Recent findings about the relationship between amyloid b-peptide (Ab) accumulation and cognitive decline in Alzheimer's disease (AD) suggest a complex role for Ab in this neurodegenerative process. The transition of Ab soluble monomers to toxic small oligomers involves an initial transition from a non organized/a-helix monomer to a b-sheet rich conformer. This early step represents a suitable target to design new potent inhibitors and obtain effective therapeutics for AD. Moreover, several chaperone molecules are though to accelerate amyloid aggregation, i.e., the enzyme acetylcholinesterase (AChE). Since most of the marketed drugs for AD are AChE inhibitors, the investigation of the inhibitory potency against the AChE-induced amyloid aggregation exerted by anti-cholinesterase agents definitively represents an interesting area of investigation for drug discovery. The in vitro Ab aggregation can be evaluated by a fluorescence-based assay by using Thioflavin T (ThT) as fluorescent dye. More in details, ThT specifically binds to amyloid fibrils (in the beta-sheet conformation) giving rise to an intense specific emission band (lem = 490 nm) in its fluorescent spectrum (1-3). Therefore the increase in the specific fluorescence emission was used to monitor amyloid fibrils formation. Two fluorescence-based assays specifically developed (4,5) for the evaluation of beta-amyloid self- and AChE-induced aggregation were applied to follow the aggregation process as well as to screen for potential inhibitors. The concomitant use of circular dichroism spectroscopy was helpful to set up the optimal experimental conditions and to draw some hypotheses on the mechanism of action of known and new inhibitors. (1) H. Naiki, K. Higuchi, K. Nakakuki, T. Takeda, Lab. Invest. 1991, 65, 104. (2) H. LeVine, Protein Sci. 1993, 2, 404. (3) H. LeVine 3rd, Methods Enzymol. 1999, 309, 274. (4) M. Bartolini, C. Bertucci, M.L. Bolognesi, A. Cavalli, C. Melchiorre, V. Andrisano, Chembiochem 2007, 8, 2152. (5) M. Bartolini, C. Bertucci, V. Cavrini, V. Andrisano, Biochem. Pharmacol. 2003, 65, 407.
M. Bartolini, C. Bertucci, V. Andrisano (2008). FLUORESCENCE BASED STUDIES ON BETA-AMYLOID MISFOLDING AND AGGREGATION. BOLOGNA : s.n.
FLUORESCENCE BASED STUDIES ON BETA-AMYLOID MISFOLDING AND AGGREGATION
BARTOLINI, MANUELA;BERTUCCI, CARLO;ANDRISANO, VINCENZA
2008
Abstract
Recent findings about the relationship between amyloid b-peptide (Ab) accumulation and cognitive decline in Alzheimer's disease (AD) suggest a complex role for Ab in this neurodegenerative process. The transition of Ab soluble monomers to toxic small oligomers involves an initial transition from a non organized/a-helix monomer to a b-sheet rich conformer. This early step represents a suitable target to design new potent inhibitors and obtain effective therapeutics for AD. Moreover, several chaperone molecules are though to accelerate amyloid aggregation, i.e., the enzyme acetylcholinesterase (AChE). Since most of the marketed drugs for AD are AChE inhibitors, the investigation of the inhibitory potency against the AChE-induced amyloid aggregation exerted by anti-cholinesterase agents definitively represents an interesting area of investigation for drug discovery. The in vitro Ab aggregation can be evaluated by a fluorescence-based assay by using Thioflavin T (ThT) as fluorescent dye. More in details, ThT specifically binds to amyloid fibrils (in the beta-sheet conformation) giving rise to an intense specific emission band (lem = 490 nm) in its fluorescent spectrum (1-3). Therefore the increase in the specific fluorescence emission was used to monitor amyloid fibrils formation. Two fluorescence-based assays specifically developed (4,5) for the evaluation of beta-amyloid self- and AChE-induced aggregation were applied to follow the aggregation process as well as to screen for potential inhibitors. The concomitant use of circular dichroism spectroscopy was helpful to set up the optimal experimental conditions and to draw some hypotheses on the mechanism of action of known and new inhibitors. (1) H. Naiki, K. Higuchi, K. Nakakuki, T. Takeda, Lab. Invest. 1991, 65, 104. (2) H. LeVine, Protein Sci. 1993, 2, 404. (3) H. LeVine 3rd, Methods Enzymol. 1999, 309, 274. (4) M. Bartolini, C. Bertucci, M.L. Bolognesi, A. Cavalli, C. Melchiorre, V. Andrisano, Chembiochem 2007, 8, 2152. (5) M. Bartolini, C. Bertucci, V. Cavrini, V. Andrisano, Biochem. Pharmacol. 2003, 65, 407.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
