The c-Raf inhibitor GW5074 provides neuroprotection in vitro and in an animal model of neurodegeneration through a MEK-ERK and Akt-independent mechanism

Cerebellar granule neurons undergo apoptosis when switched from a medium containing high potassium (HK) to one that has low potassium (LK). LK-induced cell death is blocked by GW5074 [5-Iodo-3-[(3,5-dibromo-4-hydroxyphenyl) methylene]-2-indolinone], a synthetic drug that inhibits c-Raf activity in vitro. GW5074 has no direct effect on the activities of several apoptosis-associated kinases when assayed in vitro. In contrast to its effect in vitro, treatment of neurons with GW5074 causes c-Raf activation (when measured in vitro in the absence of the drug) and stimulates the Raf-MEK-ERK pathway. Treatment of neurons with GW5074 also leads to an increase in the activity of B-Raf, which is not inhibited by GW5074 in vitro at concentrations at which the drug exerts its neuroprotective effect. PD98059 and U0126, two distinct inhibitors of MEK, block the activation of ERK by GW5074 but have no effect on its ability to prevent cell death. Overexpression of a dominant-negative form of Akt does not reduce the efficacy of GW5074, demonstrating an Akt-independent mechanism of action. Neuroprotection is inhibited by SN-50, a specific inhibitor of nuclear factor-kappa B (NF-kappaB) and by the Ras inhibitor S-trans, trans-farnesylthiosalicylic acid (FTS) implicating NF-kappaB and Ras in the neuroprotective signaling pathway activated by GW5074. In addition to preventing LK-induced apoptosis, treatment with GW5074 protects against the neurotoxic effects of MPP+ and methylmercury in cerebellar granule neurons, and glutathione depletion-induced oxidative stress in cortical neurons. Furthermore, GW5074 prevents neurodegeneration and improves behavioral outcome in an animal model of Huntington's disease. Given its neuroprotective effect on distinct types of cultured neurons, in response to different neurotoxic stimuli, and in an animal model of neurodegeneration, GW5074 could have therapeutic value against neurodegenerative pathologies in humans.     

Inhibition of neuronal apoptosis by the cyclin-dependent kinase inhibitor GW8510: identification of 3' substituted indolones as a scaffold for the development of neuroprotective drugs

Increasing evidence suggests that neuronal apoptosis is triggered by the inappropriate activation of cyclin-dependent kinases leading to an abortive re-entry of neurons into the cell cycle. Pharmacological inhibitors of cell-cycle progression may therefore have value in the treatment of neurodegenerative diseases in humans. GW8510 is a 3' substituted indolone that was developed recently as an inhibitor of cyclin-dependent kinase 2 (CDK2). We found that GW8510 inhibits the death of cerebellar granule neurons caused by switching them from high potassium (HK) medium to low potassium (LK) medium. Although GW8510 inhibits CDK2 and other CDKs when tested in in vitro biochemical assays, when used on cultured neurons it only inhibits CDK5, a cytoplasmic CDK that is not associated with cell-cycle progression. Treatment of cultured HEK293T cells with GW8510 does not inhibit cell-cycle progression, consistent with its inability to inhibit mitotic CDKs in intact cells. Neuroprotection by GW8510 is independent of Akt and MEK-ERK signaling. Furthermore, GW8510 does not block the LK-induced activation of Gsk3beta and, while inhibiting c-jun phosphorylation, does not inhibit the increase in c-jun expression observed in apoptotic neurons. We also examined the effectiveness of other 3' substituted indolone compounds to protect against neuronal apoptosis. We found that like GW8510, the VEGF Receptor 2 Kinase Inhibitors [3-(1H-pyrrol-2-ylmethylene)-1,3-dihydroindol-2-one], {(Z)-3-[2,4-Dimethyl-3-(ethoxycarbonyl)pyrrol-5-yl)methylidenyl]indol-2-one} and [(Z)-5-Bromo-3-(4,5,6,6-tetrahydro-1H-indol-2-ylmethylene)-1,3-dihydroindol-2-one], the Src family kinase inhibitor SU6656 and a commercially available inactive structural analog of an RNA-dependent protein kinase inhibitor 5-Chloro-3-(3,5-dichloro-4-hydroxybenzylidene)-1,3-dihydro-indol-2-one, are all neuroprotective when tested on LK-treated neurons. Along with our recent identification of the c-Raf inhibitor GW5074 (also a 3' substituted indolone) as a neuroprotective compound, our findings identify the 3' substituted indolone as a core structure for the designing of neuroprotective drugs that may be used to treat neurodegenerative diseases in humans.     

Phosphatidylinositol 4-kinase III beta is a target of enviroxime-like compounds for antipoliovirus activity

Enviroxime is an antienterovirus compound that targets viral protein 3A and/or 3AB and suppresses a step in enterovirus replication by unknown mechanism. To date, four antienterovirus compounds, i.e., GW5074, Flt3 inhibitor II, TTP-8307, and AN-12-H5, are known to have similar mutations in the 3A protein-encoding region causing resistance to enviroxime (a G5318A [3A-Ala70Thr] mutation in poliovirus [PV]) and are considered enviroxime-like compounds. Recently, antienterovirus activity of a phosphatidylinositol 4-kinase III beta (PI4KB) inhibitor, PIK93, was reported, suggesting that PI4KB is an important host factor targetable by antienterovirus compounds (N. Y. Hsu et al., Cell 141:799-811, 2010). In this study, we analyzed the inhibitory effects of previously identified enviroxime-like compounds (GW5074 and AN-12-H5) and a newly identified antienterovirus compound, T-00127-HEV1, on phosphoinositide (PI) kinases. We found that T-00127-HEV1 inhibited PI4KB activity with a higher specificity for than other PI kinases, in contrast to GW5074, which had a broad specificity for PI kinases. In contrast, AN-12-H5 showed no inhibitory effect on PI4KB activity and only moderate inhibitory effects on PI 3-kinase activity. Small interfering RNA (siRNA) screening targeting PI kinases identified PI4KB is a target of GW5074 and T-00127-HEV1, but not of AN-12-H5, for anti-PV activity. Interestingly, T-00127-HEV1 and GW5074 did not inhibit hepatitis C virus (HCV) replication, in contrast to a strong inhibitory effect of AN-12-H5. These results suggested that PI4KB is an enterovirus-specific host factor required for the replication process and targeted by some enviroxime-like compounds (T-00127-HEV1 and GW5074) and that enviroxime-like compounds may have targets other than PI kinases for their antiviral effect.     

Inhibition of the human deacylase Sirtuin 5 by the indole GW5074

Sirtuins are NAD⁺ consuming protein deacylases involved in many cellular processes from DNA-repair to metabolism. Their contribution to age-related and metabolic diseases makes them attractive pharmaceutical targets. Few pharmacological inhibitors have been reported yet for human Sirt5 since substrates and assays for reliable testing of its activity were unavailable until recently, and most modulators of other Sirtuins were not tested against Sirt5 and therefore have only partially characterized isoform selectivities. We used here improved substrates and assays for testing of known Sirtuin inhibitors for their effects on two activities of human Sirt5, the generic Sirtuin activity deacetylation and the more pronounced Sirt5 activity desuccinylation. Our tests show that most of the compounds have no significant effect on either Sirt5 activity. The indole GW5074, however, was found to be a potent inhibitor for Sirt5’s desuccinylation activity, identifying a first pharmacological scaffold for development into Sirt5-specific inhibitors. Interestingly, the compound showed weaker effects in Sirt5 deacetylation assays and also varying potencies against different peptide sequences, indicating a substrate-specific effect of GW5074.     

RAF associates with phosphorylated nuclear BubR1 during endoreduplication induced by JAK inhibition

The role of JAK signaling in cell cycle transit and maintenance of genomic stability was determined in HL-60 human myeloblastic leukemia cells. We have previously reported that a pan-JAK inhibitor caused ERK-dependent endoreduplication. In the current study we find that JAK inhibition caused nuclear re-localization of RAF-1, which could be inhibited by RAF inhibitor GW5074. GW5074 also inhibited JAK inhibitor-induced appearance of nuclear phosphorylated RAF-1(pS621RAF) and MEK, and it inhibited the JAK inhibitor-induced co-immunoprecipitation of nuclear RAF-1 and MEK. JAK inhibition also increased nuclear BubR1 phosphorylation, which was diminished by RAF inhibitor GW5074. RAF-1 and BubR1 in the nucleus co-immunoprecipitated; and GW5074 eliminated this. Furthermore, inhibiting RAF with GW5074 blocked the pan-JAK inhibitor-induced endoreduplication. These data thus show that JAK inhibition causes nuclear re-localization and phosphorylation of RAF and MEK where RAF binds BubR1 with ensuing nuclear RAF-dependent BubR1 phosphorylation. Inhibiting RAF inhibited this and endoreduplication. The results suggest that there is a JAK/RAF/MEK/BubR1 axis that can regulate genomic stability. In this hypothetical model JAK suppresses RAF/MEK phosphorylation and nuclear re-localization, but JAK inhibition induces the phosphorylations and re-localization with association of RAF and phosphorylated BubR1 in the nucleus leading to endoreduplication.     

Aryl azoles with neuroprotective activity--parallel synthesis and attempts at target identification

A parallel synthesis of aryl azoles with neuroprotective activity is described. All compounds obtained were evaluated in an in vitro assay using a NMDA toxicity paradigm showing a neuroprotective activity between 15% and 40%. The potential biological target of the active compounds was investigated by extensive literature searches based around similar scaffolds with reported neuroprotective activity. The most interesting molecules active in the NMDA toxicity assay (3a and 2g) showed moderate but significant activity in the inhibition of the Site 2 Sodium Channel binding assay at 10 microM. To confirm our hypothesis compounds 3a, c, f and 2g were tested in the Veratridine assay which is one of the excitotoxicity assays of relevance to NaV channels. The compounds tested showed an activity between 40% and 70%. The identification of neuroprotective small molecules and the identification of NaV channels as the potential site of action were the most important goals of this work.     

Phorbol 12-myristate 13-acetate upregulates cyclooxygenase-2 expression in human pulmonary epithelial cells via Ras, Raf-1, ERK, and NF-kappaB, but not p38 MAPK, pathways

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression and prostaglandin E2 (PGE2) release by phorbol 12-myristate 13-acetate (PMA), a protein kinase C (PKC) activator, in human pulmonary epithelial cells (A549). PMA-induced COX-2 expression was attenuated by PKC inhibitors (Go 6976 and Ro 31-8220), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), and an NF-kappaB inhibitor (PDTC), but not by a tyrosine kinase inhibitor (genistein) or a p38 MAPK inhibitor (SB 203580). PMA also caused the activation of Ras, Raf-1, and ERK1/2. PMA-induced activation of Ras and Raf-1 was inhibited by Ro 31-8220 and manumycin A. PMA-mediated activation of ERK1/2 was inhibited by Ro 31-8220, manumycin A, GW 5074, and PD 098059. Stimulation of cells with PMA caused IkappaBalpha phosphorylation, IkappaBalpha degradation, and the formation of a NF-kappaB-specific DNA-protein complex. The PMA-mediated increase in kappaB-luciferase activity was inhibited by Ro 31-8220, manumycin A, GW5074, PD 098059, and PDTC. Taken together, these results indicate that PMA might activate PKC to elicit activation of the Ras/Raf-1/ERK1/2 pathway, which in turn initiates NF-kappaB activation, and finally induces COX-2 expression and PGE2 release in A549 cells.     

RAF associates with phosphorylated nuclear BubR1 during endoreduplication induced by JAK inhibition

The role of JAK signaling in cell cycle transit and maintenance of genomic stability was determined in HL-60 human myeloblastic leukemia cells. We have previously reported that a pan-JAK inhibitor caused ERK-dependent endoreduplication. In the current study we find that JAK inhibition caused nuclear re-localization of RAF-1, which could be inhibited by RAF inhibitor GW5074. GW5074 also inhibited JAK inhibitor-induced appearance of nuclear phosphorylated RAF-1(pS621RAF) and MEK, and it inhibited the JAK inhibitor-induced co-immunoprecipitation of nuclear RAF-1 and MEK. JAK inhibition also increased nuclear BubR1 phosphorylation, which was diminished by RAF inhibitor GW5074. RAF-1 and BubR1 in the nucleus co-immunoprecipitated; and GW5074 eliminated this. Furthermore, inhibiting RAF with GW5074 blocked the pan-JAK inhibitor-induced endoreduplication. These data thus show that JAK inhibition causes nuclear re-localization and phosphorylation of RAF and MEK where RAF binds BubR1 with ensuing nuclear RAF-dependent BubR1 phosphorylation. Inhibiting RAF inhibited this and endoreduplication. The results suggest that there is a JAK/RAF/MEK/BubR1 axis that can regulate genomic stability. In this hypothetical model JAK suppresses RAF/MEK phosphorylation and nuclear re-localization, but JAK inhibition induces the phosphorylations and re-localization with association of RAF and phosphorylated BubR1 in the nucleus leading to endoreduplication.Key words: endoreduplication, JAK, genomic instability, MAPK, HL-60 cells     

The Raf-1 inhibitor GW5074 and dexamethasone suppress sidestream smoke-induced airway hyperresponsiveness in mice

Background

Sidestream smoke is closely associated with airway inflammation and hyperreactivity. The present study was designed to investigate if the Raf-1 inhibitor GW5074 and the anti-inflammatory drug dexamethasone suppress airway hyperreactivity in a mouse model of sidestream smoke exposure.

Methods

Mice were repeatedly exposed to smoke from four cigarettes each day for four weeks. After the first week of the smoke exposure, the mice received either dexamethasone intraperitoneally every other day or GW5074 intraperitoneally every day for three weeks. The tone of the tracheal ring segments was recorded with a myograph system and concentration-response curves were obtained by cumulative administration of agonists. Histopathology was examined by light microscopy.

Results

Four weeks of exposure to cigarette smoke significantly increased the mouse airway contractile response to carbachol, endothelin-1 and potassium. Intraperitoneal administration of GW5074 or dexamethasone significantly suppressed the enhanced airway contractile responses, while airway epithelium-dependent relaxation was not affected. In addition, the smoke-induced infiltration of inflammatory cells and mucous gland hypertrophy were attenuated by the administration of GW5074 or dexamethasone.

Conclusion

Sidestream smoke induces airway contractile hyperresponsiveness. Inhibition of Raf-1 activity and airway inflammation suppresses smoking-associated airway hyperresponsiveness.     

Bradykinin B2 receptor mediates NF-kappaB activation and cyclooxygenase-2 expression via the Ras/Raf-1/ERK pathway in human airway epithelial cells

In this study, we investigated the signaling pathways involved in bradykinin (BK)-induced NF-kappaB activation and cyclooxygenase-2 (COX-2) expression in human airway epithelial cells (A549). BK caused concentration- and time-dependent increase in COX-2 expression, which was attenuated by a selective B2 BK receptor antagonist (HOE140), a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), a MEK inhibitor (PD 098059), an NF-kappaB inhibitor (pyrrolidine dithiocarbate), and an IkappaB protease inhibitor (L-1-tosylamido-2-phenylethyl chloromethyl ketone). The B1 BK receptor antagonist (Lys-(Leu8)des-Arg9-BK) had no effect on COX-2 induction by BK. BK-induced increase in COX-2-luciferase activity was inhibited by cells transfected with the kappaB site deletion of COX-2 construct. BK-induced Ras activation was inhibited by manumycin A. Raf-1 phosphorylation at Ser338 by BK was inhibited by manumycin A and GW 5074. BK-induced ERK activation was inhibited by HOE140, manumycin A, GW 5074, and PD 098059. Stimulation of cells with BK activated IkappaB kinase alphabeta (IKKalphabeta), IkappaBalpha phosphorylation, IkappaBalpha degradation, p65 and p50 translocation from the cytosol to the nucleus, the formation of an NF-kappaB-specific DNA-protein complex, and kappaB-luciferase activity. BK-mediated increase in IKKalphabeta activity and formation of the NF-kappaB-specific DNA-protein complex were inhibited by HOE140, a Ras dominant-negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Our results demonstrated for the first time that BK, acting through B2 BK receptor, induces activation of the Ras/Raf-1/ERK pathway, which in turn initiates IKKalphabeta and NF-kappaB activation, and ultimately induces COX-2 expression in human airway epithelial cell line (A549).     

Peptidoglycan induces nuclear factor-kappaB activation and cyclooxygenase-2 expression via Ras, Raf-1, and ERK in RAW 264.7 macrophages

In this study, we investigated the signaling pathway involved in cyclooxygenase-2 (COX-2) expression caused by peptidoglycan (PGN), a cell wall component of the Gram-positive bacterium Staphylococcus aureus, in RAW 264.7 macrophages. PGN caused dose- and time-dependent increases in COX-2 expression, which was attenuated by a Ras inhibitor (manumycin A), a Raf-1 inhibitor (GW 5074), and an MEK inhibitor (PD 098059). Treatment of RAW 264.7 macrophages with PGN caused time-dependent activations of Ras, Raf-1, and ERK. The PGN-induced increase in Ras activity was inhibited by manumycin A. Raf-1 phosphorylation at Ser-338 by PGN was inhibited by manumycin A and GW 5074. The PGN-induced increase in ERK activity was inhibited by manumycin A, GW 5074, and PD 098059. Stimulation of cells with PGN activated IkappaB kinase alpha/beta (IKKalpha/beta), IkappaBalpha phosphorylation, IkappaBalpha degradation, and kappaB-luciferase activity. Treatment of macrophages with an NF-kappaB inhibitor (pyrrolidine dithiocarbamate), an IkappaBalpha phosphorylation inhibitor (Bay 117082), and IkappaB protease inhibitors (l-1-tosylamido-2-phenylethyl chloromethyl ketone and calpain inhibitor I) all inhibited PGN-induced COX-2 expression. The PGN-mediated increase in the activities of IKKalpha/beta and kappaB-luciferase were also inhibited by the Ras dominant negative mutant (RasN17), manumycin A, GW 5074, and PD 098059. Further studies revealed that PGN induced the recruitment of p85alpha and Ras to Toll-like receptor 2 in a time-dependent manner. Our data demonstrate for the first time that PGN activates the Ras/Raf-1/ERK pathway, which in turn initiates IKKalpha/beta and NF-kappaB activation, and ultimately induces COX-2 expression in RAW 264.7 macrophages.     

Rosiglitazone Suppresses In Vitro Seizures in Hippocampal Slice by Inhibiting Presynaptic Glutamate Release in a Model of Temporal Lobe Epilepsy

Peroxisomal proliferator-activated receptor gamma (PPARγ) is a nuclear hormone receptor whose agonist, rosiglitazone has a neuroprotective effect to hippocampal neurons in pilocarpine-induced seizures. Hippocampal slice preparations treated in Mg²⁺ free medium can induce ictal and interictal-like epileptiform discharges, which is regarded as an in vitro model of N-methyl-D-aspartate (NMDA) receptor-mediated temporal lobe epilepsy (TLE). We applied rosiglitazone in hippocampal slices treated in Mg²⁺ free medium. The effects of rosiglitazone on hippocampal CA1-Schaffer collateral synaptic transmission were tested. We also examined the neuroprotective effect of rosiglitazone toward NMDA excitotoxicity on cultured hippocampal slices. Application of 10μM rosiglitazone significantly suppressed amplitude and frequency of epileptiform discharges in CA1 neurons. Pretreatment with the PPARγ antagonist GW9662 did not block the effect of rosiglitazone on suppressing discharge frequency, but reverse the effect on suppressing discharge amplitude. Application of rosiglitazone suppressed synaptic transmission in the CA1-Schaffer collateral pathway. By miniature excitatory-potential synaptic current (mEPSC) analysis, rosiglitazone significantly suppressed presynaptic neurotransmitter release. This phenomenon can be reversed by pretreating PPARγ antagonist GW9662. Also, rosiglitazone protected cultured hippocampal slices from NMDA-induced excitotoxicity. The protective effect of 10μM rosiglitazone was partially antagonized by concomitant high dose GW9662 treatment, indicating that this effect is partially mediated by PPARγ receptors. In conclusion, rosiglitazone suppressed NMDA receptor-mediated epileptiform discharges by inhibition of presynaptic neurotransmitter release. Rosiglitazone protected hippocampal slice from NMDA excitotoxicity partially by PPARγ activation. We suggest that rosiglitazone could be a potential agent to treat patients with TLE.     

3-(N-arylsulfamoyl)benzamides, inhibitors of human sirtuin type 2 (SIRT2)

Inhibition of sirtuin 2 (SIRT2) is known to be protective against the toxicity of disease proteins in Parkinson's and Huntington's models of neurodegeneration. Previously, we developed SIRT2 inhibitors based on the 3-(N-arylsulfamoyl)benzamide scaffold, including3-(N-(4-bromophenyl)sulfamoyl)-N-(4-bromophenyl)benzamide(C2-8, 1a), which demonstrated neuroprotective effects in a Huntington's mouse model, but had low potency of SIRT2 inhibition. Here we report that N-methylation of 1a greatly increases its potency and results in excellent selectivity for SIRT2 over SIRT1 and SIRT3 isoforms. Structure-activity relationships observed for 1a analogs and docking simulation data suggest that the para-substituted amido moiety of these compounds could occupy two potential hydrophobic binding pockets in SIRT2. These results provide a direction for the design of potent drug-like SIRT2 inhibitors.     

Ultrasound stimulates NF‐κB activation and iNOS expression via the Ras/Raf/MEK/ERK signaling pathway in cultured preosteoblasts

It has been shown that ultrasound (US) stimulation accelerates fracture healing in the animal models and non‐operatively clinical uses. Nitric oxide (NO) is a crucial early mediator in mechanically induced bone formation. Here we found that US‐mediated inducible nitric oxide synthase (iNOS) expression was attenuated by Ras inhibitor (manumycin A), Raf‐1 inhibitor (GW5074), MEK inhibitor (PD98059), NF‐κB inhibitor (PDTC), and IκB protease inhibitor (TPCK). US‐induced Ras activation was inhibited by manumycin A. Raf‐1 phosphorylation at Ser³³⁸ by US was inhibited by manumycin A and GW5074. US‐induced MEK and ERK activation was inhibited by manumycin A, GW5074, and PD98059. Stimulation of preosteoblasts with US activated IκB kinase α/β (IKK α/β), IκBαphosphorylation, p65 phosphorylation at Ser²⁷⁶, p65, and p50 translocation from the cytosol to the nucleus, and κB‐luciferase activity. US‐mediated an increase of IKK α/β, IκBα, and p65 phosphorylation, κB‐luciferase activity and p65 and p50 binding to the NF‐κB element was inhibited by manumycin A, GW5074, and PD98059. Our results suggest that US increased iNOS expression in preosteoblasts via the Ras/Raf‐1/MEK/ERK/IKKαβ and NF‐κB signaling pathways. J. Cell. Physiol. 220: 196–203, 2009. © 2009 Wiley‐Liss, Inc.     

LXR and TSPO as new therapeutic targets to increase the levels of neuroactive steroids in the central nervous system of diabetic animals

Neuroactive steroid levels are decreased in the central nervous system (CNS) of streptozotocin (STZ) diabetic rats. In agreement, they exert protective effects in this experimental model, counteracting degenerative events occurring in the CNS. Therefore, an interesting therapeutic strategy could be to increase their levels directly in the CNS. In this study we have evaluated whether activation of translocator protein-18kDa (TSPO) or liver X receptors (LXRs) may affect the levels of neuroactive steroids present in the CNS of diabetic and non-diabetic animals. We observed that the treatment with either Ro5-4864 (i.e., a ligand of TSPO) or with GW3965 (i.e., a ligand of LXRs) induced an increase of neuroactive steroids in the spinal cord, the cerebellum and the cerebral cortex of STZ-rats, but not in the CNS of non-pathological animals. Interestingly, the pattern of induction was different among the three CNS areas analyzed and between the two pharmacological tools. In particular, the activation of LXRs might represent a promising neuroprotective strategy, because the treatment with GW3965, at variance to Ro5-4864 treatment, did not induce significant changes in the plasma levels of neuroactive steroids. This suggests that activation of LXRs may selectively increase the CNS levels of neuroactive steroids avoiding possible endocrine side effects exerted by the systemic treatment with these molecules. Interestingly GW3965 treatment induced an increase of dihydroprogesterone in the spinal cord of diabetic animals in association with an increase of myelin basic protein expression. Thus we demonstrated that LXR activation was able to rescue CNS symptoms of diabetes.     

Identification of naturally occurring spirostenols preventing beta-amyloid-induced neurotoxicity

22R-Hydroxycholesterol is an intermediate in the steroid biosynthesis pathway shown to exhibit a neuroprotective property against beta-amyloid (1-42) (Abeta) toxicity in rat PCl2 and human NT2N neuronal cells by binding and inactivating Abeta. In search of potent 22R-hydroxycholesterol derivatives, we assessed the ability of a series of naturally occurring entities containing the 22R-hydroxycholesterol structure to protect PC12 cells against Abeta-induced neurotoxicity, determined by measuring changes in membrane potential, mitochondrial diaphorase activity, ATP levels and trypan blue uptake. 22R-Hydroxycholesterol derivatives sharing a common spirost-5-en-3-ol or a furost-5-en-3-ol structure were tested. Although some of these compounds were neuroprotective against 0.1 microM Abeta, only three protected against the 1-10 microM Abeta-induced toxicity and, in contrast to 22R-hydroxycholesterol, all were devoid of steroidogenic activity. These entities shared a common structural feature, a long chain ester in position 3 and common stereochemistry. The neuroprotective property of these compounds was coupled to their ability to displace radiolabeled 22R-hydroxycholesterol from Abeta, suggesting that the Abeta-22R-hydroxycholesterol physicochemical interaction contributes to their beneficial effect. In addition, a 22R-hydroxycholesterol derivative inhibited the formation of neurotoxic amyloid-derived diffusible ligands. Computational docking simulations of 22R-hydroxycholesterol and its derivatives on Abeta identified two binding sites. Chemical entities, as 22R-hydroxycholesterol, seem to bind preferentially only to one site. In contrast, the presence of the ester chain seems to confer the ability to bind to both sites on Abeta, leading to neuroprotection against high concentrations of Abeta. In conclusion, these results suggest that spirost-5-en-3-ol naturally occurring derivatives of 22R-hydroxycholesterol might offer a new approach for Alzheimer's disease therapy.     

Therapeutic effect of CHF5074, a new γ-secretase modulator,in a mouse model of scrapie

In transmissible spongiform encephalopathies (TSEs) and Alzheimer disease (AD) both misfolding and aggregation of specific proteins represent key features. Recently, it was observed that PrP^C is a mediator of a synaptic dysfunction induced by Aβ oligomers. We tested a novel γ-secretase modulator (CHF5074) in a murine model of prion disease. Groups of female mice were intracerebrally or intraperitoneally infected with the mouse-adapted Rocky Mountain Laboratory prions. Two weeks prior infection, the animals were provided with a CHF5074-medicated diet (375 ppm) or a standard diet (vehicle) until they showed neurological signs and eventually died. In intracerebrally infected mice, oral administration of CHF5074 did not prolong survival of the animals. In intraperitoneally-infected mice, CHF5074-treated animals showed a median survival time of 21 d longer than vehicle-treated mice (p < 0.001). In these animals, immunohistochemistry analyses showed that deposition of PrP^Sc in the cerebellum, hippocampus and parietal cortex in CHF5074-treated mice was significantly lower than in vehicle-treated animals. Immunostaining of glial fibrillary acidic protein (GFAP) in parietal cortex revealed a significantly higher reactive gliosis in CHF5074-treated mice compared with the control group of infected animals. Although the mechanism underlying the beneficial effects of CHF5074 in this murine model of human prion disease is unclear, it could be hypothesized that the drug counteracts PrP^Sc toxicity through astrocyte-mediated neuroprotection. CHF5074 shows a pharmacological potential in murine models of both AD and TSEs thus suggesting a link between these degenerative pathologies.Key words: TSE, prion, murine model, γ-secretase modulator, therapy