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γ-Secretase: a complex target for Alzheimer's disease
g-Secretase: a complex target for Alzheimer’s disease Johan Lundkvist1 and Jan Na¨slund2 Data accumulated during the past two decades place amyloid b-peptide (Ab) at center stage as the main perpetrator in initiating the pathological cascade that eventually leads to Alzheimer’s disease. Consequently, significant resources have been allocated to identify and develop treatment strategies that alter the metabolism of Ab. The g-secretase protease has deservedly received attention as an attractive drug target, as it is directly involved in Ab biogenesis and determines the pathogenic potential of Ab by its heterogeneous catalytic action, generating peptides of various lengths. Despite the complexity of the multi-subunit g-secretase and the lack of structural information, drug discovery research has identified small-molecule compounds that inhibit or modulate activity of this enzyme and some of these have already entered clinical trials. Addresses 1 AstraZeneca CNS/Pain, Department of Molecular Pharmacology, So¨derta¨lje, SE-151 85, Sweden 2 Karolinska Institutet, Department of NVS, KI-Alzheimer Research Center, Novum, Huddinge, SE-141 57, Sweden Corresponding author: Na¨slund, Jan ([email protected])
molecular underpinnings of the disease, allowing exploration of multiple avenues for the development of diseasemodifying drugs. In this review, we highlight research aimed at achieving this objective by specifically targeting the so-called g-secretase enzyme. For other contemporary approaches, we refer the reader to some recently published reviews [2–4].
Disease pathogenesis and the amyloid cascade hypothesis Light microscopy analysis of sections from postmortem AD brain reveals the two neuropathological hallmarks of AD: an abundance of extracellular amyloid plaques in the brain parenchyma and cerebral vessels, and intraneuronal neurofibrillary tangles [5]. The major component of plaques is a fibrillar form of the 40–42 residue amyloid bpeptide (Ab). Deposited Ab contains many N-terminally truncated variants, although the C terminus appears to be more homogenous and predominantly ends with Val-40 (Ab40) or Ala-42 (Ab42). The Ab peptide is an integral part of the type I transmembrane protein APP (amyloid precursor protein), thus its formation requires the action of proteases.
Current Opinion in Pharmacology 2007, 7:112–118 This review comes from a themed issue on Neurosciences Edited by Karima Chergui, Bertil Fredholm and Per Svenningsson Available online 13th December 2006 1471-4892/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2006.10.002
Introduction Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a neurodegenerative disease characterized by progressive memory impairment, cognitive deficits and behavioral changes [1]. This year, we honour the century-old pioneering work of Alois Alzheimer in which the Bavarian psychiatrist described the clinical and pathological features of the syndrome that now bears his name. Improved life expectancy and a demographic shift towards an elderly population will probably increase the prevalence of AD in the future. This awareness, together with the enormous social and economical costs of the disease, has sparked an intense research effort to combat AD. There are currently no drugs available that can be used in the causal treatment or prevention of AD. Nevertheless, research in the past two decades has significantly increased our knowledge of the Current Opinion in Pharmacology 2007, 7:112–118
A fundamental role for Ab peptide in AD pathogenesis has been proposed by the ‘amyloid cascade hypothesis’ [6]. This working hypothesis is based on a number of observations accumulated over the years. The most straightforward evidence derives from studies of gene mutations that co-segregate with autosomal-dominant familial forms of AD. The vast majority of these mutations in the APP gene or the presenilin (PS) 1 or 2 genes either increases the production of the Ab42 peptide or enhances the Ab42/Ab40 ratio. This finding becomes significant in light of studies showing that the Ab42 peptide is more amyloidogenic and deposits earlier in the time course of the disease than the slightly shorter Ab40 variant. Moreover, transgenic mice expressing human mutant APP alone or together with mutant PS1 develop varying degrees of memory deficits and recapitulate some of the neuropathological features associated with AD. Consequently, inhibiting the production of Ab, and in particular the Ab42 peptide, constitutes a rational therapeutic approach for the treatment of AD.
APP processing and biogenesis of the Ab peptide The Ab peptide is generated from APP in the amyloidogenic pathway by the action of two proteolytic activities: b- and g-secretases [7]. Cleavage at the b-site of APP by the membrane-bound aspartyl protease BACE (b-site APP-cleaving enzyme) [8] occurs in close proximity to www.sciencedirect.com
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the membrane and results in the shedding of the luminal or extracellular domain of APP (Figure 1). The resulting APP C-terminal fragment (C99) is then further processed by g-secretase, resulting in the concomitant release of Ab and the cytosolic APP intracellular domain (AICD). Ab and AICD are generated by g-secretase using two distinct cleavage sites: one located in the approximate middle of the APP transmembrane domain (TMD; g-site), generating Ab40 and Ab42; the other located closer to the cytosolic boundary of the TMD (e site), leading to the formation of AICD. The temporal relationship between cleavage at these two sites is currently being investigated. Given its central role in the biogenesis of Ab, g-secretase has been a prioritized target for the development of antiamyloid drugs for several years.
was the PS protein (Figure 2). Inactivation of PS expression in knockout mice dramatically reduces (PS1 / ) [11] or eliminates (PS1 / , PS2 / ) [12,13] g-secretase activity. In addition, a seminal study by Wolfe et al. [14] demonstrated that two conserved aspartates in TMDs 6 and 7 of PS1 are indispensable for g-secretase activity, providing an elegant link to prior pharmacological evidence indicating that g-secretase belongs to the aspartyl protease category of enzymes [15]. Within the cell, PS is endoproteolytically cleaved into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), each of which contributes one aspartate residue. Transition-state analog g-secretase inhibitors bind directly to the NTF and CTF but not to the non-processed PS protein [16,17], providing compelling evidence that the NTF/CTF heterodimer represents the biologically active form of PS.
Can you be more complex than g-secretase? g-Secretase belongs to the family of intramembrane cleaving proteases (I-CLiPs), which all effectuate peptide bond cleavage within the interior of the lipid bilayer [9,10]. In contrast to other I-CLiPs, g-secretase requires formation of a large multi-protein complex in order to be active. The first g-secretase component to be identified
Three additional components of the complex have been identified: nicastrin, Aph-1 and Pen-2 [18–20] (Figure 2). The assembly of these four membrane proteins appears to be necessary and sufficient for g-secretase activity [21]. With the exception of PS, which indisputably is the catalytic subunit of g-secretase, the finer molecular detail
Figure 1
Schematic representation of intramembrane processing of APP and Notch. During amyloidogenic processing of APP, which generates Ab40 and Ab42, the APP holoprotein is initially cleaved by BACE, resulting in shedding of the APP ectodomain (left panel). The remaining membrane-bound C-terminal fragment, C99, is further processed by g-secretase at the g- and e-sites to produce Ab40/42 and AICD, respectively. Notch undergoes furin-like processing en route to the plasma membrane, where the heterodimeric receptor is cleaved by TACE upon ligand binding (right panel). The membrane-retained C-terminal fragment, NEXT (Notch extracellular truncated), is then cleaved by g-secretase at the S3 site, resulting in the release of Notch intracellular domain (NICD). g-Secretase cleavage has also been described at the S4 site in Notch [48]. www.sciencedirect.com
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Figure 2
Topological model of the g-secretase enzyme complex. Four different integral membrane proteins are required for g-secretase protease function: presenilin (PS), nicastrin (Nct), Aph-1 and Pen-2. The exact stoichiometry of the different components within the complex remains to be determined. PS1 contains 10 hydrophobic domains (purple barrels), of which nine form TMDs according to recent topological models [49,50]. The catalytic residues — Asp-257 in TMD6 and Asp-385 in TMD7 — are indicated by green circles. PS1 undergoes endoproteolysis within the hydrophobic domain located in the large cytosolic loop (red arrow) to yield the active NTF/CTF heterodimer.
of the function of the other components in the complex is still a subject of intense research. The exact stoichiometry of the g-secretase complex components is currently not known. Experimentally determined complex sizes range from 250 kDa to 2 MDa. Although this discrepancy, at least in part, can be explained by diverse experimental set-ups and technical limitations of the assays used, it might also indicate that one or more of the g-secretase components are present in multiple copies and/or that modulator proteins [22] are interacting with the complex.
The promiscuous nature of g-secretase: a possible show-stopper? The Notch signaling pathway is vital for controlling many cellular differentiation events during embryogenesis [23] and in the adult organism [24]. Like APP, the cell-surface Notch receptor undergoes sequential proteolytic cleavage [25]. In response to ligands, the Notch receptor is processed at the so-called S2 site by the membrane-bound TNF-a-converting enzyme (TACE) [26] before final cleavage at the intramembrane S3 site releases the Notch intracellular domain, which in turn translocates to the nucleus and regulates transcription (Figure 1). Early observations in Caernohabditis elegans and knockout mice suggested a biological link between Notch signaling and PS, and subsequent studies in cells established that S3 cleavage of Notch is performed by g-secretase [27]. It has since become apparent that a host of functionally Current Opinion in Pharmacology 2007, 7:112–118
different type I proteins (i.e. transmembrane proteins with a luminal/extracellular N terminus and a cytoplasmic C terminus) share this particular route of intramembrane processing. At present, more than 30 g-secretase substrates have been reported [28], a number that is likely to increase. For some substrates, a cellular function related to g-secretase cleavage can be ascribed; however, the physiological relevance of intramembrane proteolysis of other substrates remains to be determined. Nevertheless, the realization that g-secretase is a promiscuous enzyme capable of cleaving multiple substrates highlights that the use of g-secretase inhibitors as disease-modifying drugs might be limited owing to the risk of mechanismbased toxicity.
Early strategies to inhibit g-secretase activity The g-secretase complex imposes a considerable challenge from a drug discovery perspective. Obtaining a high-resolution X-ray crystal structure of the core fourcomponent enzyme machinery, comprising an estimated 19 TMDs, appears at present to be a daunting task. Encouragingly, the three-dimensional structure of a purified 300 kDa g-secretase complex was recently reported [29]. The 15 A˚ resolution model was reconstructed from single particle analysis of electron microscopy images of negatively stained complexes. Although this first medium-resolution model of the g-secretase complex is enthralling and informative, elucidation of www.sciencedirect.com
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the finer molecular details of the active site that will allow structure-based design of inhibitors requires crystallographic analysis. In the absence of this structural information, the development of inhibitors and the establishment of their mode of action have relied upon screenings and biochemical experiments using intact cells and solubilized cellular extracts as the source of enzyme. Included in the first generation of g-secretase inhibitors to be synthesized were transition-state analogues. Initial studies showed that Ab production could be lowered using a g-secretase cleavage site peptidomimetic [30], a conceptually important finding illustrating that pharmacological manipulation of Ab levels at the level of gsecretase is an attainable aim. Although the peptidic nature and bulky characteristics of transition-state analogues make them less feasible for in vivo studies, these compounds have been invaluable in distinguishing gsecretase as an aspartyl protease with PS at its catalytic core [16,17], and also serve as useful affinity probes for purification of active g-secretase [31].
The quest for APP-specific g-secretase inhibitors A concerted drug discovery effort in recent years has resulted in the identification of a number of new g-secretase inhibitors that have been presented in the scientific and patent literature. Many of these compounds, showing higher potency and more drug-like properties than the transition-state analogue inhibitors, are structurally related in that they contain either sulfonamide/sulfone or benzodiazepine/benzolactam moieties [32]. This second generation of g-secretase inhibitors has facilitated studies addressing such key questions as in vivo proof-of-concept and mechanism-based toxicity. For example, treatment of APP transgenic animals with LY-411,575, a benzodiazepine-containing analogue of the potent non-transitionstate inhibitor DAPT, lowered the levels of Ab40 and Ab42 in brain and plasma [33]. Disappointingly, doses of LY-411,575 that lowered Ab also affected lymphocyte development and caused profound changes in gastrointestinal tract tissue morphology. These adverse affects are probably attributable to impaired Notch signaling resulting from the inhibition of g-secretase, as LY-411,575 is also a potent inhibitor of Notch S3 cleavage in vitro (although the IC50 for APP cleavage is five-fold lower than that for Notch cleavage) [33]. Notwithstanding these discouraging findings, recent data suggest that mechanism-based toxicity associated with the use of g-secretase inhibitors is not an insurmountable problem. Similar to LY-411,575, the sulphonamide derivative BMS-299897 also inhibits g-secretase processing of both APP and Notch in vitro; however, the IC50 value for APP cleavage is 15-fold lower than that for Notch cleavage [34]. More importantly, treatment of transgenic animals with BMS-299897 lowered Ab in brain, cerebrospinal fluid www.sciencedirect.com
(CSF) and plasma, without any noticeable toxic and Notchrelated effects on lymphopoiesis or the gastrointestinal tract. Although further studies are required to ascertain the effects of long-term treatment with g-secretase inhibitors, the results obtained with BMS-299897 suggest that it could be feasible to reach a therapeutic window that retains efficacy but avoids mechanism-based toxicity. Interestingly, data recently presented by Schering-Plough demonstrated that partial, instead of near complete, inhibition of g-secretase with the sulphonamide-based inhibitor SCH 697466 reduced plasma, CSF and brain Ab in transgenic mice with reasonable efficacy, without any apparent side effects [35]. Eli Lilly and Company has recently reported the findings from a Phase II clinical trial with the benzodiazepinederived g-secretase inhibitor LY-450139 [36]. Seventy subjects with mild to moderate AD were randomly assigned to receive LY-450139 or placebo. Subjects receiving LY-450139 (30 mg QD for one week, followed by 40 mg daily for five weeks) had reduced plasma Ab140 levels (average maximum reduction = 38%). However, no significant changes were seen in the levels of Ab1-40 or Ab1-42 in CSF, raising the concern that LY-450139 had very little, or no, effect on Ab production in the brain. The dose regimen used did not result in any adverse effects that unambiguously could be related to impaired Notch signaling. Further studies should determine whether higher doses of LY-450139 will lead to a greater reduction in plasma Ab and a measurable response in CSF. In this regard, it is interesting to note that Merck recently reported results from a current Phase I trial using the g-secretase inhibitor MK-0752 [37]. The investigators found that Ab40 in CSF was significantly reduced (mean reduction = 35%) at 12 hours after a single dose of MK0752. The structure of the compound has not yet been publicly disclosed. Mechanistic detail explaining the ability of these small molecules to achieve substrate selectivity is still lacking. Mechanism of action studies have exclusively relied upon traditional radioligand and inhibitor cross-competition experiments. Results from these studies have revealed that all non-transition-state inhibitors investigated thus far target the same binding site in g-secretase, and kinetic analysis suggests a non-competitive mechanism of inhibition for this class of compounds [38–40]. This binding site is distinct from the transition-state analogue binding site. Surprisingly, transition-state analogues also inhibit gsecretase in a non-competitive manner, suggesting the existence of an initial substrate docking site in the enzyme in addition to the active site [38]. Non-transition-state inhibitors might interfere with the process of transferring substrate from the docking site to the active site. However, further studies are required to establish a more detailed mechanism of action for these compounds, and whether targeting of this process provides a feasible Current Opinion in Pharmacology 2007, 7:112–118
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strategy to develop compounds with a higher degree of substrate selectivity than those already reported.
NSAIDs alters the interaction between PS1 and APP, as well as the conformation of PS1 [45].
Modulation of g-secretase activity as an alternative to inhibition
The concept of g-secretase modulators has been further extended by the NGX series of compounds (TorreyPines Therapeutics; http://www.torreypinestherapeutics.com). These compounds, similar to the subset of NSAIDs described above, also shift g-secretase cleavage from Ab42 to shorter variants in vitro, albeit with a much higher potency (in the low nanomolar range) [46]. No effect of the compounds on Notch processing in vitro was seen at concentrations three orders of magnitude higher than those that altered APP metabolism. Interestingly, the NGX series of orally bioavailable compounds are structurally unrelated to the g-secretase-modulating NSAIDs.
In addition to the transition-state and non-transition-state inhibitors discussed above, a new class of g-secretase inhibitors, the so-called g-secretase modulators, has recently received attention. A landmark study by Weggen et al. [41] showed that the non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen, indomethacin and sulindac sulphide selectively reduce cellular secretion of the amyloidogenic Ab42 peptide, an effect that is independent of the compounds’ ability to inhibit cyclooxygenase (COX) activity. Interestingly, mass spectrometric analysis indicated that the decrease in secretion of Ab1-42 was accompanied by a dose-dependent increase in secretion of the Ab1-38 species, suggesting that some, but not all, NSAIDs subtly modulate APP metabolism by shifting gsecretase cleavage preference. Although showing only a modest potency at reducing cellular Ab42 secretion in vitro (within the low to high micromolar range), many NSAIDs have been reported to successfully lower brain Ab42 in transgenic mice [42]. One key aspect of the pharmacological profile of this subset of NSAIDs is that they do not perturb Notch signalling at therapeutic doses [41,43], thus offering an attractive therapeutic alternative to the transition-state and non-transition-state analogue g-secretase inhibitors. An additional, but still hypothetical, therapeutic benefit might result from the ability of NSAIDs to curb the cellular inflammatory response that is often seen in association with Ab plaques in human AD brain. However, the clinical merit of using NSAIDs as Ab42-lowering drugs is limited by the significant gastrointestinal and renal toxicity seen after long-term inhibition of COX [44]. The most developed g-secretase-directed clinical compound is the Ab42-lowering drug Flurizan — the Renantiomer of the NSAID flurbiprofen that shows little or no COX-inhibitory activity [42]. This compound has reached Phase III clinical trials for mild to moderate AD (Myriad Genetics; http://www.myriad.com/alzheimers/ flurizan.php). In patients with mild AD, a 12-month treatment with this drug reduced the rate of decline in activities of daily living and global function, suggesting that g-secretase modulation is a viable disease-modifying approach. The mechanism of action of the g-secretase-modulating NSAIDs is not known; however, they have been demonstrated to bind to the enzyme at an additional site separate from those already mentioned above [40]. These NSAIDs show non-competitive kinetics consistent with direct modulation of g-secretase activity by an allosteric mechanism. Indeed, a study using a fluorescence resonance energy method indicated that Ab42-lowering Current Opinion in Pharmacology 2007, 7:112–118
Conclusions Prima facie, the main hurdles associated with developing g-secretase inhibitors as AD-modifying drugs are two-fold: the sheer complexity of the enzyme itself, and the expanding number of type I proteins that are processed in a gsecretase-dependent manner. A principal challenge for drug discovery research is to develop compounds with a pronounced preference for APP rather than Notch gsecretase processing. Currently, it is not known to what degree Ab production needs to be lowered in order to achieve therapeutic efficacy; it can be hypothesized that only a modest lowering of Ab production by g-secretase inhibitors (used at concentrations where mechanismbased adverse effects are negligible) may prove beneficial. The new therapeutic concept of using g-secretase modifiers, which selectively reduce Ab42 without any effect on Notch signaling, holds great promise. Fuelling this optimism is the fact that the NSAID-derived g-secretase modulator Flurizan has reached Phase III clinical trials. The drug-like g-secretase-targeting compounds discussed in this review have all been developed and optimized in the absence of any higher order structural information on the protease itself. Given that the first high-resolution structure of an I-CLiP — the 6-TMD core catalytic domain of the Escherichia coli protease GlpG — was recently presented [47], our knowledge of this unusual proteolytic mechanism has improved and hopefully will result in the design of novel drugs aimed at inhibiting or modifying g-secretase activity. All things considered, and keeping in mind the remaining gaps in our knowledge regarding the etiology of the disease, there is reason for a cautious optimism in terms of using g-secretase-targeting compounds as future AD-modifying drugs.
Acknowledgements We gratefully acknowledge the work of past and present colleagues and apologize to the many investigators whose contributions have not been mentioned or cited owing to space restrictions. We also thank Dr Hanna Laudon for critical comments on the manuscript. JN would like to acknowledge support from Vetenskapsra˚det. www.sciencedirect.com
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48. Okochi M, Steiner H, Fukumori A, Tanii H, Tomita T, Tanaka T, Iwatsubo T, Kudo T, Takeda M, Haass C: Presenilins mediate a dual intramembranous g-secretase cleavage of Notch-1. EMBO J 2002, 21:5408-5416.
41. Weggen S, Eriksen JL, Das P, Sagi SA, Wang R, Pietrzik CU, Findlay KA, Smith TE, Murphy MP, Bulter T et al.: A subset of NSAIDs lower amyloidogenic Ab42 independently of cyclooxygenase activity. Nature 2001, 414:212-216.
49. Laudon H, Hansson EM, Melen K, Bergman A, Farmery MR, Winblad B, Lendahl U, von Heijne G, Na¨slund J: A ninetransmembrane domain topology for presenilin 1. J Biol Chem 2005, 280:35352-35360.
42. Eriksen JL, Sagi SA, Smith TE, Weggen S, Das P, McLendon DC, Ozols VV, Jessing KW, Zavitz KH, Koo EH et al.: NSAIDs and enantiomers of flurbiprofen target g-secretase and lower Ab42 in vivo. J Clin Invest 2003, 112:440-449.
50. Spasic D, Tolia A, Dillen K, Baert V, De Strooper B, Vrijens S, Annaert W: Presenilin-1 maintains a nine-transmembrane topology throughout the secretory pathway. J Biol Chem 2006, 281:26569-26977.
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molecular underpinnings of the disease, allowing exploration of multiple avenues for the development of diseasemodifying drugs. In this review, we highlight research aimed at achieving this objective by specifically targeting the so-called g-secretase enzyme. For other contemporary approaches, we refer the reader to some recently published reviews [2–4].
Disease pathogenesis and the amyloid cascade hypothesis Light microscopy analysis of sections from postmortem AD brain reveals the two neuropathological hallmarks of AD: an abundance of extracellular amyloid plaques in the brain parenchyma and cerebral vessels, and intraneuronal neurofibrillary tangles [5]. The major component of plaques is a fibrillar form of the 40–42 residue amyloid bpeptide (Ab). Deposited Ab contains many N-terminally truncated variants, although the C terminus appears to be more homogenous and predominantly ends with Val-40 (Ab40) or Ala-42 (Ab42). The Ab peptide is an integral part of the type I transmembrane protein APP (amyloid precursor protein), thus its formation requires the action of proteases.
Current Opinion in Pharmacology 2007, 7:112–118 This review comes from a themed issue on Neurosciences Edited by Karima Chergui, Bertil Fredholm and Per Svenningsson Available online 13th December 2006 1471-4892/$ – see front matter # 2006 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coph.2006.10.002
Introduction Alzheimer’s disease (AD), the most common form of dementia in the elderly, is a neurodegenerative disease characterized by progressive memory impairment, cognitive deficits and behavioral changes [1]. This year, we honour the century-old pioneering work of Alois Alzheimer in which the Bavarian psychiatrist described the clinical and pathological features of the syndrome that now bears his name. Improved life expectancy and a demographic shift towards an elderly population will probably increase the prevalence of AD in the future. This awareness, together with the enormous social and economical costs of the disease, has sparked an intense research effort to combat AD. There are currently no drugs available that can be used in the causal treatment or prevention of AD. Nevertheless, research in the past two decades has significantly increased our knowledge of the Current Opinion in Pharmacology 2007, 7:112–118
A fundamental role for Ab peptide in AD pathogenesis has been proposed by the ‘amyloid cascade hypothesis’ [6]. This working hypothesis is based on a number of observations accumulated over the years. The most straightforward evidence derives from studies of gene mutations that co-segregate with autosomal-dominant familial forms of AD. The vast majority of these mutations in the APP gene or the presenilin (PS) 1 or 2 genes either increases the production of the Ab42 peptide or enhances the Ab42/Ab40 ratio. This finding becomes significant in light of studies showing that the Ab42 peptide is more amyloidogenic and deposits earlier in the time course of the disease than the slightly shorter Ab40 variant. Moreover, transgenic mice expressing human mutant APP alone or together with mutant PS1 develop varying degrees of memory deficits and recapitulate some of the neuropathological features associated with AD. Consequently, inhibiting the production of Ab, and in particular the Ab42 peptide, constitutes a rational therapeutic approach for the treatment of AD.
APP processing and biogenesis of the Ab peptide The Ab peptide is generated from APP in the amyloidogenic pathway by the action of two proteolytic activities: b- and g-secretases [7]. Cleavage at the b-site of APP by the membrane-bound aspartyl protease BACE (b-site APP-cleaving enzyme) [8] occurs in close proximity to www.sciencedirect.com
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the membrane and results in the shedding of the luminal or extracellular domain of APP (Figure 1). The resulting APP C-terminal fragment (C99) is then further processed by g-secretase, resulting in the concomitant release of Ab and the cytosolic APP intracellular domain (AICD). Ab and AICD are generated by g-secretase using two distinct cleavage sites: one located in the approximate middle of the APP transmembrane domain (TMD; g-site), generating Ab40 and Ab42; the other located closer to the cytosolic boundary of the TMD (e site), leading to the formation of AICD. The temporal relationship between cleavage at these two sites is currently being investigated. Given its central role in the biogenesis of Ab, g-secretase has been a prioritized target for the development of antiamyloid drugs for several years.
was the PS protein (Figure 2). Inactivation of PS expression in knockout mice dramatically reduces (PS1 / ) [11] or eliminates (PS1 / , PS2 / ) [12,13] g-secretase activity. In addition, a seminal study by Wolfe et al. [14] demonstrated that two conserved aspartates in TMDs 6 and 7 of PS1 are indispensable for g-secretase activity, providing an elegant link to prior pharmacological evidence indicating that g-secretase belongs to the aspartyl protease category of enzymes [15]. Within the cell, PS is endoproteolytically cleaved into an N-terminal fragment (NTF) and a C-terminal fragment (CTF), each of which contributes one aspartate residue. Transition-state analog g-secretase inhibitors bind directly to the NTF and CTF but not to the non-processed PS protein [16,17], providing compelling evidence that the NTF/CTF heterodimer represents the biologically active form of PS.
Can you be more complex than g-secretase? g-Secretase belongs to the family of intramembrane cleaving proteases (I-CLiPs), which all effectuate peptide bond cleavage within the interior of the lipid bilayer [9,10]. In contrast to other I-CLiPs, g-secretase requires formation of a large multi-protein complex in order to be active. The first g-secretase component to be identified
Three additional components of the complex have been identified: nicastrin, Aph-1 and Pen-2 [18–20] (Figure 2). The assembly of these four membrane proteins appears to be necessary and sufficient for g-secretase activity [21]. With the exception of PS, which indisputably is the catalytic subunit of g-secretase, the finer molecular detail
Figure 1
Schematic representation of intramembrane processing of APP and Notch. During amyloidogenic processing of APP, which generates Ab40 and Ab42, the APP holoprotein is initially cleaved by BACE, resulting in shedding of the APP ectodomain (left panel). The remaining membrane-bound C-terminal fragment, C99, is further processed by g-secretase at the g- and e-sites to produce Ab40/42 and AICD, respectively. Notch undergoes furin-like processing en route to the plasma membrane, where the heterodimeric receptor is cleaved by TACE upon ligand binding (right panel). The membrane-retained C-terminal fragment, NEXT (Notch extracellular truncated), is then cleaved by g-secretase at the S3 site, resulting in the release of Notch intracellular domain (NICD). g-Secretase cleavage has also been described at the S4 site in Notch [48]. www.sciencedirect.com
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Figure 2
Topological model of the g-secretase enzyme complex. Four different integral membrane proteins are required for g-secretase protease function: presenilin (PS), nicastrin (Nct), Aph-1 and Pen-2. The exact stoichiometry of the different components within the complex remains to be determined. PS1 contains 10 hydrophobic domains (purple barrels), of which nine form TMDs according to recent topological models [49,50]. The catalytic residues — Asp-257 in TMD6 and Asp-385 in TMD7 — are indicated by green circles. PS1 undergoes endoproteolysis within the hydrophobic domain located in the large cytosolic loop (red arrow) to yield the active NTF/CTF heterodimer.
of the function of the other components in the complex is still a subject of intense research. The exact stoichiometry of the g-secretase complex components is currently not known. Experimentally determined complex sizes range from 250 kDa to 2 MDa. Although this discrepancy, at least in part, can be explained by diverse experimental set-ups and technical limitations of the assays used, it might also indicate that one or more of the g-secretase components are present in multiple copies and/or that modulator proteins [22] are interacting with the complex.
The promiscuous nature of g-secretase: a possible show-stopper? The Notch signaling pathway is vital for controlling many cellular differentiation events during embryogenesis [23] and in the adult organism [24]. Like APP, the cell-surface Notch receptor undergoes sequential proteolytic cleavage [25]. In response to ligands, the Notch receptor is processed at the so-called S2 site by the membrane-bound TNF-a-converting enzyme (TACE) [26] before final cleavage at the intramembrane S3 site releases the Notch intracellular domain, which in turn translocates to the nucleus and regulates transcription (Figure 1). Early observations in Caernohabditis elegans and knockout mice suggested a biological link between Notch signaling and PS, and subsequent studies in cells established that S3 cleavage of Notch is performed by g-secretase [27]. It has since become apparent that a host of functionally Current Opinion in Pharmacology 2007, 7:112–118
different type I proteins (i.e. transmembrane proteins with a luminal/extracellular N terminus and a cytoplasmic C terminus) share this particular route of intramembrane processing. At present, more than 30 g-secretase substrates have been reported [28], a number that is likely to increase. For some substrates, a cellular function related to g-secretase cleavage can be ascribed; however, the physiological relevance of intramembrane proteolysis of other substrates remains to be determined. Nevertheless, the realization that g-secretase is a promiscuous enzyme capable of cleaving multiple substrates highlights that the use of g-secretase inhibitors as disease-modifying drugs might be limited owing to the risk of mechanismbased toxicity.
Early strategies to inhibit g-secretase activity The g-secretase complex imposes a considerable challenge from a drug discovery perspective. Obtaining a high-resolution X-ray crystal structure of the core fourcomponent enzyme machinery, comprising an estimated 19 TMDs, appears at present to be a daunting task. Encouragingly, the three-dimensional structure of a purified 300 kDa g-secretase complex was recently reported [29]. The 15 A˚ resolution model was reconstructed from single particle analysis of electron microscopy images of negatively stained complexes. Although this first medium-resolution model of the g-secretase complex is enthralling and informative, elucidation of www.sciencedirect.com
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the finer molecular details of the active site that will allow structure-based design of inhibitors requires crystallographic analysis. In the absence of this structural information, the development of inhibitors and the establishment of their mode of action have relied upon screenings and biochemical experiments using intact cells and solubilized cellular extracts as the source of enzyme. Included in the first generation of g-secretase inhibitors to be synthesized were transition-state analogues. Initial studies showed that Ab production could be lowered using a g-secretase cleavage site peptidomimetic [30], a conceptually important finding illustrating that pharmacological manipulation of Ab levels at the level of gsecretase is an attainable aim. Although the peptidic nature and bulky characteristics of transition-state analogues make them less feasible for in vivo studies, these compounds have been invaluable in distinguishing gsecretase as an aspartyl protease with PS at its catalytic core [16,17], and also serve as useful affinity probes for purification of active g-secretase [31].
The quest for APP-specific g-secretase inhibitors A concerted drug discovery effort in recent years has resulted in the identification of a number of new g-secretase inhibitors that have been presented in the scientific and patent literature. Many of these compounds, showing higher potency and more drug-like properties than the transition-state analogue inhibitors, are structurally related in that they contain either sulfonamide/sulfone or benzodiazepine/benzolactam moieties [32]. This second generation of g-secretase inhibitors has facilitated studies addressing such key questions as in vivo proof-of-concept and mechanism-based toxicity. For example, treatment of APP transgenic animals with LY-411,575, a benzodiazepine-containing analogue of the potent non-transitionstate inhibitor DAPT, lowered the levels of Ab40 and Ab42 in brain and plasma [33]. Disappointingly, doses of LY-411,575 that lowered Ab also affected lymphocyte development and caused profound changes in gastrointestinal tract tissue morphology. These adverse affects are probably attributable to impaired Notch signaling resulting from the inhibition of g-secretase, as LY-411,575 is also a potent inhibitor of Notch S3 cleavage in vitro (although the IC50 for APP cleavage is five-fold lower than that for Notch cleavage) [33]. Notwithstanding these discouraging findings, recent data suggest that mechanism-based toxicity associated with the use of g-secretase inhibitors is not an insurmountable problem. Similar to LY-411,575, the sulphonamide derivative BMS-299897 also inhibits g-secretase processing of both APP and Notch in vitro; however, the IC50 value for APP cleavage is 15-fold lower than that for Notch cleavage [34]. More importantly, treatment of transgenic animals with BMS-299897 lowered Ab in brain, cerebrospinal fluid www.sciencedirect.com
(CSF) and plasma, without any noticeable toxic and Notchrelated effects on lymphopoiesis or the gastrointestinal tract. Although further studies are required to ascertain the effects of long-term treatment with g-secretase inhibitors, the results obtained with BMS-299897 suggest that it could be feasible to reach a therapeutic window that retains efficacy but avoids mechanism-based toxicity. Interestingly, data recently presented by Schering-Plough demonstrated that partial, instead of near complete, inhibition of g-secretase with the sulphonamide-based inhibitor SCH 697466 reduced plasma, CSF and brain Ab in transgenic mice with reasonable efficacy, without any apparent side effects [35]. Eli Lilly and Company has recently reported the findings from a Phase II clinical trial with the benzodiazepinederived g-secretase inhibitor LY-450139 [36]. Seventy subjects with mild to moderate AD were randomly assigned to receive LY-450139 or placebo. Subjects receiving LY-450139 (30 mg QD for one week, followed by 40 mg daily for five weeks) had reduced plasma Ab140 levels (average maximum reduction = 38%). However, no significant changes were seen in the levels of Ab1-40 or Ab1-42 in CSF, raising the concern that LY-450139 had very little, or no, effect on Ab production in the brain. The dose regimen used did not result in any adverse effects that unambiguously could be related to impaired Notch signaling. Further studies should determine whether higher doses of LY-450139 will lead to a greater reduction in plasma Ab and a measurable response in CSF. In this regard, it is interesting to note that Merck recently reported results from a current Phase I trial using the g-secretase inhibitor MK-0752 [37]. The investigators found that Ab40 in CSF was significantly reduced (mean reduction = 35%) at 12 hours after a single dose of MK0752. The structure of the compound has not yet been publicly disclosed. Mechanistic detail explaining the ability of these small molecules to achieve substrate selectivity is still lacking. Mechanism of action studies have exclusively relied upon traditional radioligand and inhibitor cross-competition experiments. Results from these studies have revealed that all non-transition-state inhibitors investigated thus far target the same binding site in g-secretase, and kinetic analysis suggests a non-competitive mechanism of inhibition for this class of compounds [38–40]. This binding site is distinct from the transition-state analogue binding site. Surprisingly, transition-state analogues also inhibit gsecretase in a non-competitive manner, suggesting the existence of an initial substrate docking site in the enzyme in addition to the active site [38]. Non-transition-state inhibitors might interfere with the process of transferring substrate from the docking site to the active site. However, further studies are required to establish a more detailed mechanism of action for these compounds, and whether targeting of this process provides a feasible Current Opinion in Pharmacology 2007, 7:112–118
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strategy to develop compounds with a higher degree of substrate selectivity than those already reported.
NSAIDs alters the interaction between PS1 and APP, as well as the conformation of PS1 [45].
Modulation of g-secretase activity as an alternative to inhibition
The concept of g-secretase modulators has been further extended by the NGX series of compounds (TorreyPines Therapeutics; http://www.torreypinestherapeutics.com). These compounds, similar to the subset of NSAIDs described above, also shift g-secretase cleavage from Ab42 to shorter variants in vitro, albeit with a much higher potency (in the low nanomolar range) [46]. No effect of the compounds on Notch processing in vitro was seen at concentrations three orders of magnitude higher than those that altered APP metabolism. Interestingly, the NGX series of orally bioavailable compounds are structurally unrelated to the g-secretase-modulating NSAIDs.
In addition to the transition-state and non-transition-state inhibitors discussed above, a new class of g-secretase inhibitors, the so-called g-secretase modulators, has recently received attention. A landmark study by Weggen et al. [41] showed that the non-steroidal anti-inflammatory drugs (NSAIDs) ibuprofen, indomethacin and sulindac sulphide selectively reduce cellular secretion of the amyloidogenic Ab42 peptide, an effect that is independent of the compounds’ ability to inhibit cyclooxygenase (COX) activity. Interestingly, mass spectrometric analysis indicated that the decrease in secretion of Ab1-42 was accompanied by a dose-dependent increase in secretion of the Ab1-38 species, suggesting that some, but not all, NSAIDs subtly modulate APP metabolism by shifting gsecretase cleavage preference. Although showing only a modest potency at reducing cellular Ab42 secretion in vitro (within the low to high micromolar range), many NSAIDs have been reported to successfully lower brain Ab42 in transgenic mice [42]. One key aspect of the pharmacological profile of this subset of NSAIDs is that they do not perturb Notch signalling at therapeutic doses [41,43], thus offering an attractive therapeutic alternative to the transition-state and non-transition-state analogue g-secretase inhibitors. An additional, but still hypothetical, therapeutic benefit might result from the ability of NSAIDs to curb the cellular inflammatory response that is often seen in association with Ab plaques in human AD brain. However, the clinical merit of using NSAIDs as Ab42-lowering drugs is limited by the significant gastrointestinal and renal toxicity seen after long-term inhibition of COX [44]. The most developed g-secretase-directed clinical compound is the Ab42-lowering drug Flurizan — the Renantiomer of the NSAID flurbiprofen that shows little or no COX-inhibitory activity [42]. This compound has reached Phase III clinical trials for mild to moderate AD (Myriad Genetics; http://www.myriad.com/alzheimers/ flurizan.php). In patients with mild AD, a 12-month treatment with this drug reduced the rate of decline in activities of daily living and global function, suggesting that g-secretase modulation is a viable disease-modifying approach. The mechanism of action of the g-secretase-modulating NSAIDs is not known; however, they have been demonstrated to bind to the enzyme at an additional site separate from those already mentioned above [40]. These NSAIDs show non-competitive kinetics consistent with direct modulation of g-secretase activity by an allosteric mechanism. Indeed, a study using a fluorescence resonance energy method indicated that Ab42-lowering Current Opinion in Pharmacology 2007, 7:112–118
Conclusions Prima facie, the main hurdles associated with developing g-secretase inhibitors as AD-modifying drugs are two-fold: the sheer complexity of the enzyme itself, and the expanding number of type I proteins that are processed in a gsecretase-dependent manner. A principal challenge for drug discovery research is to develop compounds with a pronounced preference for APP rather than Notch gsecretase processing. Currently, it is not known to what degree Ab production needs to be lowered in order to achieve therapeutic efficacy; it can be hypothesized that only a modest lowering of Ab production by g-secretase inhibitors (used at concentrations where mechanismbased adverse effects are negligible) may prove beneficial. The new therapeutic concept of using g-secretase modifiers, which selectively reduce Ab42 without any effect on Notch signaling, holds great promise. Fuelling this optimism is the fact that the NSAID-derived g-secretase modulator Flurizan has reached Phase III clinical trials. The drug-like g-secretase-targeting compounds discussed in this review have all been developed and optimized in the absence of any higher order structural information on the protease itself. Given that the first high-resolution structure of an I-CLiP — the 6-TMD core catalytic domain of the Escherichia coli protease GlpG — was recently presented [47], our knowledge of this unusual proteolytic mechanism has improved and hopefully will result in the design of novel drugs aimed at inhibiting or modifying g-secretase activity. All things considered, and keeping in mind the remaining gaps in our knowledge regarding the etiology of the disease, there is reason for a cautious optimism in terms of using g-secretase-targeting compounds as future AD-modifying drugs.
Acknowledgements We gratefully acknowledge the work of past and present colleagues and apologize to the many investigators whose contributions have not been mentioned or cited owing to space restrictions. We also thank Dr Hanna Laudon for critical comments on the manuscript. JN would like to acknowledge support from Vetenskapsra˚det. www.sciencedirect.com
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Current Opinion in Pharmacology 2007, 7:112–118
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