molecules Review Probing Mechanisms and Therapeutic Potential of γ-Secretase in Alzheimer’s Disease Michael S. Wolfe Department of Medicinal Chemistry, University of Kansas, 1567 Irving Hill Road, GLH-2115, Lawrence, KS 66045, USA; [email protected] Abstract: The membrane-embedded γ-secretase complex carries out hydrolysis within the lipid bilayer in proteolyzing nearly 150 different membrane protein substrates. Among these substrates, the amyloid precursor protein (APP) has been the most studied, as generation of aggregation-prone amyloid β-protein (Aβ) is a defining feature of Alzheimer’s disease (AD). Mutations in APP and in presenilin, the catalytic component of γ-secretase, cause familial AD, strong evidence for a pathogenic role of Aβ. Substrate-based chemical probes—synthetic peptides and peptidomimetics—have been critical to unraveling the complexity of γ-secretase, and small drug-like inhibitors and modulators of γ-secretase activity have been essential for exploring the potential of the protease as a therapeutic target for Alzheimer’s disease. Such chemical probes and therapeutic prototypes will be reviewed here, with concluding commentary on the future directions in the study of this biologically important protease complex and the translation of basic findings into therapeutics. Keywords: protease; amyloid; Alzheimer’s disease; inhibitors; modulators 1. Introduction Citation: Wolfe, M.S. Probing Alzheimer’s disease (AD), the most common form of dementia, is a devastating Mechanisms and Therapeutic neurodegenerative disorder that affects perhaps 30 million people worldwide, with demo- Potential of γ-Secretase in graphic projections suggesting this will increase substantially in the coming decades [1]. Alzheimer’s Disease. Molecules 2021, Cerebral neurodegeneration typically takes place first in the hippocampus, a region below 26, 388. https://doi.org/ the neocortex that is critical for consolidating long-term memories. Neuronal loss spreads 10.3390/molecules26020388 to other cortical areas, leading to progressive cognitive decline. By the end stages of the Academic Editors: Wei Li and disease, patients lose cognitive function to the point of requiring constant care, often in- Andrea Trabocchi stitutionalized. Although a number of risk factors have been associated with AD, disease Received: 21 December 2020 onset correlates best with age, and the large majority of cases occur in the elderly. Among Accepted: 10 January 2021 people over age 85, over a third are afflicted. Published: 13 January 2021 Two types of protein deposits are found in the AD brain: amyloid plaques and neurofibrillary tangles [2]. The former are extraneuronal and primarily composed of Publisher’s Note: MDPI stays neu- the 4 kDa amyloid β-peptide (Aβ), whereas the latter are intraneuronal filaments of the tral with regard to jurisdictional clai- normally microtubule-associated protein tau. Neuroinflammation is a third pathological ms in published maps and institutio- feature of AD, in which microglia—phagocytic brain immune cells that release cytokines— nal affiliations. become overactivated [3]. The role of each of these features in AD etiology and pathogenesis are not well understood. However, Aβ aggregation—in the form of oligomers, protofibrils, fibrils, and plaques—is generally observed as the earliest pathology, followed by tau tangle formation and neurodegeneration [4]. For this reason and those mentioned in Copyright: © 2021 by the author. Li- the next section, pathological Aβ is widely considered the initiator of AD, triggering censee MDPI, Basel, Switzerland. β This article is an open access article downstream tau pathology and neuroinflammation, and A has been the primary target distributed under the terms and con- for the development of AD therapeutics for over 25 years [5]. ditions of the Creative Commons At- 2. Familial AD and Genetics tribution (CC BY) license (https:// creativecommons.org/licenses/by/ As mentioned above, the “amyloid hypothesis” of AD pathogenesis has reigned for 4.0/). decades, and AD drug development has largely focused on inhibiting Aβ production, Molecules 2021, 26, 388. https://doi.org/10.3390/molecules26020388 https://www.mdpi.com/journal/molecules Molecules 2021, 26, x 2 of 17 Molecules 2021, 26, 388 2. Familial AD and Genetics 2 of 17 As mentioned above, the “amyloid hypothesis” of AD pathogenesis has reigned for decades, and AD drug development has largely focused on inhibiting Aβ production, blockingblocking Aβ aggregation, Aβ aggregation, or facilitating or facilitating Aβ clearance Aβ clearance from fromthe brain the brain[6]. The [6]. primary The primary basis basis for thisfor dogma this dogma is the isdiscovery the discovery in the in 1990s the 1990sof dominant of dominant genetic genetic mutations mutations associated associated with with early-onsetearly-onset AD [7–10]. AD [7 This–10]. familial This familial AD (FAD) AD (FAD) has a hasdisease a disease onset onsetbefore before age 60 age and 60 can and can occuroccur even evenbefore before age 30. age Other 30. Other than the than mo thenogenetic monogenetic cause causeand mid- andlife mid-life onset, onset, FAD FADis is closelyclosely similar similar to the to sporadic the sporadic AD of AD old of age old with age respect with respect to pathology, to pathology, presentation, presentation, and and progression.progression. The most The mostparsimonious parsimonious explanation explanation is that is similar that similar molecular molecular and cellular and cellular eventsevents are involved are involved in the in pa thethogenesis pathogenesis and progression and progression of both of bothforms forms of the of disease. the disease. The firstThe genetic first genetic mutations mutations associated associated with with FAD FAD were were in the in theamyloid amyloid precursor precursor pro- protein tein (APP)(APP) [7]. [7]. This This gene gene encodes encodes a asingle-pass single-pass membrane membrane protein protein that that is isinitially initially cleaved cleaved by by β-secretase,β-secretase, a membrane-tethered a membrane-tethered aspartyl aspartyl protease protease in the in pepsin the pepsin family, family, to release to release the the large largeAPP ectodomain APP ectodomain [11] (Figure [11] (Figure 1). The1). remnant The remnant C-terminal C-terminal fragment fragment (APP (APP CTF- CTF-β) is β) is then proteolyzedthen proteolyzed within within its transmembrane its transmembrane domain domain (TMD) (TMD) by γ-secretase by γ-secretase to produce to produce Aβ, Aβ, β β whichwhich is then is secreted then secreted from fromthe cell the [12]. cell Most [12]. A Mostβ is 40 A residuesis 40 residues in length in length(Aβ40), (A but40), a but β smalla portion small portion is the ismuch the muchmore moreaggregation-prone aggregation-prone 42-residue 42-residue form form(Aβ42). (A Although42). Although β β β γ Aβ42A is a42 minor is a minor Aβ variant A variant produced produced through through APP CTF- APPβ CTF- processingprocessing by γ-secretase, by -secretase, it is it is the major form deposited in the characteristic cerebral plaques of AD [13]. the major form deposited in the characteristic cerebral plaques of AD [13]. Figure 1. Amyloid precursor protein (APP) processing by β- and γ-secretases. The single-pass membrane protein APP is proteolyzed just outside the transmembrane domain (TMD) by β-secretase. FigureThe 1. Amyloid remaining precursor membrane-bound protein (APP) C-terminal processing fragment by β-(APP and γ CTF--secretases.β) is then The cleaved single-pass within the TMD membrane protein APP is proteolyzed just outside the transmembrane domain (TMD) by β-secre- to produce the amyloid β-peptide (Aβ) and the APP intracellular domain (AICD). tase. The remaining membrane-bound C-terminal fragment (APP CTF-β) is then cleaved within the TMD toThe produce 27 known the amyloid APP β mutations-peptide (A associatedβ) and the APP with intracellular FAD [14], domain each devastating (AICD). different families, are all missense mutations in and around the small Aβ region of the large APP. The 27 known APP mutations associated with FAD [14], each devastating different These mutations either alter Aβ production or increase the aggregation tendency of the families,peptide are all [15 missense]. A double mutations mutation in and just around outside the the smallN-terminus Aβ region of theof the Aβ largeregion APP. in APP Theseincreases mutations proteolysis either alter by βA-secretase,β production leading or increase to elevated the APPaggregation CTF-β and tendency therefore of the elevated peptideAβ [15].overall. A double Mutations mutation in the just TMD outside near γ the-secretase N-terminus cleavage of the sites Aβ elevate region A inβ42/A APPβ in-40, and creasesmutations proteolysis within by β the-secretase, Aβ region leading itself to make elevated the peptide APP CTF- moreβ and prone therefore to aggregation. elevated Aβ overall.FAD Mutations mutations in the were TMD then near discovered γ-secretase in cleavage presenilin-1 sites (PSEN1)elevate A [β842/A] andβ40, presenilin-2 and mutations(PSEN2) within [9], the genes Aβ encoding region itself multi-pass make the membrane peptide more proteins prone that to at aggregation. the time had no known FADfunction. mutations These missensewere then mutations—now discovered in pr withesenilin-1 over 200 (PSEN1) known [8] [14 ],and all presenilin-2 but a dozen or so (PSEN2)in PSEN1—are [9], genes encoding located multi-pass throughout membra the sequencene proteins of the that protein at the but time mostly had no within known its nine function.TMDs These [16, 17missense]. Presenilin mutations—now FAD mutations with were over soon 200 foundknown to [14], increase all but A βa 42/Adozenβ 40or [so18 –20], in PSEN1—arefurther strengthening located throughout the idea the that sequence this ratio of isthe critical protein to but pathogenesis. mostly within Moreover, its nine these TMDsfindings [16,17]. indicatedPresenilin thatFAD presenilins mutations canwere modulate soon foundγ-secretase to increase cleavage Aβ42/A ofβ40 APP [18–20], substrate, furtheras strengthening the FAD mutations the idea altered that thethis preference ratio is critical for cleavage to pathogenesis.