Alzheimer's Amyloid Precursor Protein α-Secretase Is Inhibited by Hydroxamic Acid-Based Zinc Metalloprotease Inhibitors:  Similarities to the Angiotensin Converting Enzyme Secretase

Abstract

The 4 kDa beta-amyloid peptide that forms the amyloid fibrils in the brain parenchyma of Alzheimer's disease patients is derived from the larger integral membrane protein, the amyloid precursor protein. In the nonamyloidogenic pathway, alpha-secretase cleaves the amyloid precursor protein within the beta-amyloid domain, releasing an extracellular portion and thereby preventing deposition of the intact amyloidogenic peptide. The release of the amyloid precursor protein from both SH-SY5Y and IMR-32 neuronal cells by alpha-secretase was blocked by batimastat and other related synthetic hydroxamic acid-based zinc metalloprotease inhibitors, but not by the structurally unrelated zinc metalloprotease inhibitors enalaprilat and phosphoramidon. Batimastat inhibited the release of the amyloid precursor protein from both cell lines with an I50 value of 3 microM. Removal of the thienothiomethyl substituent adjacent to the hydroxamic acid moiety or the substitution of the P2' substituent decreased the inhibitory potency of batimastat toward alpha-secretase. In the SH-SY5Y cells, both the basal and the carbachol-stimulated release of the amyloid precursor protein were blocked by batimastat. In contrast, neither the level of full-length amyloid precursor protein nor its cleavage by beta-secretase were inhibited by any of the zinc metalloprotease inhibitors examined. In transfected IMR-32 cells, the release of both the amyloid precursor protein and angiotensin converting enzyme was inhibited by batimastat, marimastat, and BB2116 with I50 values in the low micromolar range, while batimastat and BB2116 inhibited the release of both proteins from HUVECs. The profile of inhibition of alpha-secretase by batimastat and structurally related compounds is identical with that observed with the angiotensin converting enzyme secretase suggesting that the two are closely related zinc metalloproteases.

A Point Mutation in the Juxtamembrane Stalk of Human Angiotensin I-converting Enzyme Invokes the Action of a Distinct Secretase

“…As compared with untreated cells, batimastat significantly inhibited (81%) the release of wtACE into the medium with a concomitant increase in the amount of activity detected in the membrane fraction, consistent with previous results (8). Surprisingly, no ACE activity could be detected in the membranes from the cells expressing ACE NQ , and batimastat failed to significantly inhibit the release of this mutant form of the protein into the medium.…”

“…Cells-IMR-32 cells, which previously have been shown to cleave and release ACE in a batimastat-sensitive manner (8), were transfected with cDNA encoding either wtACE or ACE NQ . Both constructs were expressed in the cells as determined by metabolic labeling with [ 35 S]Met followed by immunoprecipitation from the cell lysate with the anti-ACE antibody (Fig.…”

“…The secretase responsible for the cleavage and secretion of ACE is a zinc metalloproteinase located at the cell surface (3)(4)(5)(6). It is inhibited by hydroxamate-based compounds such as batimastat and displays a remarkably similar inhibition profile to that of the ␣-secretase that cleaves the amyloid precursor protein (7,8). The precise site of cleavage in ACE has been determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometry following enzymic fragmentation of the purified protein (9).…”

The Proteolytic Processing of the Amyloid Precursor Protein Gene Family Members APLP-1 and APLP-2 Involves α-, β-, γ-, and ϵ-Like Cleavages

“…6A, lane 5). The secretion of the small peptides was also reduced by treatment with the hydroxamate-based inhibitor batimastat (55) (Fig. 6A, lane 3), suggesting that they are produced by an ␣-secretase activity and are homologous to the p3 peptides of APP.…”

Cellular prion protein regulates β-secretase cleavage of the Alzheimer's amyloid precursor protein

“…Antibody 6H4 (Prionics AG, Zurich, Switzerland) recognizes the sequence DYEDRYYRE (human PrP: amino acids 144-152). Ab54 recognizes the C-terminal region of APP, and antibody 1A9 recognizes a neoepitope on sAPP␤ formed after ␤-secretase cleavage of APP (36). Antibody 9B21 was raised to the catalytic domain of BACE1 by using BACE1-Fc fusion protein as immunogen.…”