Markus Streiff

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.

CLONING OF A HUMAN UDP-GALACTOSE :2-ACETAMIDO-2-DEOXY-D-GLUCOSE 3BETA -GALACTOSYLTRANSFERASE CATALYZING THE FORMATION OF TYPE 1 CHAINS

Biochemical evidence suggests that the galactosyltransferase activity synthesizing type 1 carbohydrate chains is separate from the well characterized enzyme that is responsible for the synthesis of type 2 chains. This was recently confirmed by the cloning, from melanoma cells, of an enzyme capable of synthesizing type 1 chains, which was shown to have no homology to other galactosyltransferases. We report here the molecular cloning and functional expression of a second human β3-galactosyltransferase distinct from the melanoma enzyme. The new β3-galactosyltransferase has homology to the melanoma enzyme in the putative catalytic domain, but has longer cytoplasmic and stem regions and a carboxyl-terminal extension. Northern blots showed that the new gene is present primarily in brain and heart. When transfected into mammalian cells, this gene directs the synthesis of type 1 chains as determined by a monoclonal antibody specific for sialyl Lewisa. A soluble version of the cloned enzyme was expressed in insect cells and purified. The soluble enzyme readily catalyzes the transfer of galactose to GlcNAc to form Gal(β1–3)GlcNAc. It also has a minor but distinct transfer activity toward Gal, LacNAc, and lactose, but is inactive toward GalNAc.

A Potent and Selective S1P1 Antagonist with Efficacy in Experimental Autoimmune Encephalomyelitis

Lymphocyte trafficking is critically regulated by the Sphingosine 1-phosphate receptor-1 (S1P(1)), a G protein-coupled receptor that has been highlighted as a promising therapeutic target in autoimmunity. Fingolimod (FTY720, Gilenya) is a S1P(1) receptor agonist that has recently been approved for the treatment of multiple sclerosis (MS). Here, we report the discovery of NIBR-0213, a potent and selective S1P(1) antagonist that induces long-lasting reduction of peripheral blood lymphocyte counts after oral dosing. NIBR-0213 showed comparable therapeutic efficacy to fingolimod in experimental autoimmune encephalomyelitis (EAE), a model of human MS. These data provide convincing evidence that S1P(1) antagonists are effective in EAE. In addition, the profile of NIBR-0213 makes it an attractive candidate to further study the consequences of S1P(1) receptor antagonism and to differentiate the effects from those of S1P(1) agonists.

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

α(1‐3)‐Galactosyltransferase Inhibition Based on a New Type of Disubstrate Analogue

How do retaining glycosyltransferases function? To answer this question, UDP-Gal and galactose were covalently linked to form disubstrate analogues 1, of which surprisingly 1β and not 1α inhibited α(1-3)-galactosyltransferases very well. An understanding of this inhibition is a key to the pharmacological prevention of hyperacute rejection in pig to primate xenotransplantation.

Structural basis of guanine nucleotide exchange mediated by the T-cell essential Vav1.

The guanine nucleotide exchange factor (GEF) Vav1 plays an important role in T-cell activation and tumorigenesis. In the GEF superfamily, Vav1 has the ability to interact with multiple families of Rho GTPases. The structure of the Vav1 DH-PH-CRD/Rac1 complex to 2.6 A resolution reveals a unique intramolecular network of contacts between the Vav1 cysteine-rich domain (CRD) and the C-terminal helix of the Vav1 Dbl homology (DH) domain. These unique interactions stabilize the Vav1 DH domain for its intimate association with the Switch II region of Rac1 that is critical for the displacement of the guanine nucleotide. Small angle x-ray scattering (SAXS) studies support this domain arrangement for the complex in solution. Further, mutational analyses confirms that the atypical CRD is critical for maintaining both optimal guanine nucleotide exchange activity and broader specificity of Vav family GEFs. Taken together, the data outline the detailed nature of Vav1s ability to contact a range of Rho GTPases using a novel protein-protein interaction network.

BZM055, an iodinated radiotracer candidate for PET and SPECT imaging of myelin and FTY720 brain distribution.

FTY720 (fingolimod, Gilenya®) is a sphingosine 1‐phosphate (S1P) receptor modulator that shows significant therapeutic efficacy after oral administration to patients of multiple sclerosis. Because FTY720 does not contain any atom whose PET or SPECT radioisotope would have a half‐life compatible with its pharmacokinetic properties, it cannot be used directly for imaging. Instead, we propose BZM055 as a surrogate tracer to study its pharmacokinetics and organ distribution in patients and, given that FTY720 accumulates in myelin sheaths, for myelin imaging. BZM055 (2 a, 2‐iodo‐FTY720) can be easily radiolabeled with 123I (for SPECT) or 124I (for PET). Not only does it closely mimic the pharmacokinetics and organ distribution of FTY720, but also its affinity, selectivity for S1P receptors, phosphorylation kinetics, and overall physicochemical properties. [123I]BZM055 is currently under development for clinical imaging.

Synthesis of Gal determinant epitopes, their glycomimetic variants, and trimeric clusters—relevance to tumor associated antigens and to discordant xenografts

Abstract The synthesis of Galα1→3Gal-quinic acid pseudo-trisaccharides and their elaboration into trimeric clusters is described.

Synthetic potential of cloned fucosyl-transferase III and VI

Abstract Two cloned fucosyl-transferases, fuc-t III and fuc-t VI, are probed with non-natural donor-sugars, in which the natural fucose moiety is replaced by mono-saccharides modified in their 2- or 6-positions. Despite their close sequence homology, the investigated transferases exhibit significant differences in their substrate recognition.