Toward the Characterization and Identification of γ‐Secretases Using Transition‐state Analogue Inhibitors

Abstract

The amyloid‐β protein (Aβ), strongly implicated in the etiology of Alzheimer's disease (AD), is formed from the amyloid‐β precursor protein (APP) through sequential proteolysis by β‐ and γ‐secretases. Cleavage by γ‐secretase takes place within the middle of the single transmembrane region of APP and results primarily in 40‐ and 42‐amino acid Aβ C‐terminal variants, Aβ40 and Aβ42. The latter form of Aβ is highly fibrillogenic, is invariably elevated in autosomal‐dominant forms of AD, and is the major Aβ component found presymptomatically in cerebral deposits. Thus, blocking production of Aβ in general and Aβ42 in particular is considered an important therapeutic goal. We have developed transition‐state analogue inhibitors of γ‐secretase as molecular probes for characterizing the active site of this enzyme, as pharmacological tools for understanding its role in biology, and as affinity labels toward its definitive identification. Specifically, we found that: (1) difluoro ketone and difluoro alcohol peptidomimetics are effective inhibitors of γ‐secretase activity in APP‐transfected cells, strongly suggesting an aspartyl protease mechanism; (2) γ‐secretases that form Aβ40 and Aβ42 are pharmacologically distinct but are nevertheless closely similar; (3) large hydrophobic P1 substituents increase the inhibitory potency of these peptidomimetics, suggesting a large complementary S1 pocket for γ‐secretases; (4) Aβ42 production is increased several fold over control by these γ‐secretase inhibitors after replacement with inhibitor‐free media; (5) a bromoacetamide derivative of one of these analogues continues to inhibit total Aβ and Aβ42 production hours after replacement with compound‐free media and should help identify the target(s) of these protease transition‐state mimics.

The presenilin hypothesis of Alzheimer's disease: Evidence for a loss-of-function pathogenic mechanism

“…In addition, observation of this phenomenon in both cell culture systems and partially purified membrane preparations makes it unlikely that increased A␤42 production is a result of inhibitory effects on other intracellular processes (36,41). Interestingly, the degree of enhancement of A␤42 production elicited by ␥-secretase inhibitors is strongly correlated with their inhibitory potency (42), suggesting that increased A␤42 generation is mechanistically related to active site-directed inhibition.…”

From Presenilinase to γ-Secretase, Cleave to Capacitate

“…Taking advantage of in vitro presenilinase and -secretase activity assays that share the same conditions for de novo generation of PS NTF and CTF and the generation of A , parallel microsomes were treated with different -secretase inhibitors. Among a number of transition-state analogue inhibitors of -secretase that were shown to inhibit A generation [96][97][98], certainsecretase inhibitors can block presenilinase activity in vitro [90]. For example, some less potent -secretase inhibitors (MW167 and CM35) reduced PS1 fragment generation.…”

The Amyloid-β Rise and γ-Secretase Inhibitor Potency Depend on the Level of Substrate Expression

“…In one study, the A␤ rise was reported in isolated membrane preparations, suggesting a direct effect of peptide aldehydes on ␥-secretase (6). Further evidence that ␥-secretase can mediate the A␤ rise comes from studies with difluoroketone-based inhibitors, which are selective for ␥-secretase and which cause a robust rise in A␤42 both in cell culture (11)(12)(13) and in isolated membrane-based assays (6). Furthermore a rise in total A␤, as well as A␤42, in response to highly selective ␥-secretase inhibitors has been observed in vivo in the plasma of guinea pigs (14) and in humans (15).…”