The identification of novel acid isostere based inhibitors of the VPS10P family sorting receptor Sortilin.

Using fragment based and structure based drug discovery strategies a series of novel Sortilin inhibitors has been identified. The inhibitors are based on the N-substituted 1,2,3-triazol-4-one/ol heterocyclic template. X-ray crystallography shows that the 1,2,3-triazol-4-one/ol acts as a carboxylic acid isostere, making a bi-dentate interaction with an arginine residue of Sortilin, an interaction which has not been previously characterised for this heterocycle.

Identification of the first small-molecule ligand of the neuronal receptor sortilin and structure determination of the receptor-ligand complex.

Sortilin is a type I membrane glycoprotein belonging to the vacuolar protein sorting 10 protein (Vps10p) family of sorting receptors and is most abundantly expressed in the central nervous system. Sortilin has emerged as a key player in the regulation of neuronal viability and has been implicated as a possible therapeutic target in a range of disorders. Here, the identification of AF40431, the first reported small-molecule ligand of sortilin, is reported. Crystals of the sortilin-AF40431 complex were obtained by co-crystallization and the structure of the complex was solved to 2.7 Šresolution. AF40431 is bound in the neurotensin-binding site of sortilin, with the leucine moiety of AF40431 mimicking the binding mode of the C-terminal leucine of neurotensin and the 4-methylumbelliferone moiety of AF40431 forming π-stacking with a phenylalanine.

Does neuroglobin protect neurons from ischemic insult? A quantitative investigation of neuroglobin expression following transient MCAo in spontaneously hypertensive rats.

Neuroglobin (NGB) is a recently characterized heme globin expressed primarily in retinal nerve cells and at very low levels in endocrine-active regions of vertebrate brains. When artificially over-expressed, NGB reduces the infarct size observed after transient Middle Cerebral Artery occlusion (tMCAo) in rats. This study addresses the post-ischemic NGB expression in vivo. Ten Spontaneously Hypertensive Rats (SHRs) were randomized to tMCAo (n = 6) or sham (n = 4), and euthanized 24 h later. NGB mRNA expression was determined by means of quantitative Reverse Transcription Polymerase Reaction (qRT-PCR). Thirteen animals subjected to either 90 min tMCAo (n = 7) or sham (n = 6) surgery, were euthanized 1 week after surgery. Post-ischemic expression of NGB and the neuronal marker NeuN was studied using free-floating immunohistochemistry. Design-based stereological quantification of NGB- and NeuN-positive cells in the striatum was performed using the optical fractionator. Significantly less NGB mRNA was expressed in the ischemic hemispheres of tMCAo animals after 24 h (P < or = 0.002). At the protein level, we found a significantly lower number of NGB- and NeuN-positive striatal neurons in tMCAo rats (P < or = 0.004). NGB expression was mainly confined to the hypothalamus and amygdala. Less than one out of every two thousand neurons expressed NGB in the striatum. In the ischemic territory we did not observe selective sparing of NGB expressing neurons. No significant change in the NGB/NeuN ratio was observed. Our data indicate that endogenous expressed NGB does not provide protection against ischemic injury induced by tMCAo in SHRs.

Chloride channel blockers inhibit iNOS expression and NO production in IFNgamma-stimulated microglial BV2 cells.

Microglial cells play an important role during neuroinflammation in the central nervous system. Among other factors, activated microglia produce nitric oxide (NO), which is toxic to neurons and excessive microglial activation and NO production contribute to the pathology of neurodegenerative diseases. Chloride channels have previously been shown to participate in microglial activation. Here we investigate the effects of established chloride channel blockers with different chemical structures on interferon-gamma (IFNgamma)-induced activation of the murine microglial cell line, BV2. IFNgamma-induced NO production was effectively reduced by NPPB, IAA-94, tamoxifen, NS3728 and NS1652, with NS1652 being the most potent. In contrast, DIDS reduced NO production only at cytotoxic concentrations. Furthermore, NS1652 reduced the protein and mRNA levels of inducible nitric oxide synthase (iNOS), without altering STAT1 phosphorylation. These observations suggest a microglial chloride conductance as a critical permissive factor downstream in the IFNgamma-induced iNOS cascade. The nature of the underlying channel is unknown, but the pharmacological profile appears incompatible with the involvement of the volume activated anion conductance (VRAC).