β-secretase and γ-Secretase Inhibitors

2018-08-18

Proteolytic Processing of APP 

Following discovery of the full-length APP cDNA clone, numerous studies were undertaken to detect the APP protein in cells and tissues. Full-length, membrane-bound forms of APP were readily detected by Western blotting, and it soon became apparent that a large, soluble, N-terminal fragment of APP (sAPPα) is released by the action of a putative "α-secretase" into conditioned tissue culture medium, cerebrospinal fluid, serum, and tissues such as brain. Esch et al. and Anderson et al. showed that this was due to cleavage of APP at the Lys16-Leu17 bond in the middle of the Aβ sequence, which would preclude formation of the intact Aβ peptide. This led to speculation that the production of Aβ from APP must be a purely pathological event. However, it soon became apparent that C-terminally truncated forms of secreted APP completely lacking Aβ immunoreactivity could also be detected, along with C-terminal membrane-associated fragments of APP apparently containing the entire Aβ sequence. Seubert et al. Demonstrated the existence of a form of secreted APP (sAPPβ) that terminates at the Met596 residue immediately prior to the N terminus of the Aβ sequence. This was demonstrated by means of a specific monoclonal antibody (termed "92") to residues 591-596 of APP695, the reaction of which depended on the presence of the free carboxyl-terminal Met596. These observations suggested the presence of an alternative "β-secretase" activity that cleaves APP to release the N terminus of the Aβ peptide. The detection of Aβ itself in culture medium from cells, and in body fluids (cerebrospinal fluid, blood, urine) from normal individuals, showed that this peptide is, in fact, a product of the normal metabolism of APP. These findings also inferred the action of a third "γ-secretase" activity that acts within the membrane-spanning domain of APP to produce the C-terminus of Aβ. The detection of "short" (predominantly Aβ40) and "long" (predominantly Aβ 42) forms of Aβ (see, e.g., ref. 38) was also important, given later data on the effects of familial AD mutations on APP processing. The Aβ peptide may be physiologically active in brain, as in its soluble form it has weak neurotrophic properties.

The identity of the α-, β-, and γ-secretases is unknown, although it is likely that α-secretase is a zinc metalloproteinase. There are numerous reports claiming identification of β-secretase and fewer reports claiming the identification of γ-secretase, but in no case for the various candidates in the litreature is there strong evidence that they are actually β- or γ-secretase. As far as β-secretase is concerned, the multicatalytic proteinase or "proteasome" has been implicated, as have several chymotrypsin-like serine proteinases. The metallopeptidase thimet has been proposed, but has always been an unlikely candidate, as it seems not to tolerate large substrates such as APP, and can now be discounted. Cathepsin D (an aspartyl proteinase) has received considerable attention as a potential β-secretase due to its ability to cleave peptide substrates containing the APP Swedish mutant sequence at a much faster rate than the normal sequence. However, the fact that cathepsin D knockout mice still produce Aβ indicates that this enzyme cannot be β-secretase.

A number of small peptide aldehydes of the type known to inhibit both cysteine and serine proteinases have been shown to inhibit Aβ formation from cultured cells, probably through inhibition of the γ-secretase pathway. The activity of these compounds as inhibitors of γ-secretase cleavage has been shown to correlate with their potency as inhibitors of the chymotrypsin-like activity of the proteasome, suggesting that the latter may be involved, either directly or indirectly, in the γ-secretase cleavage event. Further candidates for γ-secretase include prolyl endopeptidase, and cathepsin D.

β-Secretase and Inhibitors

Because Aβ is so closely associated with AD, the proteases that produce this peptide, β- and γ-secretases, have been top targets for therapeutic development. β-Secretase (also called BACE1) is a membrane-anchored aspartyl protease in the pepsin family, and a soluble recombinant form of this protease has been cocrystallised with an active site-directed inhibitor. The search for β-secretase inhibitors has been greatly facilitated by its resemblance to other aspartyl protease targets, particularly renin and HIV protease, and the ability to do structure-based design. Despite these advantages, the development of β-secretase inhibitors as practical drug candidates has been challenging. One reason is the nature of β-secretase active site, which is relatively long, shallow and hydrophilic, making difficult the discovery of small, potent inhibitors that penetrate biological membranes. The other problem is the tendency of β-secretase inhibitors to be substrates for P-glycoprotein, which transports the compounds out of the brain.

γ-Secretase Inhibitors and Modulators

γ-Secretase is considerably more complicated than β-secretase. While a single protein is responsible for β-secretase activity, γ-secretase is a complex of integral membrane proteins that includes presenilin. Pharmacological evidence suggested an aspartyl protease mechanism for this enzyme: peptide analogues that mimic an intermediate in aspartyl protease catalysis inhibit gsecretase. Moreover, knockout of presenilin abolishes γ-secretase activity, and two conserved transmembrane aspartates are critical for γ-secretase activity, consistent with the fact that this enzyme cleaves within the transmembrane domain of APP. However, presenilin does not act alone. This protein enters high molecular weight complexes and itself is cut into two pieces that remain associated. These heterodimers are metabolically stable, and their formation is tightly regulated, suggesting that they are the bioactive form of presenilin. Indeed, active site-directed inhibitors of γ-secretase bind directly to presenilin heterodimers, strong evidence that the catalytic centre of this protease resides at the heterodimeric interface. It is now clear that the γ-secretase complex consists of presenilin heterodimers, a single-pass membrane protein called nicastrin, and two other integral membrane proteins called aph-1 and pen-2.

References:

Westermark, P., Bellotti, V., Obici, L., Kisilevsky, R., Merlini, G., Sipe, J. D., ... & Proteoglycans, E. M. H. S. (2005). Amyloid proteins: the beta sheet conformation and disease.

Martínez, A. (Ed.). (2010). Emerging Drugs and Targets for Alzheimer's Disease: Volume 1: Beta-Amyloid, Tau Protein and Glucose Metabolism. Royal Society of Chemistry.

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