Biomedical subjects
Publications and source records attributed to Thomas L Emmons.
We describe an optimized series of acyclic hydroxyethylamine transition state isosteres of beta-secretase that incorporates a variety of P(2) side chains that yield potent inhibitors with excellent cellular activity. A 2.2A crystal structure of compound 13 is shown.
We describe a novel series of potent inhibitors of human beta-secretase. These compounds possess the hydroxyethyl amine transition state isostere. A 2.5A crystal structure of inhibitor 32 bound to BACE is provided.
The 43-amino acid Alzheimer's amyloid-beta peptide (Abeta peptide) retains a predominantly alpha-helix and beta-strand structure in sodium dodecyl sulfate (SDS) solution. This conformer has a high tendency to aggregate during conventional SDS-polyacrylamide gel electrophoresis (PAGE). Both the secondary structure and the proclivity for aggregation are obviated by the use of urea-SDS-PAGE: In 8M urea-with or without SDS-the Abeta peptide becomes 100% random coil and remains monomeric. However, during electrophoresis in this medium, the peptide and its truncated variants do not obey the law of mass/mobility relationship that most proteins-including Abeta peptides-follow in conventional SDS-PAGE. Rather, the smaller carboxy-terminally truncated peptides migrate slower than the larger full-length peptide, while the amino terminally truncated peptide does migrate faster than the full-length Abeta peptide. Thus, despite their small size (2-4kDa) and minor differences between their lengths, the Abeta peptides display a wide separation in this low-porosity (12% acrylamide) gel. We found that this unusual electrophoretic mobility in 8M urea is due to the fact that the quantity of [35S]SDS bound to the Abeta peptides, instead of being proportional to the total number of amino acids, is rather proportional to the sum of the hydrophobicity consensus indices of the constituent amino acids. It is then their hydrophobicity and, hence, the net negative charges contributed by the peptide-bound SDS that plays a major role in determining the mobility of Abeta peptides in 8M urea-SDS-PAGE. The high selectivity of the 8M urea-SDS-PAGE method allowed us to detect the presence of hitherto unknown Abeta peptide variants that were secreted in the conditioned medium by cultured HeLa cells.
The involvement of beta-secretase (BACE1; beta-site APP-cleaving enzyme) in producing the beta-amyloid component of plaques found in the brains of Alzheimer's patients, has fueled a major research effort to characterize this protease. Here, we describe work toward understanding the substrate specificity of BACE1 that began by considering the natural APP substrate and its Swedish mutant, APPSw, and proceeded on to include oxidized insulin B chain and ubiquitin substrates. From these findings, and the study of additional synthetic peptides, we determined that a decapeptide derived from APP in which the P3-P2' sequence, ...VKM--DA..., was replaced by ...ISY--EV... (-- = beta site of cleavage), yielded a substrate that was cleaved by BACE1 seven times faster than the corresponding APPSw peptide, SEVNL--DAEFR. The expanded peptide, GLTNIKTEEISEISY--EVEFRWKK, was cleaved an additional seven times faster than its decapeptide counterpart (boldface), and provides a substrate allowing assay of BACE1 at picomolar concentrations. Several APP mutants reflecting these beta-site amino acid changes were prepared as the basis for cellular assays. The APPISYEV mutant proved to be a cellular substrate that was superior to APPSw. The assay based on APPISYEV is highly specific for measuring BACE1 activity in cells; its homolog, BACE2, barely cleaved APPISYEV at the beta-site. Insertion of the optimized ISY--EV motif at either the beta-site (Asp1) or beta'-site (Glu11) directs the rate of cellular processing of APP at these two accessible sites. Thus, we have identified optimal BACE1 substrates that will be useful to elucidate the cellular enzymatic actions of BACE1, and for design of inhibitors that might be of therapeutic benefit in Alzheimer's disease.