batimastat and Alzheimer-Disease

Alzheimer's disease is characterised by the progressive deposition of the 4 kDa beta-amyloid peptide (A beta) in extracellular senile plaques in the brain. A beta is derived by proteolytic cleavage of the amyloid precursor protein (APP) by various proteinases termed secretases. alpha-Secretase is inhibited by hydroxamate-based zinc metalloproteinase inhibitors such as batimastat with I50 values in the low micromolar range, and displays many properties in common with the secretase that releases angiotensin converting enzyme. A cell impermeant biotinylated derivative of one such inhibitor completely blocked the release of APP from the surface of neuronal cells, indicating that alpha-secretase cleaves APP at the cell-surface. A range of hydroxamate-based compounds have been used to distinguish between alpha-secretase and tumour necrosis factor-alpha convertase, a member of the ADAMs (a disintegrin and metalloproteinase-like) family of zinc metalloproteinases. Recent data suggests that the presenilins may be aspartyl proteinases with the specificity of gamma-secretase. Although APP and the presenilins are present in detergent-insoluble, cholesterol- and glycosphingolipid-rich lipid rafts, they do not behave as typical lipid raft proteins, and thus it is unclear whether these membrane domains are the sites for proteolytic processing of APP.

Topics: ADAM Proteins; ADAM17 Protein; Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Animals; Aspartic Acid Endopeptidases; Biotinylation; Endopeptidases; Humans; Hydroxamic Acids; Membrane Lipids; Membrane Proteins; Metalloendopeptidases; Nerve Tissue Proteins; Phenylalanine; Presenilin-1; Presenilin-2; Protease Inhibitors; Rats; Thiophenes

Aberrant production of amyloid-beta peptides by processing of the beta-amyloid precursor protein leads to the formation of characteristic extracellular protein deposits which are thought to be the cause of Alzheimer's disease. Therefore, inhibiting the key enzymes responsible for amyloid-beta peptide generation, beta- and gamma-secretase may offer an opportunity to intervene with the progression of the disease. In human brain and cell culture systems a heterogeneous population of amyloid-beta peptides with various truncations is detected and at present, it is unclear how they are produced. We have used a combination of surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) and a specific inhibitor of gamma-secretase to investigate whether the production of all amyloid-beta peptide species requires the action of gamma-secretase. Using this approach, we demonstrate that the production of all truncated amyloid-beta peptides except those released by the action of the nonamyloidogenic alpha-secretase enzyme or potentially beta-site betaAPP cleaving enzyme 2 depends on gamma-secretase activity. This indicates that none of these peptides are generated by a separate enzyme entity and a specific inhibitor of the gamma-secretase enzyme should havethe potential to block the generation of all amyloidogenicpeptides. Furthermore in the presence of gamma-secretase inhibitors, the observation of increased cleavage of the membrane-bound betaAPP C-terminal fragment C99 by alpha-secretase suggests that during its trafficking C99 encounters compartments in which alpha-secretase activity resides.

Topics: Alzheimer Disease; Amino Acid Sequence; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Antibodies, Monoclonal; Antibody Specificity; Aspartic Acid Endopeptidases; Cell Line; Culture Media, Conditioned; Endopeptidases; Enzyme Activation; Enzyme Inhibitors; Fluorobenzenes; Humans; Kidney; Mass Spectrometry; Metalloendopeptidases; Molecular Sequence Data; Neuroblastoma; Pentanoic Acids; Peptide Fragments; Peptides; Phenylalanine; Recombinant Proteins; Thiophenes; Transfection

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.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Angiotensin-Converting Enzyme Inhibitors; Animals; Aspartic Acid Endopeptidases; Drug Screening Assays, Antitumor; Endopeptidases; Enzyme Inhibitors; Humans; Hydroxamic Acids; Metalloendopeptidases; Microvilli; Neuroblastoma; Peptidyl-Dipeptidase A; Phenylalanine; Swine; Tetrazolium Salts; Thiophenes; Tumor Cells, Cultured; Zinc