Carmen Barske

Inhibitory Activity of Myelin-Associated Glycoprotein on Sensory Neurons Is Largely Independent of NgR1 and NgR2 and Resides within Ig-Like Domains 4 and 5

Myelin-associated glycoprotein (MAG) is a sialic acid binding Ig-like lectin (Siglec) which has been characterized as potent myelin-derived inhibitor of neurite outgrowth. Two members of the Nogo-receptor (NgR) family, NgR1 and NgR2, have been identified as neuronal binding proteins of MAG. In addition, gangliosides have been proposed to bind to and confer the inhibitory activity of MAG on neurons. In this study, we investigated the individual contribution of NgRs and gangliosides to MAG-mediated inhibition of sensory neurons derived from dorsal root ganglia (DRG) of ngr1, ngr2 or ngr1/ngr2 deletion mutants. We found no disinhibition of neurite growth in the absence of either NgR1 or NgR2. Sensory neurons deficient for both NgR proteins displayed only a moderate reduction of MAG-mediated inhibition of neurite growth. If treated with Vibrio cholerae neuraminidase (VCN), inhibition by MAG is further attenuated but still not annulled. Thus, disrupting all known protein and ganglioside receptors for MAG in sensory neurons does not fully abolish its inhibitory activity pointing to the existence of as yet unidentified receptors for MAG. Moreover, by employing a variety of protein mutants, we identified the Ig-like domains 4 or 5 of MAG as necessary and sufficient for growth arrest, whereas abolishing MAGs ability to bind to sialic acid did not interfere with its inhibitory activity. These findings provide new insights into the inhibitory function of MAG and suggest similarities but also major differences in MAG inhibition between sensory and central nervous system (CNS) neurons.

Design and Synthesis of Selective and Potent Orally Active S1P5 Agonists

The immunomodulatory drug fingolimod (FTY720, 2-amino-2[2-(4-octylphenyl)ethyl]propane-1,3-diol), derived from a fungal metabolite (ISP-1, myriocin), is phosphorylated in vivo by sphingosine kinases to produce (R)-FTY720-phosphate (FTY720-P). 2] FTY720-P activates sphingosine-1-phosphate (S1P) receptors S1P1, S1P3, S1P4, and S1P5 at low nanomolar concentrations and is inactive toward the S1P2 receptor. The FTY720-P-mediated activation of the S1P1 receptor on lymphocytes induces receptor internalization, which attenuates T-cell response to S1P gradients, preventing their egress from secondary lymphoid tissues. In addition to playing a role in the immune system, all S1P receptors except S1P4 are also found differentially expressed in the central nervous system and on various tumor cell types. 6] Although the precise regulation of these receptors by locally released S1P remains unclear, S1P receptors are thought to play a role in such events as astrocyte migration, oligodendrocyte differentiation, and cell survival and neurogenesis. To assess the relevance of individual S1P receptor subtypes for the activity of FTY720-P, selective agonists are required. Because S1P5 receptors are expressed on oligodendrocytes, and S1P5 receptors are thought to play a role in oligodendrocyte differentiation and survival, we focused on the development of S1P5 agonists. By using a highthroughput screening calcium mobilization assay with GPCR priming and FLIPR technology, we discovered benzamide 1, which has good in vitro potency toward the S1P5 receptor (EC50=270 nm), but has modest selectivity against S1P1 (EC50=3140 nm) and S1P4 (EC50=100 nm). Herein we report our studies of various benzamide modifications carried out to improve the selectivity, bioactivity, pharmacokinetic properties, and ancillary profile of 1, ultimately resulting in the discovery of potent and very selective S1P5 agonists. To guide the optimization process, homology models of all S1P receptors were built from a crystal structure of bovine rhodopsin (PDB ID: 1F88). Docking experiments of 1 into these models revealed a possible location of the binding site, some essential features of the interactions, and indicated potential regions for gaining selectivity and improving potency. In these complexes (Figure 1), 1 adopts a twisted conformation with the aniline ring, ~708 out of the benzamide plane and stabilized by a hydrogen bond between the aniline NH group and the amide carbonyl. In the S1P5 receptor complex, the amide group forms a hydrogen bond with OG1-Thr120. The benzamide phenyl ring lies in a large hydrophobic pocket surrounded by Phe196, Phe201, Phe268, Leu119, Trp264, Leu267, and Leu271. The aniline ring undergoes a T-shaped interaction with Phe116 and hydrophobic contacts with Leu271 and Leu292. The ortho-methyl substituents fill a small pocket formed by Tyr89, Val115, and Leu292 on one side, and sit at the face of Phe196 on the other side. Inspection of sequence alignments (Figure 2) revealed two positions, one in transmembrane (TM) helix TM3 (115, S1P5 sequence) and one in TM5 (192), where S1P5 has smaller residues lining the binding site, thus creating putative pockets. We hypothesized that filling these pockets with atoms from our ligands should lead to high selectivity for the S1P5 receptor. Position 2 on the benzamide core, which was closest to the hypothesized pocket around Val115, was therefore extensively modified. Syntheses of derivative 1A–L (Scheme 1) began with 3-fluorobromobenzene 2, which was converted into acid 3 by reaction with lithium diisopropylamide (LDA) and carbon dioxide. Nucleophilic substitution of the fluorine atom with trimethylaniline at 78 8C yielded 4. This intermediate was then used in various ways. Copper-catalyzed nucleophilic substitution of the bromine atom with various alcohols yielded ethers 5D–N, which were amidated with ammonia using chlorodimethoxytriazine for activation to yield 1D–J. Palladium-catalyzed substitution of the bromine atom in acid 4 with various alkylstannanes yielded 6A–C, which were amidated as described above to yield 1A–C. Alternatively, palladium-catalyzed substitution of the bromine atom with tributyl-(1-ethoxyvinyl)stannane yielded 9, which was cyclized to 1L by reaction with hydrazine. Acid 4 was also amidated with allylamine, using chlorodimethoxytriazine for activation, to yield allylamide 10. Palladiumcatalyzed cyclization of this intermediate led to 1K. All compounds were assayed for S1P5 activation in GTPgS assays, which gave more reliable structure–activity results than the FLIPR assays, at concentrations up to 10 mm. EC50 values were determined for all compounds (Table 1). Disrupting the intramolecular hydrogen bond by introducing small alkyl substituents at position 2 (compounds 1A–C), led to a [a] Dr. H. Mattes, Prof. Dr. K. K. Dev, Dr. R. Bouhelal, Dr. C. Barske, Dr. F. Gasparini, Dr. D. Guerini, Dr. A. K. Mir, Dr. D. Orain, M. Osinde, A. Picard, C. Dubois, E. Tasdelen, S. Haessig Novartis Institute for Biomedical Research WKL-122 4002 Basel (Switzerland) Fax: (+41)61 696 2455 E-mail : henri.mattes@novartis.com [b] Prof. Dr. K. K. Dev Molecular Neuropharmacology, Department of Physiology Trinity College Institute of Neuroscience (TCIN) Medical School Trinity College Dublin, Dublin 2 (Ireland) Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201000253.

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.

Transgenic expression of β1 antibody in brain neurons impairs age-dependent amyloid deposition in APP23 mice

Heterologous expression of the functional amyloid beta (Aβ) antibody β1 in the central nervous system was engineered to maximize antibody exposure in the brain and assess the effects on Aβ production and accumulation in these conditions. A single open reading frame encoding the heavy and light chains of β1 linked by the mouth and foot virus peptide 2A was expressed in brain neurons of transgenic mice. Two of the resulting BIN66 transgenic lines were crossed with APP23 mice, which develop severe central amyloidosis. Brain concentrations at steady-state 5 times greater than those found after peripheral β1 administration were obtained. Similar brain and plasma β1 concentrations indicated robust antibody efflux from the brain. In preplaque mice, β1 formed a complex with Aβ that caused a modest Aβ increase in brain and plasma. At 11 months of age, β1 expression reduced amyloid by 97% compared with age-matched APP23 mice. Interference of β1 with β-secretase cleavage of amyloid precursor protein was relatively small. Our data suggest that severely impaired amyloid formation was primarily mediated by a complex of β1 with soluble Aβ, which might have prevented Aβ aggregation or favored transport out of the brain.