P2-335: Supplementing α-secretase cleavage of beta-amyloid by targeted proteolytic antibody...
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P2-335 SUPPLEMENTING -SECRETASE CLEAVAGE OF BETA-AMYLOID BY TARGETED PROTEOLYTIC ANTIBODY FRAGMENTS USING YEAST DISPLAY TECHNOLOGY Srinath Kasturirangan, Mike Sierks, Arizona State University, Tempe, AZ, USA. Contact e-mail: [email protected] Background: Deposition of beta-amyloid (A ) is considered an important early event in the pathogenesis of Alzheimer’s Disease (AD), and clearance of A represents a potential therapeutic approach. The recombinant antibody light chain fragment, mK18 was identified to have -secretase-like activity, producing the 1-16 and 17-40 amino acid fragments of A 40 as a primary product. In order to improve the targeting specificity of the proteolytic anti- body for A , we have constructed a second generation library from a parent single chain antibody fragment (scFv) containing the variable domain of the proteolytic light chain and a random heavy chain. Methods: The scFv library was created by introducing random mutations in the CDR3 region of the heavy chain and subsequently expressing the library on the surface of yeast. Biotin- ylated covalently reactive analogs based on the -secretase site sequence were synthesized, immobilized on streptavidin beads, and used to select scFvs with increased specificity for A using magnetic bead enrichment. Collected cells were individually screened for proteolytic activity using 96-well plates and an internally quenched fluorogenic substrate that contains the -secretase site of A. Several individual clones with increased proteolytic activity were identi- fied from 750 screened clones. Kinetic parameters were determined and compared. In-vitro studies using Thioflavin-T fluorescence, cytotoxicity using SHSY5Y cells and AFM imaging will be used to determine the effects of the second generation scFv on beta amyloid aggregation. Results: Second gener- ation yeast display library was constructed in yeast. Two clones with highest binding affinity compared to the original clone were identified. Analysis of kinetic parameters (k cat and K M ) of purified soluble scFv showed one clone had a 10-fold increase in activity (k cat /K M ) toward the synthetic A substrate compared to the original scFv. Conclusions: Results on construction of the library, synthesis of analogs, the panning protocol and catalytic specificity and activity of selected clones will be presented. Catalytic activity toward A oligomers and fibrils and subsequent effects on aggregation and toxicity will be discussed. P2-336 BIS-STYRYLPYRIDINE AND BIS-STYRYLBENZENE DERIVATIVES AS INHIBITORS FOR BETA- AMYLOID FIBRIL FORMATION Young Soo Kim 1 , Seong Rim Byeon 1 , Hye Yun Kim 1 , Ji Hoon Lee 1 , Ji-Hoon Sohn 2 , Inhee Mook-Jung 2 , Dong Jin Kim 1 , 1 Korea Institute of Science and Technology, Seoul, Republic of Korea; 2 Seoul National University, 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 treat- ment 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. Pathomorpho- logically important hallmarks in AD are senile plaques (SP) composed of amyloid (A) and neurofibrillary tangles (NFTs). Therefore, reducing deposition of A plaques in the brain is believed to be pivotal to treat the AD patients. Based on A hypothesis, A (1-42) monomers released from APP by- and-secretases are gradually aggregated to oligomers, proto- fibrils, and fibrils. A (1-42) 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 evaluation of novel bis-styrylpyridine and bis-styrylbenzene derivatives as A aggregation inhibitors. The 34 com- pounds were evaluated by A fibril formation inhibitory assay using thioflavin T. Results: Most of the synthesized compounds in this study inhibited A (1-42) fibril formation near sub- M range of IC 50 (0.1-2.7 M), better than curcumin (0.8 M), without cytotoxicities on HT-22 neuronal cells by MTT assay at 1, 10, and 50 M. Conclusions: In particular, KMS80013, KMS80019, KMS80029, and KMS80030 exhibited the best data of inhibition activities and cytotoxicities and are promising AD drug candidates. P2-337 CHARACTERIZATION OF THE AFFINITY OF GSI-953 FOR BINDING TO GAMMA-SECRETASE AND COMPARISON TO BENCHMARK GAMMA- SECRETASE INHIBITORS Anthony F. Kreft, Magid A. Abou-Gharbia, Ann E. Aulabaugh, Kevin P. Atchison, George Diamantidis, Boyd L. Harrison, Warren D. Hirst, Xinyi Huang, Dennis M. Kubrak, Kerri Lipinski, Ronald L. Magolda, Robert L. Martone, Michael K. May, Scott C. Mayer, Menelas N. Pangalos, Alex M. Porte, Peter H. Reinhart, Lynn Resnick, Hua Zhou, J. Steven Jacobsen, Wyeth Research, Princeton, NJ, USA. Contact e-mail: [email protected] Background: Blocking the synthesis of the AD pathogen beta amyloid peptide () through inhibition of APP processing by gamma secretase (GS) is a promising therapeutic strategy that may provide the first disease- modifying anti-Alzheimer’s drug (DMAAD). In animal models, GSI-953 is a Notch-sparing gamma secretase inhibitor (GSI) and this compound has subsequently entered clinical trials. Our objective was to characterize the relative affinity of GSI-953 for binding to GS and compare it to the potency of GS inhibition. Methods: A cell-free GS binding assay was developed employing membranes isolated from human neuroblastoma SY5Y cells T471 Poster Presentations P2:
Transcript of P2-335: Supplementing α-secretase cleavage of beta-amyloid by targeted proteolytic antibody...
P2-335 SUPPLEMENTING �-SECRETASE CLEAVAGE OFBETA-AMYLOID BY TARGETED PROTEOLYTICANTIBODY FRAGMENTS USING YEAST DISPLAYTECHNOLOGY
Srinath Kasturirangan, Mike Sierks, Arizona State University, Tempe,AZ, USA. Contact e-mail: [email protected]
Background: Deposition of beta-amyloid (A�) is considered an importantearly event in the pathogenesis of Alzheimer’s Disease (AD), and clearance ofA� represents a potential therapeutic approach. The recombinant antibodylight chain fragment, mK18 was identified to have �-secretase-like activity,producing the 1-16 and 17-40 amino acid fragments of A�40 as a primaryproduct. In order to improve the targeting specificity of the proteolytic anti-body for A�, we have constructed a second generation library from a parentsingle chain antibody fragment (scFv) containing the variable domain of theproteolytic light chain and a random heavy chain. Methods: The scFv librarywas created by introducing random mutations in the CDR3 region of the heavychain and subsequently expressing the library on the surface of yeast. Biotin-ylated covalently reactive analogs based on the �-secretase site sequence weresynthesized, immobilized on streptavidin beads, and used to select scFvs withincreased specificity for A� using magnetic bead enrichment. Collected cellswere individually screened for proteolytic activity using 96-well plates and aninternally quenched fluorogenic substrate that contains the �-secretase site ofA�. Several individual clones with increased proteolytic activity were identi-fied from 750 screened clones. Kinetic parameters were determined andcompared. In-vitro studies using Thioflavin-T fluorescence, cytotoxicity usingSHSY5Y cells and AFM imaging will be used to determine the effects of thesecond generation scFv on beta amyloid aggregation. Results: Second gener-ation yeast display library was constructed in yeast. Two clones with highestbinding affinity compared to the original clone were identified. Analysis ofkinetic parameters (kcat and KM) of purified soluble scFv showed one clonehad a 10-fold increase in activity (kcat/KM) toward the synthetic A� substratecompared to the original scFv. Conclusions: Results on construction of thelibrary, synthesis of analogs, the panning protocol and catalytic specificity andactivity of selected clones will be presented. Catalytic activity toward A�
oligomers and fibrils and subsequent effects on aggregation and toxicity willbe discussed.
P2-336 BIS-STYRYLPYRIDINE AND BIS-STYRYLBENZENEDERIVATIVES AS INHIBITORS FOR BETA-AMYLOID FIBRIL FORMATION
Young Soo Kim1, Seong Rim Byeon1, Hye Yun Kim1, Ji Hoon Lee1,Ji-Hoon Sohn2, Inhee Mook-Jung2, Dong Jin Kim1, 1Korea Institute ofScience and Technology, Seoul, Republic of Korea; 2Seoul NationalUniversity, Seoul, Republic of Korea. Contact e-mail: [email protected]
Background: Since cholinergic deficit in the central nervous system hasbeen observed in AD patients, inhibitors for acetylcholine esterase (AchE)such as tacrin, donepezil, and rivastigmine are used for the clinical treat-ment of AD symptoms in current therapeutic approaches. Although theseare the only FDA-approved AD therapies on the market, they cannot solvefundamental problems but can be only palliative solutions. Pathomorpho-logically important hallmarks in AD are senile plaques (SP) composed ofamyloid � (A�) and neurofibrillary tangles (NFTs). Therefore, reducingdeposition of A� plaques in the brain is believed to be pivotal to treat theAD patients. Based on A� hypothesis, A�(1-42) monomers released fromAPP by �- and �-secretases are gradually aggregated to oligomers, proto-fibrils, and fibrils. A�(1-42) aggregates such as oligomers and fibrilsdeposit in the brain causing strong neuronal toxicity. In addition, it wasrecently reported that oligomers might be more toxic than fibrils, and theoligomers are becoming the target for the development of AD treatments.Methods:
Here we report the synthesis and evaluation of novel bis-styrylpyridine andbis-styrylbenzene derivatives as A� aggregation inhibitors. The 34 com-pounds were evaluated by A� fibril formation inhibitory assay usingthioflavin T. Results: Most of the synthesized compounds in this studyinhibited A�(1-42) fibril formation near sub-�M range of IC50 (0.1-2.7�M), better than curcumin (0.8 �M), without cytotoxicities on HT-22neuronal cells by MTT assay at 1, 10, and 50 �M. Conclusions: Inparticular, KMS80013, KMS80019, KMS80029, and KMS80030 exhibitedthe best data of inhibition activities and cytotoxicities and are promisingAD drug candidates.
P2-337 CHARACTERIZATION OF THE AFFINITY OFGSI-953 FOR BINDING TO GAMMA-SECRETASEAND COMPARISON TO BENCHMARK GAMMA-SECRETASE INHIBITORS
Anthony F. Kreft, Magid A. Abou-Gharbia, Ann E. Aulabaugh,Kevin P. Atchison, George Diamantidis, Boyd L. Harrison,Warren D. Hirst, Xinyi Huang, Dennis M. Kubrak, Kerri Lipinski,Ronald L. Magolda, Robert L. Martone, Michael K. May,Scott C. Mayer, Menelas N. Pangalos, Alex M. Porte, Peter H. Reinhart,Lynn Resnick, Hua Zhou, J. Steven Jacobsen, Wyeth Research,Princeton, NJ, USA. Contact e-mail: [email protected]
Background: Blocking the synthesis of the AD pathogen beta amyloidpeptide (�) through inhibition of APP processing by gamma secretase(GS) is a promising therapeutic strategy that may provide the first disease-modifying anti-Alzheimer’s drug (DMAAD). In animal models, GSI-953is a Notch-sparing gamma secretase inhibitor (GSI) and this compound hassubsequently entered clinical trials. Our objective was to characterize therelative affinity of GSI-953 for binding to GS and compare it to the potencyof GS inhibition. Methods: A cell-free GS binding assay was developedemploying membranes isolated from human neuroblastoma SY5Y cells
T471Poster Presentations P2:
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