- Email: [email protected]
Disaggregation of β-amyloid plaques by KMSB600
Poster Presentations P3 calcium influx were completely uneffective on Ab levels. Experiments aiming at defining the impact of selected DHPs on APP cleavage as well as their ability to alter intracellular amyloid transport are currently ongoing. Conclusions: Results obtained to date emphasise the relevance of DHP-like structures as potential candidates in the development of an Ab-targeted pharmacology. P3-265
KMS88009 AND KMS88016 AS SMALL MOLECULE INHIBITORS FOR IN VIVO Ab FIBRIL FORMATION
YoungSoo Kim1, Ji Hun Byun1, Jiyeon Ryu1, Hana Hwang1, Ji Hoon Lee1, Jeiwon Cho1, HyeYun Kim1, Young-Gil Ahn2, Young Hoon Kim2, Maeng Sup Kim2, Gwan Sun Lee2, Inhee Mook-Jung3, Kyung Ho Yoo1, Dong Jin Kim1, 1Korea Institute of Science and Technology, Seoul, Republic of Korea; 2Hanmi Research Center, Hanmi Pharm. Co., Ltd., Seoul, Republic of Korea; 3Seoul National University College of Medicine, Seoul, Republic of Korea. Contact e-mail: [email protected] Background: Since cholinergic deficit in the central nervous system has been observed in AD patients, inhibitors for acetylcholine esterase (AchE) such as tacrin, donepezil, and rivastigmine are used for the clinical treatment of AD symptoms in current therapeutic approaches. Although these are the only FDA-approved AD therapies on the market, they cannot solve fundamental problems but can be only palliative solutions. Ab aggregates such as oligomers and fibrils deposit in the brain causing strong neuronal toxicity. In addition, it was recently reported that oligomers might be more toxic than fibrils, and the oligomers are becoming the target for the development of AD treatments. Methods: Here we report the synthesis and in vitro/vivo evaluations of a novel KMS88 series with potent inhibitory activities for Ab fibril formation. Thioflavin T assays, in vitro MTT cytotoxicity assays, transmission electron microscopy, pahrmacokinetics, behavior studies of transgenic mouse models, immunohistochemistry studies, and ex vivo brain imaging were performed to exam activities on Ab aggregation cascade in AD. Results: All 23 synthetic compounds in this study inhibited Ab(1-42) fibril formation near sub-mM range of IC50 (0.07-5.44 mM), better than curcumin (0.8 mM), without cytotoxicities on HT-22 neuronal cells by MTT assay. Then, KMS88009 (BA 64%, BBB >100%) and KMS88016 (BA 69%, BBB >100%) were selected as primary drug candidates for further in vivo studies. In the brains of AD transgenic mice, the amount of Ab fibrils were notably decreased after 7 months of oral administration. Prevention of cognitive impairment was also observed in behavior tests. Conclusions: KMS88009 and KMS88016 are promising AD therapeutics candidates which inhibit formation of Ab fibrils in AD brains and prevent cognitive impairment.
Background: The formation of Amyloid beta (Ab) oligomers and fibrils plays an important role in Alzheimer’s disease (AD). Among the wide variety of attempts to interpret amyloidosis mechanism, interactions of Ab and sulfate moieties of glycosaminoglycans (GAGs) or proteoglycans (PGs) have been reported as a critical factor in Ab oligomer/fibril formation. Negatively charged sulfate moieties of GAGs bind to Ab and induce a conformational change in Ab from a random coil to a b-sheet. Methods: Via thioflavin T (ThT) aggregation assays, SDS-PAGE with PICUP chemistry, and transmission electron microscopic studies, we examined effects of KMSB600 on Ab fibril formation. Results: As a result, KMSB600 inhibited the assembly Ab from monomers to oligomers/fibrils and also disaggregated preformed Ab fibrils. In addition, the excellent cell viability was observed due to KMSB600 at inhibits and reverses amyloidogenesis. A GAG mimicking organic acid compound, KMSB600 with a sulfate moiety, was found as a promising small molecule to inhibit Ab aggregation and to disaggregate Ab fibrils in vitro. Conclusions: Although the mechanism by KMSB600 on Ab fibril formation in vitro is still unclear, it could serve as a key scaffold for the development of more competent molecules to treat AD in vivo.
P3-267
DISAGGREGATION OF b-AMYLOID PLAQUES BY KMSB600
HyeYun Kim1, YoungSoo Kim1, GyoonHee Han2, Dong Jin Kim1, 1Korea Institute of Science & Technology (KIST), Seoul, Republic of Korea; 2Yonsei University, Seoul, Republic of Korea. Contact e-mail: cookietomato@ hotmail.com
REGULATION OF GENE EXPRESSION BY SAPPa AND RER DERIVATIVE IN DIFFERENTIATED NEUROBLASTOMA CELLS
Margaret M. Ryan, Gary Morris, Katie Bourne, Bruce G. Mockett, Wickliffe C. Abraham, Warren P. Tate, Joanna M. Williams, University of Otago, Dunedin, New Zealand. Contact e-mail: [email protected] Background: The amyloid precursor protein (APP) is cleaved by two opposing pathways, the most well understood of which yields the neurotoxic peptide, amyloid-b, while the second mutually exclusive pathway yields a secreted molecule, sAPPa, a 612 amino acid peptide. Infusion of sAPPa in vivo enhances the LTP-model of memory and inhibition of endogenous sAPPa synthesis in vivo by infusion of TAPI, a metalloprotease inhibitor, results in a reduced retention of spatial memories. This effect, however, can be rescued by co-infusion of recombinant sAPPa. LTP persistence and memory storage are dependent on new gene transcription and sAPPa is known to regulate gene expression, in particular it can up regulate expression of insulinlike growth factor-2 (IGF2) and IGF-binding protein-2 (IGFBP2). As short 3’ motifs of sAPPa have been shown to protect against amyloid-b-induced memory loss, we have investigated whether the RER-sAPPa motif can regulate gene expression in a manner similar to sAPPa. Methods: Retinoic acid-differentiated SH-SY5Y neuroblastoma cells were treated with either saline, recombinant sAPPa (1 or 10 nM) or RER (10 nM) for 2 h. The relative levels of IGF2 and IGFBP2 in the drug-treated vs. control groups were determined by extraction of total RNA from 6 x 105 cells, conversion to cDNA and a SYBR Green quantitative PCR assay, where HPRT levels were used as the normaliser. All assays were carried out in triplicate and the data presented is an average of three separate experiments (n¼3). Results: We found that sAPPa treatment resulted in an increase in the expression of both IGF2 (1 nM: 3 fold; 10 nM:1.5 fold) and IGFBP2 (1 nM: 3.6 fold; 10 nM: 1.7 fold) and preliminary results show that this effect was similar with the RER motif (IGF2: 7.5 fold; IGFBP2: 1.88 fold). Conclusions: These data show that the both sAPPa and the RER motif can induce gene expression changes in differentiated neuroblastoma cells. These results suggest that the RER motif may potentially be vital to the neuroprotective and neuromodulatory properties of sAPPa. P3-268
P3-266
P421
AMYLOID-BETA (Aß) 42 PREFIBRILLAR OLIGOMER-SPECIFIC MIMOTOPE-PEPTIDE WHICH INHIBITS Aß42 BUT NOT Aß40 FIBRIL FORMATION
Koichi Tanaka, Masaaki Nishimura, Sho Kitamoto, Sho Takiguchi, Makoto Yamaguchi, Hiroko Nagao, Shuhei Hashiguchi, Yuji Ito, Kazuhisa Sugimura, Kagoshima University, Kagoshima, Japan. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is characterized by abnormal amyloid-ß (Aß) aggregation in brain including fibrils and oligomers. The Aß
KMS88009 AND KMS88016 AS SMALL MOLECULE INHIBITORS FOR IN VIVO Ab FIBRIL FORMATION
YoungSoo Kim1, Ji Hun Byun1, Jiyeon Ryu1, Hana Hwang1, Ji Hoon Lee1, Jeiwon Cho1, HyeYun Kim1, Young-Gil Ahn2, Young Hoon Kim2, Maeng Sup Kim2, Gwan Sun Lee2, Inhee Mook-Jung3, Kyung Ho Yoo1, Dong Jin Kim1, 1Korea Institute of Science and Technology, Seoul, Republic of Korea; 2Hanmi Research Center, Hanmi Pharm. Co., Ltd., Seoul, Republic of Korea; 3Seoul National University College of Medicine, Seoul, Republic of Korea. Contact e-mail: [email protected] Background: Since cholinergic deficit in the central nervous system has been observed in AD patients, inhibitors for acetylcholine esterase (AchE) such as tacrin, donepezil, and rivastigmine are used for the clinical treatment of AD symptoms in current therapeutic approaches. Although these are the only FDA-approved AD therapies on the market, they cannot solve fundamental problems but can be only palliative solutions. Ab aggregates such as oligomers and fibrils deposit in the brain causing strong neuronal toxicity. In addition, it was recently reported that oligomers might be more toxic than fibrils, and the oligomers are becoming the target for the development of AD treatments. Methods: Here we report the synthesis and in vitro/vivo evaluations of a novel KMS88 series with potent inhibitory activities for Ab fibril formation. Thioflavin T assays, in vitro MTT cytotoxicity assays, transmission electron microscopy, pahrmacokinetics, behavior studies of transgenic mouse models, immunohistochemistry studies, and ex vivo brain imaging were performed to exam activities on Ab aggregation cascade in AD. Results: All 23 synthetic compounds in this study inhibited Ab(1-42) fibril formation near sub-mM range of IC50 (0.07-5.44 mM), better than curcumin (0.8 mM), without cytotoxicities on HT-22 neuronal cells by MTT assay. Then, KMS88009 (BA 64%, BBB >100%) and KMS88016 (BA 69%, BBB >100%) were selected as primary drug candidates for further in vivo studies. In the brains of AD transgenic mice, the amount of Ab fibrils were notably decreased after 7 months of oral administration. Prevention of cognitive impairment was also observed in behavior tests. Conclusions: KMS88009 and KMS88016 are promising AD therapeutics candidates which inhibit formation of Ab fibrils in AD brains and prevent cognitive impairment.
Background: The formation of Amyloid beta (Ab) oligomers and fibrils plays an important role in Alzheimer’s disease (AD). Among the wide variety of attempts to interpret amyloidosis mechanism, interactions of Ab and sulfate moieties of glycosaminoglycans (GAGs) or proteoglycans (PGs) have been reported as a critical factor in Ab oligomer/fibril formation. Negatively charged sulfate moieties of GAGs bind to Ab and induce a conformational change in Ab from a random coil to a b-sheet. Methods: Via thioflavin T (ThT) aggregation assays, SDS-PAGE with PICUP chemistry, and transmission electron microscopic studies, we examined effects of KMSB600 on Ab fibril formation. Results: As a result, KMSB600 inhibited the assembly Ab from monomers to oligomers/fibrils and also disaggregated preformed Ab fibrils. In addition, the excellent cell viability was observed due to KMSB600 at inhibits and reverses amyloidogenesis. A GAG mimicking organic acid compound, KMSB600 with a sulfate moiety, was found as a promising small molecule to inhibit Ab aggregation and to disaggregate Ab fibrils in vitro. Conclusions: Although the mechanism by KMSB600 on Ab fibril formation in vitro is still unclear, it could serve as a key scaffold for the development of more competent molecules to treat AD in vivo.
P3-267
DISAGGREGATION OF b-AMYLOID PLAQUES BY KMSB600
HyeYun Kim1, YoungSoo Kim1, GyoonHee Han2, Dong Jin Kim1, 1Korea Institute of Science & Technology (KIST), Seoul, Republic of Korea; 2Yonsei University, Seoul, Republic of Korea. Contact e-mail: cookietomato@ hotmail.com
REGULATION OF GENE EXPRESSION BY SAPPa AND RER DERIVATIVE IN DIFFERENTIATED NEUROBLASTOMA CELLS
Margaret M. Ryan, Gary Morris, Katie Bourne, Bruce G. Mockett, Wickliffe C. Abraham, Warren P. Tate, Joanna M. Williams, University of Otago, Dunedin, New Zealand. Contact e-mail: [email protected] Background: The amyloid precursor protein (APP) is cleaved by two opposing pathways, the most well understood of which yields the neurotoxic peptide, amyloid-b, while the second mutually exclusive pathway yields a secreted molecule, sAPPa, a 612 amino acid peptide. Infusion of sAPPa in vivo enhances the LTP-model of memory and inhibition of endogenous sAPPa synthesis in vivo by infusion of TAPI, a metalloprotease inhibitor, results in a reduced retention of spatial memories. This effect, however, can be rescued by co-infusion of recombinant sAPPa. LTP persistence and memory storage are dependent on new gene transcription and sAPPa is known to regulate gene expression, in particular it can up regulate expression of insulinlike growth factor-2 (IGF2) and IGF-binding protein-2 (IGFBP2). As short 3’ motifs of sAPPa have been shown to protect against amyloid-b-induced memory loss, we have investigated whether the RER-sAPPa motif can regulate gene expression in a manner similar to sAPPa. Methods: Retinoic acid-differentiated SH-SY5Y neuroblastoma cells were treated with either saline, recombinant sAPPa (1 or 10 nM) or RER (10 nM) for 2 h. The relative levels of IGF2 and IGFBP2 in the drug-treated vs. control groups were determined by extraction of total RNA from 6 x 105 cells, conversion to cDNA and a SYBR Green quantitative PCR assay, where HPRT levels were used as the normaliser. All assays were carried out in triplicate and the data presented is an average of three separate experiments (n¼3). Results: We found that sAPPa treatment resulted in an increase in the expression of both IGF2 (1 nM: 3 fold; 10 nM:1.5 fold) and IGFBP2 (1 nM: 3.6 fold; 10 nM: 1.7 fold) and preliminary results show that this effect was similar with the RER motif (IGF2: 7.5 fold; IGFBP2: 1.88 fold). Conclusions: These data show that the both sAPPa and the RER motif can induce gene expression changes in differentiated neuroblastoma cells. These results suggest that the RER motif may potentially be vital to the neuroprotective and neuromodulatory properties of sAPPa. P3-268
P3-266
P421
AMYLOID-BETA (Aß) 42 PREFIBRILLAR OLIGOMER-SPECIFIC MIMOTOPE-PEPTIDE WHICH INHIBITS Aß42 BUT NOT Aß40 FIBRIL FORMATION
Koichi Tanaka, Masaaki Nishimura, Sho Kitamoto, Sho Takiguchi, Makoto Yamaguchi, Hiroko Nagao, Shuhei Hashiguchi, Yuji Ito, Kazuhisa Sugimura, Kagoshima University, Kagoshima, Japan. Contact e-mail: [email protected] Background: Alzheimer’s disease (AD) is characterized by abnormal amyloid-ß (Aß) aggregation in brain including fibrils and oligomers. The Aß