Structure−Activity Relationship of Hydroxamate-Based Inhibitors on the Secretases that Cleave the Amyloid Precursor Protein, Angiotensin Converting Enzyme, CD23, and Pro-Tumor Necrosis Factor-α

Abstract

Multiple proteins are proteolytically shed from the membrane, including the amyloid precursor protein (APP) involved in Alzheimer's disease, the blood pressure regulating angiotensin converting enzyme (ACE), the low affinity IgE receptor CD23, and the inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). The inhibitory effect of a range of hydroxamic acid-based compounds on the secretases involved in cleaving and releasing these four proteins has been examined to build up a structure-activity relationship. Compounds have been identified that can discriminate between TNF-alpha convertase and the other three secretases (compound 15), between the shedding of CD23 and the shedding of APP and ACE (compound 21), and between the secretases and matrix metalloproteinase-1 (compound 22). The structure-activity relationship for the APP alpha-secretase and the ACE secretase were remarkably similar, and both secretases were activated in whole cell systems by the serine proteinase inhibitor 3,4-dichloroisocoumarin. The basal and carbachol-stimulated shedding of APP and ACE from human SH-SY5Y neuroblastoma cells could not be differentiated by any of the hydroxamate compounds, implying that the same or very similar activities are involved in the constitutive and regulated shedding of these two proteins. By utilizing a key discriminatory compound (compound 15) that potently inhibits TNF-alpha convertase but not alpha-secretase, we show that TNF-alpha convertase is not involved in the regulated shedding of APP from human neuronal cells. The compounds reported here will be useful in future studies aimed at identifying and validating candidate secretases.

Oxidation of Peptides by Methyl(trifluoromethyl)dioxirane: The Protecting Group Matters

“…The β-CH bond of valine in Ac-Val-Ala-OMe (11) reacted to a lower extent, as in the simpler case 8, N-Ac-Val(β-OH)-Ala-OMe (11a) being produced in 12% yield (entry 5). N-Ac-Leu-Val-OMe (12) and…”

Dual Mechanisms for Shedding of the Cellular Prion Protein

“…For stable transfections, 30 g of DNA was introduced to cells by electroporation, and selection was performed in normal growth medium containing 500 g/ml neomycin selection antibiotic. SB 244000 and all other hydroxamate-based inhibitor compounds were synthesized at GlaxoSmithKline Pharmaceuticals (Harlow, UK) and were used at 20 M. Structural details of these compounds have been published previously (25). Phorbol myristate acetate (PMA), filipin, and M␤CD (Sigma) were used at 1 M, 10 nM, and 5 mM, respectively.…”

“…APP is expressed endogenously in SH-SY5Y cells; therefore, its shedding can be compared directly with that of PrP overexpressed in the same cells. Cells were incubated in the absence or presence of a range of zinc metalloprotease hydroxamate-based inhibitors (25). Conditioned media were immunoblotted with the APP monoclonal antibody 6E10 (Fig.…”

The Kallikrein-Kinin System: Current and Future Pharmacological Targets

“…Although the ACE secretase has not yet been identified, studies with a range of hydroxamic acid-based inhibitors have shown that it has a remarkably similar inhibition profile to the amyloid precursor protein α-secretase, leading to the conclusion that the two secretases are, at the very least, closely related (65). Human plasma ACE originates from endothelial cells, while in other body fluids ACE originates from epithelial, endothelial, or germinal cells (66).…”