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• W2568951569 abstract "•Developed next-generation γ-secretase inhibitor photoprobes with cleavable linkers•Mapped the attachment site of a photoprobe on presenilin-1•Built a model of the structure of the γ-secretase complex with inhibitor•Novel insights into the mechanism of arylsulfonamide inhibitors are provided γ-Secretase, a four-subunit transmembrane aspartic proteinase, is a highly valued drug target in Alzheimer's disease and cancer. Despite significant progress in structural studies, the respective molecular mechanisms and binding modes of γ-secretase inhibitors (GSIs) and modulators (GSMs) remain uncertain. Here, we developed biotinylated cleavable-linker photoprobes based on the BMS-708163 GSI to study its interaction with γ-secretase. Comparison of four cleavable linkers indicated that the hydrazine-labile N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker was cleaved most efficiently to release photolabeled and affinity-captured presenilin-1 (PS1), the catalytic subunit of γ-secretase. Peptide mapping showed that the BMS-708163-based probe photoinserted at L282 of PS1. This insertion site was consistent with the results of molecular dynamics simulations of the γ-secretase complex with inhibitor. Taken together, this work reveals the binding site of a GSI and offers insights into the mechanism of action of this class of inhibitors. γ-Secretase, a four-subunit transmembrane aspartic proteinase, is a highly valued drug target in Alzheimer's disease and cancer. Despite significant progress in structural studies, the respective molecular mechanisms and binding modes of γ-secretase inhibitors (GSIs) and modulators (GSMs) remain uncertain. Here, we developed biotinylated cleavable-linker photoprobes based on the BMS-708163 GSI to study its interaction with γ-secretase. Comparison of four cleavable linkers indicated that the hydrazine-labile N-1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl (Dde) linker was cleaved most efficiently to release photolabeled and affinity-captured presenilin-1 (PS1), the catalytic subunit of γ-secretase. Peptide mapping showed that the BMS-708163-based probe photoinserted at L282 of PS1. This insertion site was consistent with the results of molecular dynamics simulations of the γ-secretase complex with inhibitor. Taken together, this work reveals the binding site of a GSI and offers insights into the mechanism of action of this class of inhibitors. γ-Secretase is an intramembrane aspartic proteinase composed of four subunits: Aph-1, nicastrin (Nct), Pen-2, and presenilin (PS) (De Strooper, 2003De Strooper B. Aph-1, Pen-2, and nicastrin with presenilin generate an active gamma-secretase complex.Neuron. 2003; 38: 9-12Abstract Full Text Full Text PDF PubMed Scopus (831) Google Scholar), which is the catalytic subunit of the enzyme (Ahn et al., 2010Ahn K. Shelton C.C. Tian Y. Zhang X. Gilchrist M.L. Sisodia S.S. Li Y.-M. Activation and intrinsic γ-secretase activity of presenilin 1.Proc. Natl. Acad. Sci. USA. 2010; 107: 21435-21440Crossref PubMed Scopus (123) Google Scholar). Endoproteolysis of PS, which results in the formation of PS1-NTF (N-terminal fragment) and CTF (C-terminal fragment) heterodimer, is required for γ-secretase activation (Thinakaran et al., 1996Thinakaran G. Borchelt D.R. Lee M.K. Slunt H.H. Spitzer L. Kim G. Ratovitsky T. Davenport F. Nordstedt C. Seeger M. et al.Endoproteolysis of presenilin 1 and accumulation of processed derivatives in vivo.Neuron. 1996; 17: 181-190Abstract Full Text Full Text PDF PubMed Scopus (939) Google Scholar). γ-Secretase cleaves amyloid precursor protein (APP), Notch, and many other substrates. Aberrant cleavage of APP contributes to the pathogenesis of Alzheimer's disease (AD), and abnormal Notch signaling promotes tumor growth (Crump et al., 2013Crump C.J. Johnson D.S. Li Y.-M. Development and mechanism of γ-secretase modulators for Alzheimer’s disease.Biochemistry. 2013; 52: 3197-3216Crossref PubMed Scopus (139) Google Scholar). Multiple classes of small molecules that target γ-secretase have been developed, including both inhibitors (GSIs) and modulators (GSMs). These small molecules bind either to the active site or to allosteric sites (Crump et al., 2013Crump C.J. Johnson D.S. Li Y.-M. Development and mechanism of γ-secretase modulators for Alzheimer’s disease.Biochemistry. 2013; 52: 3197-3216Crossref PubMed Scopus (139) Google Scholar), but the precise locations of these binding sites have been elusive. A recent 3.4 Å cryo-electron microscopy (cryo-EM) structure of γ-secretase (Bai et al., 2015bBai X.C. Yan C. Yang G. Lu P. Ma D. Sun L. Zhou R. Scheres S.H. Shi Y. An atomic structure of human gamma-secretase.Nature. 2015; 525: 212-217Crossref PubMed Scopus (393) Google Scholar) has shed light on the way in which the enzyme's structure may account for its highly regulated yet variable function (Gertsik et al., 2014Gertsik N. Chiu D. Li Y.M. Complex regulation of gamma-secretase: from obligatory to modulatory subunits.Front Aging Neurosci. 2014; 6: 342PubMed Google Scholar), but many questions about the active site of γ-secretase and its interactions with small molecules remain. Recently, we developed a series of chemical probes based on BMS-708163 (avagacestat), an arylsulfonamide GSI that was in clinical trials for AD (Coric et al., 2012Coric V. van Dyck C.H. Salloway S. Andreasen N. Brody M. Richter R.W. Soininen H. Thein S. Shiovitz T. Pilcher G. et al.Safety and tolerability of the gamma-secretase inhibitor avagacestat in a phase 2 study of mild to moderate Alzheimer disease.Arch. Neurol. 2012; 69: 1430-1440Crossref PubMed Scopus (266) Google Scholar), and characterized their interaction with γ-secretase (Crump et al., 2012Crump C.J. Castro S.V. Wang F. Pozdnyakov N. Ballard T.E. Sisodia S.S. Bales K.R. Johnson D.S. Li Y.M. BMS-708,163 targets presenilin and lacks notch-sparing activity.Biochemistry. 2012; 51: 7209-7211Crossref PubMed Scopus (81) Google Scholar, Crump et al., 2016Crump C.J. Murrey H.E. Ballard T.E. Am Ende C.W. Wu X. Gertsik N. Johnson D.S. Li Y.M. Development of sulfonamide photoaffinity inhibitors for probing cellular gamma-secretase.ACS Chem. Neurosci. 2016; 7: 1166-1173Crossref PubMed Scopus (18) Google Scholar). Here, we develop cleavable BMS-708163-based photoprobes and use them to identify a probe-labeled peptide within the intracellular loop of PS1 near the endoproteolytic site. In addition, we provide a model of the binding site for BMS-708163 within the γ-secretase complex based on the insertion site of the photoprobe and molecular dynamics (MD) simulations. We synthesized four biotinylated 163-BP3-based BMS-708163 probes with various cleavable linkers (Figure 1A) by Cu-catalyzed azide-alkyne cycloaddition (CuAAC) of 163-BP3 with the requisite biotin-azide (see Supplemental Information). The four linkers can be cleaved through different mechanisms: Dde-, diol-, azo-, and DADPS-linkers are cleaved with hydrazine (N2H4) (Yang and Verhelst, 2013Yang Y. Verhelst S.H. Cleavable trifunctional biotin reagents for protein labelling, capture and release.Chem. Commun. (Camb). 2013; 49: 5366-5368Crossref PubMed Scopus (31) Google Scholar), sodium periodate (NaIO4) (Yang et al., 2013Yang Y. Hahne H. Kuster B. Verhelst S.H. A simple and effective cleavable linker for chemical proteomics applications.Mol. Cell Proteomics. 2013; 12: 237-244Crossref PubMed Scopus (40) Google Scholar), sodium hydrosulfite (Na2S2O4) (Blum et al., 2013Blum G. Bothwell I.R. Islam K. Luo M. Profiling protein methylation with cofactor analog containing terminal alkyne functionality.Curr. Protoc. Chem. Biol. 2013; 5: 67-88Crossref PubMed Scopus (16) Google Scholar), and formic acid (CH2O2) (Szychowski et al., 2010Szychowski J. Mahdavi A. Hodas J.J. Bagert J.D. Ngo J.T. Landgraf P. Dieterich D.C. Schuman E.M. Tirrell D.A. Cleavable biotin probes for labeling of biomolecules via azide-alkyne cycloaddition.J. Am. Chem. Soc. 2010; 132: 18351-18360Crossref PubMed Scopus (147) Google Scholar), respectively. As a control, we also included 163-BP3-PEG-biotin, which can be eluted with heat (Crump et al., 2016Crump C.J. Murrey H.E. Ballard T.E. Am Ende C.W. Wu X. Gertsik N. Johnson D.S. Li Y.M. Development of sulfonamide photoaffinity inhibitors for probing cellular gamma-secretase.ACS Chem. Neurosci. 2016; 7: 1166-1173Crossref PubMed Scopus (18) Google Scholar). All five probes are potent γ-secretase inhibitors with half maximal inhibitory concentration (IC50) for Aβ40 in the low nanomolar range, similar to the parent compound BMS-708163 (Figures 1A and S1A). However, the IC50 values of the probes varied slightly, with 163-BP3-diol-biotin being the most potent inhibitor of Aβ40 production (IC50 = 1.1 nM) and 163-BP3-DADPS-biotin being the least potent (IC50 = 8.0 nM). Next, we performed a side-by-side comparison of the efficiency of PS1-NTF photolabeling by all probes (Figure S1B). First, we examined labeling efficiency by photolabeling HeLa membrane with 20 nM of each probe in the presence of 0.25% CHAPSO, affinity-capturing probe-labeled proteins on streptavidin, eluting them with 2 mM biotin in 2× SDS-PAGE sample buffer at 70°C for 10 min, and analyzing the eluate by western blot for PS1-NTF. The photolabeling efficiencies of the probes correlated with their respective IC50 values, with the more potent probes labeling PS1-NTF more efficiently (Figure S1B). We also determined the labeling efficiency in HeLa membranes after adjusting the concentration of each probe to take into account its IC50 value (each probe was used at 50× its own IC50). We observed close to equal labeling using these IC50-adjusted concentrations, although labeling of PS1-NTF by 163-BP3-azo-biotin was weaker than that achieved with the other probes (Figure 1B). We used samples labeled under these IC50-adjusted concentrations to optimize elution conditions for all the cleavable probes (Figures 2 and S2). HeLa membrane was labeled with an IC50-adjusted concentration of a photoprobe, and the labeled proteins were pulled down with streptavidin beads and eluted using two rounds of treatment with probe-specific cleavage conditions (elutions I and II). We then washed samples with 0.1% SDS and eluted a third time under harsh conditions with 2 mM biotin in 2× SDS-PAGE sample buffer at 70°C for 10 min (elution III) to recover any probe-labeled proteins that had not been cleaved from the resin. Equivalent amounts of each elution were loaded onto SDS-PAGE and analyzed by western blot, probing for PS1-NTF. Cleavage of 163-BP3-Dde-biotin-labeled proteins from streptavidin with 2% hydrazine + 0.05% SDS (elutions I and II) resulted in exhaustive elution of PS1-NTF, such that subsequent treatment with 2 mM biotin in 2× SDS-PAGE sample buffer at 70°C (elution III) did not result in the elution of additional target protein (Figure 2A). In contrast, cleavage-specific elution of 163-BP3-diol-biotin, 163-BP3-azo-biotin, and 163-BP3-DADPS-biotin showed partial elution of probe-labeled PS1-NTF (Figures S2A and S2B, elutions I and II; Figure S2C, elution I), as notable quantities of PS1-NTF were also present in the subsequent heat-eluted samples (Figures S2A and S2B, elution III; Figure S2C, elution II). The cleavage of 163-BP3-diol-biotin with sodium periodate resulted in a shift in the probe-labeled PS1-NTF band on the western blot, which suggested that the cleavage conditions caused modification of PS1 and that this probe is not suitable for identifying the probe-labeled peptide(s) (Figure 2B). It should also be noted that it was necessary to add 0.01% BSA to the formic acid cleavage cocktail in order to recover probe-labeled PS1-NTF when labeled by the DADPS linker probe. Presumably, PS1 is not compatible with the formic acid conditions and BSA serves as a carrier protein to help reduce PS1-NTF aggregation/instability. Taken together, the data suggested that 163-BP3-Dde-biotin was the most efficient probe for photolabeling and eluting PS1-NTF. To confirm this, we labeled HeLa membrane side by side with 163-BP3-Dde-biotin, 163-BP3-diol-biotin, and 163-BP3-azo-biotin, treated each sample with appropriate cleavage-specific conditions, and ran equivalent amounts of the eluate from each sample on SDS-PAGE followed by western blot for PS1-NTF (Figure 2B). The sample labeled by 163-BP3-Dde-biotin showed ∼4 times more PS1-NTF than the 163-BP3-diol-biotin-labeled sample and ∼3 times more PS1-NTF than the 163-BP3-azo-biotin-labeled sample, confirming that 163-BP3-Dde-biotin was superior to the other probes at capturing and eluting PS1-NTF. More generally, these findings suggest that the Dde linker could be a preferred choice for developing cleavable chemical probes in the studies of membrane proteins and soluble proteins. Next, we performed a large-scale photolabeling experiment with 163-BP3-Dde-biotin using membrane preparations from ANPP cells, in which all four subunits of γ-secretase are overexpressed (Kim et al., 2003Kim S.H. Ikeuchi T. Yu C. Sisodia S.S. Regulated hyperaccumulation of presenilin-1 and the “gamma-secretase” complex. Evidence for differential intramembranous processing of transmembrane substrates.J. Biol. Chem. 2003; 278: 33992-34002Crossref PubMed Scopus (96) Google Scholar), to map its binding site on PS1. The ANPP cell membrane was photolabeled with 100 nM 163-BP3-Dde-biotin in the absence and presence of 10 μM parent compound BMS-708163. Labeled proteins were captured with streptavidin, eluted by linker cleavage with 2% hydrazine containing 0.05% SDS, and concentrated ∼24-fold to give 60 μL of sample. Western blot analysis using 1 μL of sample showed that the labeling of PS1-NTF was specific, as it was blocked in the presence of BMS-708163 (data not shown). The remaining sample was prepared for proteomic analysis, digested with LysC/trypsin, and analyzed by nano-liquid chromatography(LC)-mass spectrometry (MS) using a Q Exactive spectrometer. When seeking modified peptides that could reveal sites of photoinsertion, we limited our proteomics search space by assembling a database containing the sequences of the four recognized γ-secretase subunits together with those of the most abundant proteins detected by LC-MS/MS, such as streptavidin, keratins, and ribosomal proteins. Modification corresponding to the anticipated product of photolabeling by 163-BP3-Dde-biotin followed by hydrazinolysis was permitted in the form of a C31ClF3H32N6O5S (692.179 Da) adduct on different subsets of amino acids residues. This search strategy carried an increased risk of false positives but reduced the risk of missing a genuine modified peptide due to low scoring caused by atypical fragmentation in the MS/MS spectrum. Searching by this method led to identification of a probe-modified semitryptic fragment of PS1 with the sequence NETLFPALIYSST (residues 279–291 of PS1) (Figure S3A). Comparison of extracted ion traces for this peptide showed that it was present in the digest of protein labeled without competition (Figure 3A, pane 2) but undetectable when competitor was present (Figure 3A, pane 4). The doubly charged precursor ion leading to the identification had m/z = 1074.460 (monoisotopic), while the theoretical value for [M + 2H]2+ of the proposed modified peptide was m/z = 1074.462 (mass discrepancy 1.9 ppm). Simulation of the peptide spectrum based on the product's proposed elementary analysis closely matched the experiment (Figure S3B). In the tandem mass spectrum, the site of modification was confined to L282 by detection of b2, b3, y8, and y9 ions lacking any modification by hydrazine-cleaved 163-BP3-Dde-biotin (Figures 3B and S3C). Dehydrated b5, b7, b8, and b9 ions bearing the modification were also fully consistent with modification having occurred at L282, with the prevalence of dehydration tentatively attributed to loss of water at the previously reactive carbon of the benzophenone. Next, we performed MD simulations utilizing the recent cryo-EM structures of γ-secretase to model the probe binding site (PDB: 5FN2; Bai et al., 2015aBai X.C. Rajendra E. Yang G. Shi Y. Scheres S.H. Sampling the conformational space of the catalytic subunit of human gamma-secretase.Elife. 2015; 4https://doi.org/10.7554/eLife.11182Crossref Scopus (384) Google Scholar, Bai et al., 2015bBai X.C. Yan C. Yang G. Lu P. Ma D. Sun L. Zhou R. Scheres S.H. Shi Y. An atomic structure of human gamma-secretase.Nature. 2015; 525: 212-217Crossref PubMed Scopus (393) Google Scholar). Modeling studies using a combination of MOE Site Finder for initial ligand placement, our in-house docking program (AGDOCK), and the DESMOND MD program, revealed a binding pocket and preferred ligand orientation in excellent agreement with the experimental labeling of L282 (Figures 4 and S4, see Supplemental Information for details). We observed that the probe forms primary hydrophobic contacts with L150, L282, and F283 of PS1, with the sulfonamide interacting primarily through water molecules to R278 and the backbone carbonyl of P284. The amino NH of the ligand makes sustained contact with the backbone carbonyl of G382, and the carbonyl of the benzophenone accepts a hydrogen bond from S170 (Figures 4 and S4). Based on our analysis, we suggest that the combination of experimentally determined attachment site data and computational modeling is more likely to give reliable binding site results than computational modeling and docking alone. For example, a recent docking study suggested that BMS-708163 interacts directly with the catalytic residues and the chlorophenyl moiety points away from the aspartates (Somavarapu and Kepp, 2016Somavarapu A.K. Kepp K.P. The dynamic mechanism of presenilin-1 function: sensitive gate dynamics and loop unplugging control protein access.Neurobiol. Dis. 2016; 89: 147-156Crossref PubMed Scopus (39) Google Scholar), which is not congruent with our results. The cleavable arylsulfonamide photoprobe, 163-BP3-Dde-biotin, developed herein led us to a novel finding that BMS-708163 engages with the inhibitory loop of PS1 within the γ-secretase complex. The probe-labeled residue, L282, is close to the site of PS1 endoproteolysis that occurs between T291 and A299 (Podlisny et al., 1997Podlisny M.B. Citron M. Amarante P. Sherrington R. Xia W. Zhang J. Diehl T. Levesque G. Fraser P. Haass C. et al.Presenilin proteins undergo heterogeneous endoproteolysis between Thr291 and Ala299 and occur as stable N- and C-terminal fragments in normal and Alzheimer brain tissue.Neurobiol. Dis. 1997; 3: 325-337Crossref PubMed Scopus (273) Google Scholar) and is required for activation of γ-secretase. The endoproteolytic loop is highly functional and, when intact, maintains PS1 as a zymogen (Li et al., 2000Li Y.M. Xu M. Lai M.T. Huang Q. Castro J.L. DiMuzio-Mower J. Harrison T. Lellis C. Nadin A. Neduvelil J.G. et al.Photoactivated gamma-secretase inhibitors directed to the active site covalently label presenilin 1.Nature. 2000; 405: 689-694Crossref PubMed Scopus (865) Google Scholar). Moreover, a clinical mutation in which the entire endoproteolysis site is deleted (residues 290–319, known as PS1ΔE9), results in an active γ-secretase and causes AD (Steiner et al., 1999Steiner H. Romig H. Grim M.G. Philipp U. Pesold B. Citron M. Baumeister R. Haass C. The biological and pathological function of the presenilin-1 Deltaexon 9 mutation is independent of its defect to undergo proteolytic processing.J. Biol. Chem. 1999; 274: 7615-7618Crossref PubMed Scopus (114) Google Scholar). Previous studies have proposed two different mechanisms of action for GSIs, one that involves blocking the substrate from entering the active site (Fukumori et al., 2010Fukumori A. Fluhrer R. Steiner H. Haass C. Three-amino acid spacing of presenilin endoproteolysis suggests a general stepwise cleavage of gamma-secretase-mediated intramembrane proteolysis.J. Neurosci. 2010; 30: 7853-7862Crossref PubMed Scopus (80) Google Scholar) and the other where GSIs position the catalytic aspartates in an orientation that prevents catalysis (Ahn et al., 2010Ahn K. Shelton C.C. Tian Y. Zhang X. Gilchrist M.L. Sisodia S.S. Li Y.-M. Activation and intrinsic γ-secretase activity of presenilin 1.Proc. Natl. Acad. Sci. USA. 2010; 107: 21435-21440Crossref PubMed Scopus (123) Google Scholar). The second model is supported by the finding that full-length, catalytically inactive D257A and D385A PS1 mutants can still bind substrates (Xia et al., 2000Xia W. Ray W.J. Ostaszewski B.L. Rahmati T. Kimberly W.T. Wolfe M.S. Zhang J. Goate A.M. Selkoe D.J. Presenilin complexes with the C-terminal fragments of amyloid precursor protein at the sites of amyloid beta-protein generation.Proc. Natl. Acad. Sci. USA. 2000; 97: 9299-9304Crossref PubMed Scopus (132) Google Scholar) but not the active site-directed inhibitor JC-8 (Ahn et al., 2010Ahn K. Shelton C.C. Tian Y. Zhang X. Gilchrist M.L. Sisodia S.S. Li Y.-M. Activation and intrinsic γ-secretase activity of presenilin 1.Proc. Natl. Acad. Sci. USA. 2010; 107: 21435-21440Crossref PubMed Scopus (123) Google Scholar). Whether BMS-708163 is preventing substrate entry, separating the catalytic aspartates or both, remains to be investigated. The finding that the 163-BP3-Dde-biotin attachment site on PS1-NTF is in a region that forms the inhibitory loop of PS1 suggests that BMS-708163 is in fact a pan inhibitor of γ-secretase, which is consistent with previous reports that BMS-708163 has little Notch-sparing activity and blocks the photoinsertion of four active site-directed probes to PS1-NTF (Crump et al., 2012Crump C.J. Castro S.V. Wang F. Pozdnyakov N. Ballard T.E. Sisodia S.S. Bales K.R. Johnson D.S. Li Y.M. BMS-708,163 targets presenilin and lacks notch-sparing activity.Biochemistry. 2012; 51: 7209-7211Crossref PubMed Scopus (81) Google Scholar). Furthermore, modeling indicates that the inhibitor potentially interacts with the backbone carbonyl of G382, which is proximal to the catalytic residue D385, and could also contribute to the mode of inhibition. In addition, cryo-EM analysis of γ-secretase complexed with DAPT suggested that DAPT resides in a pocket formed by TM2, TM3, TM5, TM6, and TM7 of PS1, with M146, M233, W165, F283, and G384 likely interacting with DAPT, although the orientation of the inhibitor could not be determined (Bai et al., 2015aBai X.C. Rajendra E. Yang G. Shi Y. Scheres S.H. Sampling the conformational space of the catalytic subunit of human gamma-secretase.Elife. 2015; 4https://doi.org/10.7554/eLife.11182Crossref Scopus (384) Google Scholar). Our studies indicate that S170, R278, L282, F283, and G382 residues are involved in the binding of BMS-708163, suggesting that the two binding sites partially overlap. This proposal is supported by our finding that DAPT inhibits photolabeling of PS1 by 163-BP3-Dde-biotin (Figure S4C). In previous work, we have shown that distinct classes of GSIs and GSMs bind to non-overlapping regions on PS1, but the precise binding sites have yet to be elucidated (Crump et al., 2013Crump C.J. Johnson D.S. Li Y.-M. Development and mechanism of γ-secretase modulators for Alzheimer’s disease.Biochemistry. 2013; 52: 3197-3216Crossref PubMed Scopus (139) Google Scholar). BMS-708163 (avagacestat) was reported as a Notch-sparing inhibitor (Gillman et al., 2010Gillman K.W. Starrett Jr., J.E. Parker M.F. Xie K. Bronson J.J. Marcin L.R. McElhone K.E. Bergstrom C.P. Mate R.A. Williams R. et al.Discovery and evaluation of BMS-708163, a potent, selective and orally bioavailable gamma-secretase inhibitor.ACS Med. Chem. Lett. 2010; 1: 120-124Crossref PubMed Scopus (151) Google Scholar), but failed in AD clinical trials partially due to Notch-mediated toxicity (Coric et al., 2012Coric V. van Dyck C.H. Salloway S. Andreasen N. Brody M. Richter R.W. Soininen H. Thein S. Shiovitz T. Pilcher G. et al.Safety and tolerability of the gamma-secretase inhibitor avagacestat in a phase 2 study of mild to moderate Alzheimer disease.Arch. Neurol. 2012; 69: 1430-1440Crossref PubMed Scopus (266) Google Scholar). Our study provides mechanistic evidence that BMS-708163 is a pan inhibitor of γ-secretase, reconciling reports that were heretofore seemingly disparate. Furthermore, it breaks ground for investigating the molecular mechanism and binding mode of GSMs, which specifically target toxic Aβ species and are at the forefront of AD research because of their potential as disease-modifying agents (Crump et al., 2013Crump C.J. Johnson D.S. Li Y.-M. Development and mechanism of γ-secretase modulators for Alzheimer’s disease.Biochemistry. 2013; 52: 3197-3216Crossref PubMed Scopus (139) Google Scholar). In addition, this work develops an effective approach for mapping the interaction site of small molecules with target proteins, including membrane proteins. Identification of small-molecule binding sites within the γ-secretase complex has been a formidable challenge in both structural and chemical biology studies due to the nature of this multi-subunit transmembrane protein complex. In this report, we have developed a new generation of BMS-708163-based probes with cleavable linkers and ultimately found that 163-BP3-Dde-biotin was the most efficient at labeling and eluting PS1-NTF. Taking advantage of this probe's efficiency, we mapped the binding site of 163-BP3-Dde-biotin on γ-secretase for the first time. We were able to localize 163-BP3-Dde-biotin binding to amino acids 279–291 of PS1-NTF and identified Leu282 as the probe-modified residue. Identification of a site of labeling combined with MD simulations based on the γ-secretase structure allowed us to confidently model the binding site for the BMS-708163 series of GSIs, offering a molecular basis for inhibition of γ-secretase. This work paves the way for mapping the binding sites of other clinically relevant GSIs/GSMs in order to better understand the mechanism by which these compounds alter γ-secretase activity. For full experimental procedures, see the Supplemental Information. ANPP membrane (Kim et al., 2003Kim S.H. Ikeuchi T. Yu C. Sisodia S.S. Regulated hyperaccumulation of presenilin-1 and the “gamma-secretase” complex. Evidence for differential intramembranous processing of transmembrane substrates.J. Biol. Chem. 2003; 278: 33992-34002Crossref PubMed Scopus (96) Google Scholar) (6.4 mg) was photolabeled with 100 nM 163-BP3-Dde-biotin with or without 10 μM BMS-708163. The reactions were incubated at 37°C for 1 hr and UV irradiated at 350 nm for 30 min on a cold block. The labeled samples were centrifuged at 100,000 × g, and pellets were resuspended in 500 μL of RIPA buffer using TissuLyser (QIAGEN). The solubilized samples were spun down at 16,000 × g for 10 min and ensuing supernatants were combined and pulled down with 30 μL of streptavidin beads. Beads were washed three times with RIPA and three times with PBS and then eluted twice with 40 μL of 2% hydrazine + 0.05% SDS and twice with 40 μL of water, and these eluates were combined and frozen. When all samples had been eluted, they were combined and concentrated approximately 24-fold (from 1,440 μL down to ∼60 μL) on 10 kDa Amicon Ultra Centricon. A fraction (1 μL) of these samples was run on a western blot, blotting for PS1-NTF, and the rest was processed for LC-MS/MS. The concentrated sample (60 μL) was treated with 60 μL of water and 480 μL of acetone and incubated at −20°C overnight. The samples were then centrifuged at 4°C for 5 min at 15,000 × g, and the residues were dried. They were next redissolved in 40 μL of 8 M urea containing 5 mM DTT and incubated at 60°C for 1 hr, after which they were allowed to cool to room temperature, treated with iodoacetamide (10 mM), and then incubated for 1 hr at room temperature. The samples were then treated with 40 μL of 0.05 M NH4HCO3 containing 0.2 μg of LysC (Wako) and incubated overnight at 37°C, after which they were treated with 160 μL of 0.05 M NH4HCO3 containing 0.2 μg of trypsin (Promega sequencing grade), and incubated for 4 hr at 37°C. Following this, each sample was acidified to pH < 3 by addition of trifluoroacetic acid and subjected to solid-phase extraction of peptides using StageTips (Rappsilber et al., 2007Rappsilber J. Mann M. Ishihama Y. Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips.Nat. Protoc. 2007; 2: 1896-1906Crossref PubMed Scopus (2569) Google Scholar). The resulting samples were dried in the centrifugal evaporator, redissolved in 40 μL of 0.1% trifluoroacetic acid, and 7.5 μL portions were analyzed by LC-MS using a Waters nanoAcquity system operating at 250 nL/min and a Q Exactive mass spectrometer (Thermo Fisher Scientific). Specific searches for products of photolabeling were conducted against a custom-built small database containing the sequences of human γ-secretase subunits together with the sequences of known abundant contaminants. Theoretical peptide masses and MS/MS fragmentations were calculated using GPMAW v. 9.5 (Lighthouse data, Odense, Denmark) and ChemBioDraw Ultra v. 13.0 (PerkinElmer). N.G. conducted experiments, analyzed data, and wrote the paper. C.W.A. and P.M. designed and synthesized compounds. K.F.G., C.N., and U.S. performed MS experiments and analyzed data. S.M. conducted molecular dynamics simulations. D.S.J. and Y.-M.L. conceived the project and wrote the paper. Y.-M.L serves as the Lead Contact. We thank Dr. David Iaea for discussion of this work and Dr. Sam Sisodia's generosity for providing the ANPP cell line. This work is supported by NIH grant R01AG026660 (Y.-M.L.), R01NS076117 (Y.-M.L.), R01NS096275 (Y.-M.L.) Alzheimer Association IIRG-12-242137 (Y.-M.L.), the JPB Foundation (Y.-M.L.), the MetLife Foundation (Y.-M.L.). We also acknowledge the MSK Cancer Center Support Grant/Core Grant (P30 CA008748), Mr. William H. Goodwin and Mrs. Alice Goodwin, and the Commonwealth Foundation for Cancer Research, the Experimental Therapeutics Center of MSKCC, and the William Randolph Hearst Fund in Experimental Therapeutics. C.W.A., P.M., K.F.G., C.N., U.S., S.M., and D.S.J. are employees at Pfizer. Y.-M.L. is a consultant for Pfizer. Download .pdf (1.04 MB) Help with pdf files Document S1. Supplemental Experimental Procedures and Figures S1–S4" @default.
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