate the immunomodulatory role of mobilized autologous HSCs, via target of the CXCR4-CXL12 axis, to promote engraftment of islet cell transplantation. Islets from BALB/c mice were transplanted beneath the kidney capsule of hyperglycemic C57BL/6 mice, and treatment of recipients with CXCR4 antagonist resulted in mobilization of HSCs and in prolongation of islet graft survival. Addition of rapamycin to anti-CXCR4 therapy further promoted HSC mobilization and islet allograft survival, inducing a robust and transferable host hyporesponsiveness, while administration of an ACK2 (anti-CD117) mAb halted CXCR4 antagonist-mediated HSC release and restored allograft rejection. Mobilized HSCs were shown to express high levels of the negative costimulatory molecule programmed death ligand 1 (PD-L1), and HSCs extracted from wild-type mice, but not from PD-L1 knockout mice, suppressed the in vitro alloimmune response. Moreover, HSC mobilization in PD-L1 knockout mice failed to prolong islet allograft survival. Targeting the CXCR4–CXCL12 axis thus mobilizes autologous HSCs and promotes long-term survival of islet allografts via a PD-L1–mediated mechanism.

Novel glycodendrimers self-assemble to nanoparticles which function as polyvalent ligands in vitro and in vivo

The recognition of oligosaccharides by proteins represents the basis of many biologically important events.[1] Individual protein±carbohydrate interactions are generally weak (KD1⁄4 10 3±10 4m 1).[2] To overcome this, such processes often involve polyvalent binding, which is characterized by the simultaneous contact of multiple ligands (oligosaccharides) on one biological entity to multiple receptors (proteins) on another.[3] Polyvalent carbohydrate±protein interactions occur frequently in recognition events on cellular membranes. Collectively, they can be much stronger than corresponding monovalent interactions rendering it difficult to control them with individual small molecules.[4] Therefore, complex macromolecules have been used as polyvalent antagonists, however, both characterization and preparation of these nonuniform entities is demanding.[4] Here we present an alternative concept for the polyvalent presentation of ligands based on the supramolecular chemistry[5] of small molecules that fulfil single-molecule entity criteria (Figure 1). Novel dendrons capped with carbohydrate ligands (glycodendrimers[6]) were found to self-assemble to noncovalent nanoparticles which function as polyvalent ligands. We demonstrate that these particles–not the individual molecules–efficiently inhibit polyvalent interactions, such as IgM binding (IgM1⁄4 immunoglobulin), to the aGal-epitope[7] (a-d-Gal-(1!3)-b-d-Gal(1!4)-d-GlcNAc), both in vitro and in vivo. As self-assembly is dynamic, optimization of size and shape of the polyvalent ligand could occur utilizing the receptor as a template. Dendrimer cores were prepared by a convergent TMoutsidein∫ approach[8] based on a single building block 1a which was obtained frommethyl 3,5-diaminobenzoate and 4-(tert-butoxycarbonylaminomethyl)benzoic acid (Scheme 1a). Selective deprotection furnished 1b and 1c (first-generation dendrimer core, two end-groups). A one-pot procedure comprising coupling of 1c (1 equiv) and 1b (0.5 equiv) followed by methyl ester cleavage gave 2c (second-generation dendrimer core, four end groups).[9] The third-generation dendrimer 3c (eight end groups) was obtained from 2c (1 equiv) and 1b (0.5 equiv).[9] Applying the same procedure repetitively gave dendrimers with up to 64 end groups (4c, fourth generation, 16 end groups; 5c, fifth generation, 32 end groups; 6c, sixth generation, 64 end groups). Dendrimers 1c±6c were deprotected (!1d±6d) and transformed into their chloroacetamide derivatives (1e±6e) to allow subsequent introduction of thiolated oligosaccharides such as aGal-SH[10] and Lac-SH (Figure 2b) furnishing water-soluble glycodendrimers 1 f±6 f and 3g which were purified by ultrafiltration. Compound 7, which is similar to 2 f but contains butylene chains instead of the disubstituted aromatic rings, was also prepared (Scheme 1a). The integrity of all compounds was established by 1H NMR spectroscopy. Accordingly, the firstto third-generation dendrimers exist as single molecules (purity > 95%). The fourthto sixth-generation dendrimers possibly contain minute quantities of smaller fragments. The 500 MHz 1H NMR spectra of compound 3 f in [D6]DMSO demonstrates the remarkable purity of these compounds (Figure 2). The first indication that our glycodendrimers were aggregating in water came from 1H NMR spectroscopy of 2 f in D2O. At ambient temperature, we observed very broad signals which sharpened at elevated temperatures. The aggregation was quantified using multiangle light scattering (MALS; Table 1). The first-generation dendrimer 1 f forms small aggregates (50 kDa) whereas 2 f forms large particles of 7000 kDa (more than 1500 individual molecules per particle). Interestingly, the particle weight obtained for 3 f±6 f drops (2200 to 200 kDa) with increasing mass of the individual molecule. The root-mean-square radii of the particles formed by 2 f±6 f showed the same trend (for 2 f, 3 f, and 4 f 49, 34, and 12 nm, respectively; for 5 f and 6 f the radii were below the detection limit of 10 nm). Core-modified second-generation glycodendrimer 7 (4 iaGal), which is of comparable size and lipophilicity as 2 f but contains butylene chains instead of the disubstituted aromatic rings, does not form aggregates. The third-generation compounds 3g (8 iLac; 1900 kDa) and 3 f ZUSCHRIFTEN

Synthesis and biological evaluation of a sialyl Lewis X mimic with significantly improved E-selectin inhibition

The synthesis of the highly potent E-selectin inhibitor 5 is described. Sialyl Lewis X mimic 5 was rationally designed by combining two previously disclosed beneficial sLe(x) modifications in a single molecule. The compound was found to be 30-fold more potent than sLe(x) in a static, cell-free equilibrium assay. Furthermore, compound 5 was highly active (IC50 = 10 microM) in a dynamic non-equilibrium assay in which sLe(x) did not inhibit neutrophil rolling at up to 1000 microM.

Selective inhibitors of the Janus Kinase Jak3 – are they effective?

Jak3, together with Jak1, is involved in signal transduction initiated by cytokines signaling through the common gamma chain which are important in immune homeostasis and immune pathologies. Based on genetic evidence Jak3 has been considered to be an attractive target for immunosuppression. The Jak inhibitor tofacitinib (CP-690,550) which is an approved drug for rheumatoid arthritis was originally introduced as a selective Jak3 inhibitor, however, it also inhibits Jak1 and Jak2. The search for new selective Jak3 inhibitors has yielded several compounds whose profiles will be reviewed here. Implications on Jak3 as a therapeutic target are also discussed.

Polymers bearing sLex-mimetics are superior inhibitors of E-selectin-dependent leukocyte rolling in vivo

Selectins mediate leukocyte rolling and may represent good anti‐inflammatory drug targets. Detailed knowledge regarding the structure of selectin ligands has permitted development of selectin antagonists with varying specificities and activity. Efficacy of monovalent selectin antagonists may be increased by presenting them on a polymer backbone. We have synthesized a range of multivalent selectin antagonists and characterized their activity by using intravital microscopy of the mouse cremaster muscle. The monovalent inhibitor CGP77175A inhibited E‐selectin‐dependent leukocyte rolling at a dose of 3 mg/kg. Multivalent presentation of CGP77175A on a modified polylysine backbone (degree of polymerization = 1200; 50% of the polylysines carry the inhibitor) greatly enhanced in vivo activity giving an inhibitor that produced an equivalent effect at 0.1 mg/kg. The polylysine conjugate was also longer acting than the monovalent antagonist. In spite of greatly enhanced activity against E‐selectin compared with monovalent inhibitor, the multivalent inhibitor had no measurable effect on P‐ or L‐selectin‐dependent leukocyte rolling.

Potent E-Selectin Antagonists

In the search for drugs that could control excessive leukocyte extravasation, we now report on modifications of the already known potent E-selectin antagonist 3 containing a cyclohexyllactic acid residue and a glucal-derived building block. Thus, we describe the synthesis and biological evaluation of a series of derivatives 6 with modified glucal-derived moieties (CH2NR1R2 instead of CH2OH in 3) to explore a hypothetical potential complementary interaction with E-selectin. However, similar activity profiles of most derivatives 6 and compound 3 do not support such an interaction, but rather indicate topological-structure changes of 6 (and 3) in the orientation of the neighboring fucose and galactose due to intramolecular steric interactions. The most potent E-selectin antagonist 6v showed >50-fold improved E-selectin inhibition compared to the weak selectin ligand sialyl Lewisx (sLex, 1; IC50=1000 – 1500 μM), but only a 2-fold improvement compared to 3. Compound 6x was tested in vivo in a murine model of acute inflammation and found to be as potent as 3 (ED50=15 mg/kg).