Targeting ySecretase for Alzheimers Disease

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Michael S. Wolfe1

Y-Secretase is responsible for the final proteolysis that produces the amyloid p-peptide (Ap) from its precursor, amyloid precursor protein (APP), and has been considered a potential therapeutic target for Alzheimer's disease (AD) since the early 1990s, even before anything was known about its character or identity. This protease activity, which takes place within the transmembrane domain of APP, generates heterogeneity at the C-terminus of Ap peptides, forming longer, minor variants, especially the 42-residue variant (Ap42), which is highly prone to aggregation and represents the major species of Ap found deposited in the characteristic cerebral plaques of AD (Hardy and Selkoe 2002). The first hint to the identity of Y-secretase was the discovery that AD-associated missense mutations in the presenilin genes, presenilin-1 and presenilin-2, cause increases in the ratio of Ap42 to the less aggregation prone 40-residue variant (Citron et al. 1997; Duff et al. 1996; Lemere et al. 1996; Scheuner et al. 1996). Subsequently, two major clues were the finding that knockout of presenilin-1 dramatically reduces Ap production at the level of Y-secretase (De Strooper et al. 1998) and the observation that aspartyl protease transition-state mimics can likewise inhibit Y-secretase activity in cultured cells (Wolfe et al. 1999b).

Connecting these clues led to the hypothesis that presenilin might be a novel membrane-embedded aspartyl protease and the discovery that two conserved transmembrane aspartates in the presenilins are indeed critical for Y-secretase activity (Wolfe et al. 1999a, 1999c). Further support for this hypothesis soon followed. First, the aspartyl protease transition-state mimicking inhibitors of Y-secretase were found to directly interact with presenilin-1 (Esler et al. 2000; Li et al. 2000). Second, presenilin consistently came along with Y-secretase activity through biochemical purification steps as part of a high molecular weight complex (Esler et al. 2002; Kimberly et al. 2003; Li et al. 2000). Third, a distantly related presenilin homolog was discovered to be the protease signal peptide peptide (Weihofen et al. 2002). It is now clear that Y-secretase is a complex of four different integral membrane proteins, with presenilin ostensibly being the catalytic component (Edbauer et al. 2003; Fraering et al. 2004b; Kimberly et al. 2003; Takasugi et al. 2003; Fig. 1). During assembly of this complex, presenilin undergoes cleavage into two subunits (Thinakaran et al. 1996) (likely through autopro-teolysis; Wolfe et al. 1999c)), each of which contributes one of the key aspartates to the active site (Wolfe et al. 1999c). Because the active site contains water and two aspartates, it is likely sequestered from the hydrophobic lipids (Wolfe et al. 1999a). Indeed, the enzyme apparently contains an initial docking site for substrate that is distinct from the

1 Center for Neurologic Diseases, Harvard Medical School and Brigham and Women's Hospital, 77 Avenue Louis Pasteur, H.I.M. 754, Boston, MA 02115 U.S.A.

Presenilin Nicastrin Aph-1 Pen-2

Presenilin Nicastrin Aph-1 Pen-2

Fig. 1. Components of the y-secretase complex. y-Secretase is composed of four different integral membrane proteins: presenilin, nicastrin, Aph-1, and Pen-2. Presenilin undergoes endoproteoly-sis into an N-terminal fragment (NTF) and a C-terminal fragment (CTF) that remain associated. Two conserved aspartates within adjacent transmembrane domains are essential for both presenilin endoproteolysis and y-secretase activity

Fig. 1. Components of the y-secretase complex. y-Secretase is composed of four different integral membrane proteins: presenilin, nicastrin, Aph-1, and Pen-2. Presenilin undergoes endoproteoly-sis into an N-terminal fragment (NTF) and a C-terminal fragment (CTF) that remain associated. Two conserved aspartates within adjacent transmembrane domains are essential for both presenilin endoproteolysis and y-secretase activity active site (Esler et al. 2002), and evidence suggests that this docking site is also at the interface between the two presenilin subunits (Kornilova et al. 2005). Thus, substrate passes, in whole or in part, between these subunits to access the internal active site.

In parallel with the discoveries connecting presenilin to APP processing and AD were studies revealing a role of presenilin in the Notch signaling pathway of developmental biology (Selkoe and Kopan 2003). This revelation proved critical for identifying other members of the protease complex, two of which were discovered via genetic screens using Notch-deficient phenotypes as a read-out (Francis et al. 2002; Goutte et al. 2002). Notch, like APP, was found to be cleaved within its transmembrane domain, and this proteolysis is necessary for Notch signaling and cell fate determinations (Schroeter et al. 1998). Presenilin is necessary for this transmembrane cleavage (De Strooper et al. 1999), and knockout of presenilin-1 results in a lethal phenotype similar to that seen upon knockout of Notch1 (Shen et al. 1997; Wong et al. 1997). These findings began to raise concerns about y-secretase as a target for AD: inhibition of this protease, while lowering Ap production, might cause severe toxicities due to blocking critical cell differentiation events. The remainder of this chapter provides a current assessment of the therapeutic potential of targeting y-secretase, especially strategies for lowering Ap without affecting Notch signaling.

Although y-secretase has in many ways been an attractive target for Alzheimer therapeutics, interference with Notch processing and signaling may lead to toxicities that preclude clinical use of inhibitors of this protease. Knockout of Notch1 or presenilin-1 is lethal in embryonic mice (Shen et al. 1997; Wong et al. 1997), but Notch signaling and y-secretase activity are crucial in adulthood as well, because Notch plays a critical role in many cell differentiation events (Selkoe and Kopan 2003). Indeed, long-term

Targeting y-Secretase for Alzheimer's Disease

Targeting y-Secretase for Alzheimer's Disease

Fig. 2. y-secretase inhibitor (left) and modulator (right) currently in clinical trials for the treatment of AD

LY450139 R-Flurbiprofen

Fig. 2. y-secretase inhibitor (left) and modulator (right) currently in clinical trials for the treatment of AD

treatment with y-secretase inhibitors causes severe gastrointestinal toxicity and interferes with the maturation of B- and T-lympocytes in mice, effects that are indeed due to inhibition of Notch processing and signaling (Searfoss et al. 2003; Wong et al. 2004). Nevertheless, hope remains that a y-secretase inhibitor might lower Ap production in the brain enough to prevent Ap oligomerization and fibril formation while leaving enough Notch signaling intact to avoid toxic effects. Presently, the Eli Lilly compound. LY450139 (Fig. 2), is in Phase II clinical trials in the US, with the dose being cautiously increased to that needed for Ap lowering. Compounds in this general structural class have not displayed selective inhibition of APP processing with respect to that of Notch (Wong et al. 2004). So far, LY450139 has been shown to lower Ap in plasma but not in cerebral spinal fluid, while signs of toxicity have been minimal (Siemers et al. 2006).

In contrast, compounds that can modulate the enzyme to alter or block Ap production with little or no effect on Notch would bypass this potential roadblock to therapeutics. Recent studies suggest that the protease complex contains allosteric binding sites that can alter substrate selectivity and the sites of substrate proteolysis. Certain non-steroidal anti-inflammatory drugs (NSAIDs; e.g., ibuprofen, indomethacin, and sulindac sulfide) can reduce the production of the highly aggregation-prone Ap42 peptide and increase a 38-residue form of Ap, a pharmacological property independent of inhibition of cyclooxygenase (Weggen et al. 2001). The alteration of the proteolytic cleavage site is observed with isolated or purified y-secretase (Fraering et al. 2004b; Weggen et al. 2003), indicating that the compounds can interact directly with the protease complex to exert these effects. Enzyme kinetic studies and displacement experiments suggest the selective NSAIDs can be noncompetitive with respective to APP substrate and to a transition-state analogue inhibitor, suggesting interaction with a site distinct from the active site and the docking site (Beher et al. 2004). The site of cleavage within the Notch transmembrane domain is similarly affected, but this subtle change does not inhibit the release of the intracellular domain and thus does not affect Notch signaling (Okochi et al. 2006). For this reason, these agents maybe safer as Alzheimer therapeutics than inhibitors that block the active site or the docking site. Indeed, one compound, R-flurbiprofen or Flurizan (Fig. 2), has recently advanced to Phase III clinical trials in the US. However, the potency of this drug candidate (Eriksen et al. 2003) and other NSAIDs toward Ap42 lowering raises questions about efficacy.

Another type of allosteric modulator includes the compounds that resemble kinase inhibitors and interact with a nucleotide binding site on the y-secretase complex. The discovery that adenosine triphosphate (ATP) can increase Ap production in membrane preparations prompted the testing of a variety of compounds known to interact with ATP binding sites on other proteins (Netzer et al. 2003). In this focused screen, the Abl kinase inhibitor Gleevec emerged as a selective inhibitor of Ap production in cells without affecting the proteolysis of Notch. In light of these findings, ATP and other nucleotides were tested for effects on purified y-secretase preparations and found to selectively increase the proteolytic processing of a purified recombinant APP-based substrate without affecting the proteolysis of a Notch counterpart (Fraering et al. 2005). Furthermore, certain compounds known to interact with ATP binding sites were found to selectively inhibit APP processing vis-à-vis Notch in purified protease preparations. The y-secretasecomplex couldbepulleddownwith beads containing immobilized ATP, and the presenilin-1 CTF was specifically photolabeled by 8-azido-ATP. This labeling was not blocked by a transition-state analogue inhibitor or by purified, recombinant APP- and Notch-based substrates; however, the APP-selective inhibitors could prevent photolabeling by 8-azido-ATP. Taken together, these results suggest that the y-secretase complex contains a nucleotide binding site, to which the presenilin-1 CTF is at least a contributor, and that this site allows allosteric regulation of y-secretase processing of APP with respect to Notch. Whether this regulation is physiologically important is unclear, but the pharmacological relevance is profound and may lead to new therapeutic candidates for AD. This hope is tempered by the fact that y-secretase cleaves numerous other type I membrane protein stubs that result from ectodomain shedding (Kopan and Ilagan 2004). Agents selective for APP versus Notch may reveal new long-term toxicities due to blocking proteolysis of these other substrates, toxicities that are masked by the severe Notch-related effects with nonselective inhibitors.

In conclusion, our knowledge of y-secretase and its role in AD and in biology has increased dramatically in the past ten years. The identification, purification and characterization of the full protease complex leave structural biology as the next frontier toward an intimate understanding of how this enzyme carries out hydrolysis within the boundaries of the hydrophobic environment of the lipid bilayer. Meanwhile, the discovery that the protease complex can be modulated to block or alter Ap production without affecting Notch proteolysis or signaling suggests that the toxicities associated with nonselective inhibitors can be overcome. Ultimately, these paths should intersect, allowing structure-based design of selective y-secretase modulators for the treatment of AD.

Bart De Strooper

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