Facile Modulation of Antibody Fucosylation with Small Molecule Fucostatin Inhibitors and Cocrystal Structure with GDP-Mannose 4,6-Dehydratase.

The efficacy of therapeutic antibodies that induce antibody-dependent cellular cytotoxicity can be improved by reduced fucosylation. Consequently, fucosylation is a critical product attribute of monoclonal antibodies produced as protein therapeutics. Small molecule fucosylation inhibitors have also shown promise as potential therapeutics in animal models of tumors, arthritis, and sickle cell disease. Potent small molecule metabolic inhibitors of cellular protein fucosylation, 6,6,6-trifluorofucose per-O-acetate and 6,6,6-trifluorofucose (fucostatin I), were identified that reduces the fucosylation of recombinantly expressed antibodies in cell culture in a concentration-dependent fashion enabling the controlled modulation of protein fucosylation levels. 6,6,6-Trifluorofucose binds at an allosteric site of GDP-mannose 4,6-dehydratase (GMD) as revealed for the first time by the X-ray cocrystal structure of a bound allosteric GMD inhibitor. 6,6,6-Trifluorofucose was found to be incorporated in place of fucose at low levels (<1%) in the glycans of recombinantly expressed antibodies. A fucose-1-phosphonate analog, fucostatin II, was designed that inhibits fucosylation with no incorporation into antibody glycans, allowing the production of afucosylated antibodies in which the incorporation of non-native sugar is completely absent-a key advantage in the production of therapeutic antibodies, especially biosimilar antibodies. Inhibitor structure-activity relationships, identification of cellular and inhibitor metabolites in inhibitor-treated cells, fucose competition studies, and the production of recombinant antibodies with varying levels of fucosylation are described.

Defucosylated anti-CCR4 monoclonal antibody exerts potent ADCC against primary ATLL cells mediated by autologous human immune cells in NOD/Shi-scid, IL-2R gamma(null) mice in vivo.

There is a lack of suitable small animal models to evaluate human Ab-dependent cellular cytotoxicity (ADCC) in vivo, because of the species incompatibility between humans and animals or due to nonspecific allogeneic immune reactions. To overcome these problems, we established a human tumor-bearing mouse model, using NOD/Shi-scid, IL-2Rgamma(null) (NOG) mice as recipients, in which autologous human immune cells are engrafted and mediate ADCC but in which endogenous murine cells are unable to mediate ADCC. In the present study, we used NOG mice bearing primary adult T cell leukemia/lymphoma (ATLL) cells and a therapeutic chimeric anti-CCR4 mAb, the Fc region of which is defucosylated to enhance ADCC. We report significant antitumor activity in vivo associated with robust ADCC mediated by autologous effector cells from the same patients. The present study is the first to report a mouse model in which a potent antitumor effect of the therapeutic mAb against primary tumor cells is mediated by autologous human immune cells. Human autologous ADCC in mice in vivo was confirmed by the depletion of human immune cells before ATLL PBMC inoculation. In addition, NOG mice bearing primary ATLL cells presented features identical with patients with ATLL. In conclusion, this approach makes it possible to model the human immune system active in Ab-based immunotherapy in vivo, and thus to perform more appropriate preclinical evaluations of novel therapeutic mAb. Furthermore, the potent ADCC mediated by defucosylated anti-CCR4 mAb, observed here in vivo in humanized mice, will be exploited in clinical trials in the near future.

Design and synthesis of 5a-carbaglycopyranosylamime glycosidase inhibitors.

5a-Carba-alpha-D-glucopyranosylamine, validamine, and analogous compounds valienamine and valiolamine, have proved to be important lead compounds for development of clinically useful medicines, including the very strong alpha-glucosidase inhibitor, voglibose, N-(1,3-dihydroxyprop-2-yl)valiolamine, now used widely as a clinically important antidiabetic agent. In this review, we describe recent advances in development of glycosidase inhibitors on the basis of the ground-state mimics of the postulated glycopyranosyl cation, considered to be formed during hydrolysis of glycopyranosides, and introduce a new type of highly potent alpha-fucosidase inhibitor, 5a-carba-alpha-L-fucopyranosylamine, alpha-fuco validamine. Interestingly, the corresponding beta-anomer, and in particular its D-enantiomer, has been shown to possess very strong cross-inhibitory activity toward beta-galactosidase and beta-glucosidase. Structure and inhibitory activity relationships concerning these alpha,beta-fuco derivatives, as well as parent alpha,beta-galacto validamines are discussed here with reference to our results.

Store-operated calcium channels trigger exocytosis of the sea urchin sperm acrosomal vesicle.

The acrosome reaction (AR) of sperm is a prerequisite for fusion with the egg. In sea urchins, the complete AR (CAR) consists of exocytosis of the acrosomal vesicle (AV) and polymerization of acrosomal actin to form the approximately 1 micro m long acrosomal process. The fucose sulfate polymer (FSP) of egg jelly stimulates Ca(2+) entry through two distinct Ca(2+) channels and induces the CAR. Here we report that the second channel is blocked by SKF96365 (SKF), an inhibitor of store-operated channels. SKF also blocks the thapsigargin (TG), trifluoperazine (TFP), and calmidizolium (CMZ) stimulated Ca(2+) entry into sperm. These data indicate that the second Ca(2+) channel is a store-operated channel (SOC) that may be regulated by calmodulin. The TG, TFP, and CMZ-induced intracellular Ca(2+) elevations are similar to those induced by FSP, but the sperm acrosomal process does not polymerize. An antibody to bindin, the major protein of the AV, showed that in a significant percentage of these drug-treated sperm, the AV had undergone exocytosis. When NH(4)Cl was added to increase intracellular pH, the TG-treated sperm polymerized actin to form the acrosomal process. We conclude that the second Ca(2+) channel of sea urchin sperm is a SOC that triggers AV exocytosis.

Inhibition of alpha(1-6)-linked fucose decreases inner retinal cells and increases photoreceptors in chicken retinal reaggregates.

We investigated the developmental role of alpha(1-6)-linked fucose, applying Aleuria aurantia lectin to a specific retinal regeneration system. Thereby, dissociated retinal cells of chicken embryos reaggregate, proliferate, and differentiate in vitro into histotypical spheres, so-called retinospheroids. Under the influence of A. aurantia lectin, processes of proliferation, differentiation and histogenesis of retinospheroids were disturbed. Extending these in vitro studies, we here show that A. aurantia lectin treatment decreases cells of the inner half retina and their processes into inner plexiform layer areas, as revealed by quantitative enzyme histochemistry for butyryl- and acetylcholinesterase, and immunohistochemistry using antibodies to acetylcholinesterase, Pax-6, calbindin-D, and F11. Concomitantly, the number of rod and red/green photoreceptors dramatically increases, using the antibodies rho4D2 and CERN901 (both specific for rods) and CERN906 (specific for red/green cones). These findings show that glycoproteins exhibiting fucose in alpha(1-6)-linkage are involved in processes determining retinal cell fate, strongly shifting the relative ratio of cells of the inner towards cells of the outer retina.

Supplemental myo-inositol prevents L-fucose-induced diabetic neuropathy.

Nerve myo-inositol depletion, which has been implicated in the pathogenesis of acute experimental diabetic neuropathy, can be reproduced in normal rats by feeding diets enriched in L-fucose, a competitive inhibitor of sodium-dependent myo-inositol transport. Previously, we reported that L-fucose feeding for 6 weeks reproduces the effect of experimental diabetes on nerve Na+-K+-ATPase activity and conduction velocity, which can be prevented by simultaneous dietary myo-inositol supplementation. To further validate this model of myo-inositol depletion, we examined the effects of long-term (24-week) L-fucose feeding and dietary myo-inositol supplementation on nerve Na+-K+-ATPase, nerve conduction velocity, and myelinated nerve fiber pathology. After 24 weeks of L-fucose enriched (10 or 20%) diets, nerve myo-inositol levels and Na+-K+-ATPase activity decreased significantly (P < 0.05) and were associated with a 25-30% reduction in nerve conduction velocity, all of which were completely prevented by 1% dietary myo-inositol. Twenty percent L-fucose diet resulted in significant axonal atrophy, paranodal swelling (P < 0.001), and paranodal demyelination (P < 0.005), without increasing Wallerian degeneration or nerve fiber loss, a pattern qualitatively similar to that seen in early murine diabetic neuropathy. Dietary myo-inositol supplementation prevented these structural changes and increased nodal remyelination, supporting a role of myo-inositol depletion in the genesis of early diabetic neuropathy. The L-fucose model system may therefore serve as an experimental tool to elucidate the pathophysiological role of isolated myo-inositol depletion and its consequences in the multifactorial pathogenesis of diabetic neuropathy.

2-Deoxy-D-galactose, a substrate for the galactose-transport system of Escherichia coli.

The following observations showed that 2-deoxy-D-galactose is a useful tool for the isolation and elucidation of the activity of one system for galactose uptake into Escherichia coli. 1. 2-Deoxygalactose, which is not a substrate for growth of E. coli, was transported into strains of the organism induced for galactose transport. 2. By using appropriate mutants it was shown that 2-deoxygalactose is a much better substrate for the galactose-transport system than for the methyl galactoside-transport system. This was confirmed by the results of mutual inhibition studies with substrates of each transport system. 3. The glucose-, arabinose- or lactose-transport systems did not effect significant transport of 2-deoxygalactose. 4. Like other substrates of the galactose-transport system, 2-deoxygalactose promoted effective proton uptake into de-energized suspensions of appropriate E. coli strains. 5. The S183 series of E. coli mutants were found to contain a constitutive galactose-transport system, if 2-deoxygalactose transport is used as one criterion for such activity.