New precision medicine avenues to the prevention of Alzheimer's disease from insights into the structure and function of γ-secretases.

Two phase-III clinical trials with anti-amyloid peptide antibodies have met their primary goal, i.e. slowing of Alzheimer's disease (AD) progression. However, antibody therapy may not be the optimal therapeutic modality for AD prevention, as we will discuss in the context of the earlier small molecules described as "γ-secretase modulators" (GSM). We review here the structure, function, and pathobiology of γ-secretases, with a focus on how mutations in presenilin genes result in early-onset AD. Significant progress has been made in generating compounds that act in a manner opposite to pathogenic presenilin mutations: they stabilize the proteinase-substrate complex, thereby increasing the processivity of substrate cleavage and altering the size spectrum of Aß peptides produced. We propose the term "γ-secretase allosteric stabilizers" (GSAS) to distinguish these compounds from the rather heterogenous class of GSM. The GSAS represent, in theory, a precision medicine approach to the prevention of amyloid deposition, as they specifically target a discrete aspect in a complex cell biological signalling mechanism that initiates the pathological processes leading to Alzheimer's disease.

Cell-free protein production of a gamma secretase homolog.

Cleavage of the transmembrane domain (TMD) of amyloid-ß precursor protein (APP) by γ-secretase, an intramembrane aspartyl protease, generates Aß peptides of various lengths that form plaques in the brains of Alzheimer's disease patients. Although the debate has not been finally resolved whether these plaques trigger the onset of Alzheimer's or are side products, disease-related mutations suggest their implication in the etiology of the dementia. These occur both in presenilin, the catalytic subunit of γ-secretase, and in the TMD of APP. Despite two seminal cryo-electron microscopy structures that show the complex of γ-secretase with its substrates APP and Notch, the mechanism of γ-secretase is not yet fully understood. Especially on which basis it selects its substrates is still an enigma. The presenilin homolog PSH from the archaeon Methanoculleus marisnigri JR1 (MCMJR1) is catalytically active without accessory proteins in contrast to γ-secretase making it an excellent model for studies of the basic cleavage process. We here focused on the cell-free expression of PSH screening a range of conditions. Cleavage assays to verify the activity show that not only the yield, but mainly the activity of the protease depends on the careful selection of expression conditions. Optimal results were found for a cell-free expression at relatively low temperature, 20 °C, employing cell lysates prepared from E. coli Rosetta cells. To speed up protein preparation for immediate functional assays, a crude purification protocol was developed. This allows to produce ready-made PSH in a fast and efficient manner in less than two days.

Permissive Conformations of a Transmembrane Helix Allow Intramembrane Proteolysis by γ-Secretase.

The intramembrane protease γ-secretase activates important signaling molecules, such as Notch receptors. It is still unclear, however, how different elements within the primary structure of substrate transmembrane domains (TMDs) contribute to their cleavability. Using a newly developed yeast-based cleavage assay, we identified three crucial regions within the TMDs of the paralogs Notch1 and Notch3 by mutational and gain-of-function approaches. The AAAA or AGAV motifs within the N-terminal half of the TMDs were found to confer strong conformational flexibility to these TMD helices, as determined by mutagenesis coupled to deuterium/hydrogen exchange. Crucial amino acids within the C-terminal half may support substrate docking into the catalytic cleft of presenilin, the enzymatic subunit of γ-secretase. Further, residues close to the C-termini of the TMDs may stabilize a tripartite ß-sheet in the substrate/enzyme complex. NMR structures reveal different extents of helix bending as well as an ability to adopt widely differing conformational substates, depending on the sequence of the N-terminal half. The difference in cleavability between Notch1 and Notch3 TMDs is jointly determined by the conformational repertoires of the TMD helices and the sequences of the C-terminal half, as suggested by mutagenesis and building molecular models. In sum, cleavability of a γ-secretase substrate is enabled by different functions of cooperating TMD regions, which deepens our mechanistic understanding of substrate/non-substrate discrimination in intramembrane proteolysis.

Structural basis for membrane-proximal proteolysis of substrates by ADAM10.

The endopeptidase ADAM10 is a critical catalyst for the regulated proteolysis of key drivers of mammalian development, physiology, and non-amyloidogenic cleavage of APP as the primary α-secretase. ADAM10 function requires the formation of a complex with a C8-tetraspanin protein, but how tetraspanin binding enables positioning of the enzyme active site for membrane-proximal cleavage remains unknown. We present here a cryo-EM structure of a vFab-ADAM10-Tspan15 complex, which shows that Tspan15 binding relieves ADAM10 autoinhibition and acts as a molecular measuring stick to position the enzyme active site about 20 Å from the plasma membrane for membrane-proximal substrate cleavage. Cell-based assays of N-cadherin shedding establish that the positioning of the active site by the interface between the ADAM10 catalytic domain and the bound tetraspanin influences selection of the preferred cleavage site. Together, these studies reveal the molecular mechanism underlying ADAM10 proteolysis at membrane-proximal sites and offer a roadmap for its modulation in disease.

GS-SMD server for steered molecular dynamics of peptide substrates in the active site of the γ-secretase complex.

Despite recent advances in research, the mechanism of Alzheimer's disease is not fully understood yet. Understanding the process of cleavage and then trimming of peptide substrates, can help selectively block γ-secretase (GS) to stop overproduction of the amyloidogenic products. Our GS-SMD server (https://gs-smd.biomodellab.eu/) allows cleaving and unfolding of all currently known GS substrates (more than 170 peptide substrates). The substrate structure is obtained by threading of the substrate sequence into the known structure of GS complex. The simulations are performed in an implicit water-membrane environment so they are performed rather quickly, 2-6 h per job, depending on the mode of calculations (part of GS complex or the whole structure). It is also possible to introduce mutations to the substrate and GS and pull any part of the substrate in any direction using the steered molecular dynamics (SMD) simulations with constant velocity. The obtained trajectories are visualized and analyzed in the interactive way. One can also compare multiple simulations using the interaction frequency analysis. GS-SMD server can be useful for revealing mechanisms of substrate unfolding and role of mutations in this process.

High throughput and targeted screens for prepilin peptidase inhibitors do not identify common inhibitors of eukaryotic gamma-secretase.

INTRODUCTION: Prepilin peptidases (PPP) are essential enzymes for the biogenesis of important virulence factors, such as type IV pili (T4P), type II secretion systems, and other T4P-related systems of bacteria and archaea. PPP inhibitors could be valuable pharmaceuticals, but only a few have been reported. Interestingly, PPP share similarities with presenilin enzymes from the gamma-secretase protease complex, which are linked to Alzheimer's disease. Numerous gamma-secretase inhibitors have been reported, and some have entered clinical trials, but none has been tested against PPP. OBJECTIVE: The objective of this study is to develop a high-throughput screening (HTS) method to search for inhibitors of PPP from various chemical libraries and reported gamma-secretase inhibitors. METHOD: More than 15,000 diverse compounds, including 13 reported gamma-secretase inhibitors and other reported peptidase inhibitors, were screened to identify potential PPP inhibitors. RESULTS: The authors developed a novel screening method and screened 15,869 compounds. However, the screening did not identify a PPP inhibitor. Nevertheless, the study suggests that gamma-secretase is sufficiently different from PPP that specific inhibitors may exist in a larger chemical space. CONCLUSION: The authors believe that the HTS method that they describe has numerous advantages and encourage others to consider its application in the search for PPP inhibitors.

In silico Study of Acetylcholinesterase and Beta-secretase Inhibitors: Potential Multitarget Anti-Alzheimer's Agents.

BACKGROUND: Alzheimer's disease is a progressive neurodegenerative process with multifactorial characteristics. This disease follows the natural aging process, affecting mainly people over 65 years. Pharmacotherapeutic treatment currently combats symptoms related to cognitive function. Several targets have begun to attract the interest of the scientific community to develop new drug candidates which have better pharmacokinetic and lower toxicity parameters. OBJECTIVE: The present study aims to design new candidates for acetylcholinesterase/ß-secretase (AChE/BACE1) multitarget inhibitor drugs. METHODS: 17 natural products were selected from the literature with anticholinesterase activity and 1 synthetic molecule with inhibitory activity for BACE1. Subsequently, the molecular docking study was performed, followed by the derivation of the pharmacophoric pattern and prediction of pharmacokinetic and toxicological properties. Finally, the hybrid prototype was designed. RESULTS: All selected molecules showed interactions with their respective target enzymes. Derivation of the pharmacophoric pattern from molecules that interacted with the AChE enzyme resulted in 3 pharmacophoric regions: an aromatic ring, an electron-acceptor region and a hydrophobic region. The molecules showed good pharmacokinetic and toxicological results, showing no warnings of mutagenicity and/or carcinogenicity. After the hybridization process, three hybrid molecules were obtained, which showed inhibitory activity for both targets. CONCLUSION: It is concluded that research in the field of medicinal chemistry is advancing towards the discovery of new drug candidates that bring a better quality of life to patients with AD.

Identification of potential lead compounds against BACE1 through in-silico screening of phytochemicals of Medhya rasayana plants for Alzheimer's disease management.

Alzheimer's disease is a progressive and irreversible neurodegenerative disease that accounts for 70-80% of dementia in the elderly. According to recent clinical data, the incidence of the disease is exponentially increasing with age. Beta-site amyloid precursor protein cleaving enzyme1 (BACE1) is an important molecule involved in the pathogenesis of Alzheimer's disease due to its early role in the amyloid cascade. Cleavage of amyloid precursor protein by BACE1 is the rate-limiting step leading to the production and aggregation of amyloid-beta plaques. A number of natural products are being identified as non-competitive BACE1 inhibitors. In Ayurveda, Medhya rasayana is a group of medicinal herbs, specifically used for managing neurological disorders and is known to be effective in improving cognitivity and intellect. This study aimed to analyze the pharmacological activity of bio-active compounds in Medhya rasayana plants against BACE1, employing structure-based docking approach. 11 compounds out of 876 were identified as potential hits, based on docking scores, binding energies, and interactions with the critical residues of BACE1. Possible neurological activities of these compounds were predicted using PASS server. Out of the 11 compounds screened, two compounds, 'Convolidine' from the plant Convolvulus pleuricaulis Choisy and 'N-(4-hydroxybutyl) phthalimide' from Glycyrrhiza glabra satisfied the pharmacological parameters of Lipinski rule of filtering and ADMET prediction. The binding stability of these compounds against BACE1 was confirmed by molecular dynamic simulation and post dynamic MM/GBSA calculations. Detailed analysis of the interaction with the critical amino acids in the active site revealed the possible inhibitory potential of these compounds of medicinal plant origin against BACE1.

Seeing your partner: Structural elucidation of the first C8 tetraspanin protein.

Tetraspanins are proteins that organize cell membranes via interactions with partner proteins mediated by their large ectodomain. In this issue of Structure, Lipper et al., 2022 have elucidated the structure of the first C8 tetraspanin and expand functional insight into how C8 tetraspanins regulate substrate specificity for the transmembrane protease ADAM10.

Aminoimidazo[1,2-a]pyridine Bearing Different Pyrazole Moieties as the Structural Scaffold for the Development of BACE1 Inhibitor; Synthesis, Structural Characterization, in vitro and in silico Studies.

Regarding the critical role of amyloid-ß plaques in the pathogenesis of Alzheimer's disease, a series of aminoimidazo[1,2-a]pyridine derivatives were designed and synthesized as potential anti-BACE1 agents targeting the production of amyloid-ß plaques. In vitro biological results demonstrated that compounds 7b and 7f exhibited the best inhibitory potency against BACE1 with IC50 values of 22.48 ± 2.06 and 30.61 ± 3.48 µM, respectively. Also, the ligandprotein docking evaluations revealed that compounds 7b and 7f could effectively bind with the different pockets of BACE1 through different interactions with the residue of the active site. The results of current studies underline the potential role of aminoimidazo[1,2-a] pyridine-containing pyrazole derivatives for developing novel BACE1 inhibitors.

Unravelling the molecular basis of AM-6494 high potency at BACE1 in Alzheimer's disease: an integrated dynamic interaction investigation.

ß-amyloid precursor protein cleaving enzyme1 (BACE1) has prominently been an important drug design target implicated in Alzheimer's disease pathway. The failure rate of most of the already tested drugs at different clinical phases remains a major concern. Recently, AM-6494 was reported as a novel potent, highly selective, and orally effective inhibitor against BACE1. AM-6494 displayed no alteration of skin/fur colour in animal studies, an adverse effect common to previous BACE1 inhibitors. However, the atomistic molecular mechanism of BACE1 inhibition by AM-6494 remains unclear. To elucidate the binding mechanism of AM-6494 relative to umibecestat (CNP-520) as well as the structural changes when bound to BACE1, advanced computational techniques such as accelerated MD simulation and principal component analysis have been utilised. The results demonstrated higher binding affinity of AM-6494 at BACE1 with van der Waals as dominant energy contributor compared to umibecestat. Conformational monitoring of the ß-hairpin flap covering the active site revealed an effective flap closure when bound with AM-6494 compared to CNP-520, which predominantly alternates between semi-open and closed conformations. The observed effective flap closure of AM-6494 explains its higher inhibitory power towards BACE1. Besides the catalytic Asp32/228 dyad, Tyr14, Leu30, Tyr71 and Gly230 represent critical residues in the potency of these inhibitors at BACE1 binding interface. The findings highlighted in this research provide a basis to explain AM-6494 high inhibitory potency and might assist in the design of new inhibitors with improved selectivity and potency for BACE1.

In-silico evaluations of the isolated phytosterols from polygonum hydropiper L against BACE1 and MAO drug targets.

Our previous anti-Alzheimer's studies on crude extracts, essential oils and isolated compounds including ß-sitostrol from Polygonum hydropiper L, motivated us for further studies against beta amyloid cleaving enzyme 1 (BACE1) and monoamine oxidases (MAO-A), (MAO-B) enzymes. Before performing detailed studies on the compounds using animal models and immunohistochemistry, molecular docking study was performed against three vital enzymes implicated in several neurological disorders including Alzheimer's disease (AD), Parkinson's disease (PD), depression and anxiety to predict their inhibitory potential against important enzymes. Beta amyloid cleaving enzyme 1 (BACE1) is important enzyme that catalyze pathological amyloidogenic pathway of processing amyloid precursor proteins to form neurotoxic amyloid plaques. Subsequently, BACE1 inhibitors are considered an important tool in the management of AD. MAOs have been categorized in two well-known groups MAO-A and MAO-B, based on their differential affinity for various monoamines substrates. MAO-A has more affinity for norepinephrine and 5-HT, whereas, MAO-B mainly catalyze the breakdown of dopamine and 2-phenylathylamine (PEA) and other monoamines. Subsequently, they have divergent behavioral outcomes and play a significant role in pathophysiology of several neurodegenerative disorders like AD, depression, drug abuse, migraines, schizophrenia, Attention Deficit Disorder (ADD) and Parkinson's disease (PD). Molecular docking was carried out to predict the binding modes of ß-sitosterol and stigmasterol in the binding pockets of BACE1 (beta-sectretase 1) and MAO (monoamine oxidase A, B) enzymes. The 3 D structure of BACE1 (PDB ID: 2QP8), MAO A (PDB ID: 2ZPX) and MAO B (PDB ID: 2XFN) were downloaded from protein databank. The 3 D structures were then subjected to protonation and energy minimization using default parameters of MOE. Three dimensional structures of ß-sitosterol and stigmasterol were built by using Molecular Builder Module program implemented in MOE and saved as a (.mdb) file for molecular docking. Subsequently, the energy of both the compounds were minimized up to 0.05 Gradient using MMFF 94 s force field implemented in MOE. Both the compounds were docked into the active site of proteins using the Triangular Matching docking method (default) and 10 different conformations for each compound were generated. To obtain minimum energy structures the ligands were allowed to be flexible during docking. At the end of docking, the predicted ligand-protein complexes were analyzed for molecular interactions. Overall the docking results showed that these compounds showed good interaction with active site residues of BACE1 as compare to MAO-A and MAO-B. Furthermore, ß-sitosterol showed good interaction with BACE1 as compare to stigmasterol.Communicated by Ramaswamy H. Sarma.

Probing the intermolecular interactions, binding affinity, charge density distribution and dynamics of silibinin in dual targets AChE and BACE1: QTAIM and molecular dynamics perspective.

Alzheimer's disease (AD) is the grievous neurodegenerative disorder. Reportedly, many enzymes are responsible for this disease, in which notably, acetylcholinesterase (AChE) and ß-secretase (BACE1) are largely involved for AD. An experimental study reports that silibinin molecule inhibits both AChE and BACE1 enzymes. Present study aims to understand the dual binding mechanism of silibinin in the active site of AChE and BACE1 from the intermolecular interactions, conformational flexibility, charge density distribution, binding energy and the stability of molecule. To obtain the above information, the molecular docking, molecular dynamics (MD) and QTAIM (quantum theory of atoms in molecules) calculations have been performed. The molecular docking reveals that silibinin molecule is forming strong and weak intermolecular interactions with the catalytic site of both enzymes. The QTAIM analysis for the binding pockets of both complexes shows the charge density distribution of intermolecular interactions. The electrostatic potential map displays the electronegative/positive regions at the interaction zone of silibinin with AChE and BACE1 complexes. The MD simulation confirms that the silibinin molecule is stable in the active site of AChE and BACE1 enzymes. The binding free energies of silibinin with both enzymes are more favorable to have the interactions.Communicated by Ramaswamy H. Sarma.

Conformational Models of APP Processing by Gamma Secretase Based on Analysis of Pathogenic Mutations.

Proteolytic processing of amyloid precursor protein (APP) plays a critical role in the pathogenesis of Alzheimer's disease (AD). Sequential cleavage of APP by ß and γ secretases leads to the generation of Aß40 (non-amyloidogenic) and Aß42 (amyloidogenic) peptides. Presenilin-1 (PS1) or presenilin-2 (PS2) play the role of a catalytic subunit of γ-secretase. Multiple familial AD (FAD) mutations in APP, PS1, or PS2 result in an increased Aß42:Aß40 ratio and the accumulation of toxic Aß42 oligomers and plaques in patient brains. In this study, we perform molecular modeling of the APP complex with γ-secretase and analyze potential effects of FAD mutations in APP and PS1. We noticed that all FAD mutations in the APP transmembrane domain are predicted to cause an increase in the local disorder of its secondary structure. Based on structural analysis of known γ-secretase structures, we propose that APP can form a complex with γ-secretase in 2 potential conformations-M1 and M2. In conformation, the M1 transmembrane domain of APP forms a contact with the perimembrane domain that follows transmembrane domain 6 (TM6) in the PS1 structure. In conformation, the M2 transmembrane domain of APP forms a contact with transmembrane domain 7 (TM7) in the PS1 structure. By analyzing the effects of PS1-FAD mutations on the local protein disorder index, we discovered that these mutations increase the conformational flexibility of M2 and reduce the conformational flexibility of M1. Based on these results, we propose that M2 conformation, but not M1 conformation, of the γ secretase complex with APP leads to the amyloidogenic (Aß42-generating) processing of APP. Our model predicts that APP processing in M1 conformation is favored by curved membranes, such as the membranes of early endosomes. In contrast, APP processing in M2 conformation is likely to be favored by relatively flat membranes, such as membranes of late endosomes and plasma membranes. These predictions are consistent with published biochemical analyses of APP processing at different subcellular locations. Our results also suggest that specific inhibitors of Aß42 production could be potentially developed by selectively targeting the M2 conformation of the γ secretase complex with APP.

Concatenation of molecular docking and molecular simulation of BACE-1, γ-secretase targeted ligands: in pursuit of Alzheimer's treatment.

INTRODUCTION: Alzheimer's disease (AD), the most predominant cause of dementia, has evolved tremendously with an escalating frequency, mainly affecting the elderly population. An effective means of delaying, preventing, or treating AD is yet to be achieved. The failure rate of dementia drug trials has been relatively higher than in other disease-related clinical trials. Hence, multi-targeted therapeutic approaches are gaining attention in pharmacological developments. AIMS: As an extension of our earlier reports, we have performed docking and molecular dynamic (MD) simulation studies for the same 13 potential ligands against beta-site APP cleaving enzyme 1 (BACE-1) and γ-secretase as a therapeutic target for AD. The In-silico screening of these ligands as potential inhibitors of BACE-1 and γ-secretase was performed using AutoDock enabled PyRx v-0.8. The protein-ligand interactions were analyzed in Discovery Studio 2020 (BIOVIA). The stability of the most promising ligand against BACE-1 and γ-secretase was evaluated by MD simulation using Desmond-2018 (Schrodinger, LLC, NY, USA). RESULTS: The computational screening revealed that the docking energy values for each of the ligands against both the target enzymes were in the range of -7.0 to -10.1 kcal/mol. Among the 13 ligands, 8 (55E, 6Z2, 6Z5, BRW, F1B, GVP, IQ6, and X37) showed binding energies of ≤-8 kcal/mol against BACE-1 and γ-secretase. For the selected enzyme targets, BACE-1 and γ-secretase, 6Z5 displayed the lowest binding energy of -10.1 and -9.8 kcal/mol, respectively. The MD simulation study confirmed the stability of BACE-6Z5 and γ-secretase-6Z5 complexes and highlighted the formation of a stable complex between 6Z5 and target enzymes. CONCLUSION: The virtual screening, molecular docking, and molecular dynamics simulation studies revealed the potential of these multi-enzyme targeted ligands. Among the studied ligands, 6Z5 seems to have the best binding potential and forms a stable complex with BACE-1 and γ-secretase. We recommend the synthesis of 6Z5 for future in-vitro and in-vivo studies.

Nanoscale organization of Nicastrin, the substrate receptor of the γ-secretase complex, as independent molecular domains.

Alterations in the canonical processing of Amyloid Precursor Protein generate proteoforms that contribute to the onset of Alzheimer's Disease. Modified composition of γ-secretase or mutations in its subunits has been directly linked to altered generation of Amyloid beta. Despite biochemical evidence about the role of γ-secretase in the generation of APP, the molecular origin of how spatial heterogeneity in the generation of proteoforms arises is not well understood. Here, we evaluated the localization of Nicastrin, a γ-secretase subunit, at nanometer sized functional zones of the synapse. With the help of super resolution microscopy, we confirm that Nicastrin is organized into nanodomains of high molecular density within an excitatory synapse. A similar nanoorganization was also observed for APP and the catalytic subunit of γ-secretase, Presenilin 1, that were discretely associated with Nicastrin nanodomains. Though Nicastrin is a functional subunit of γ-secretase, the Nicastrin and Presenilin1 nanodomains were either colocalized or localized independent of each other. The Nicastrin and Presenilin domains highlight a potential independent regulation of these molecules different from their canonical secretase function. The collisions between secretases and substrate molecules decide the probability and rate of product formation for transmembrane proteolysis. Our observations of secretase nanodomains indicate a spatial difference in the confinement of substrate and secretases, affecting the local probability of product formation by increasing their molecular availability, resulting in differential generation of proteoforms even within single synapses.

Design of Transmembrane Mimetic Structural Probes to Trap Different Stages of γ-Secretase-Substrate Interaction.

The transmembrane domain (TMD) of the amyloid precursor protein of Alzheimer's disease is cut processively by γ-secretase through endoproteolysis and tricarboxypeptidase "trimming". We recently developed a prototype substrate TMD mimetic for structural analysis-composed of a helical peptide inhibitor linked to a transition-state analogue-that simultaneously engages a substrate exosite and the active site and is pre-organized to trap the carboxypeptidase transition state. Here, we developed variants of this prototype designed to allow visualization of transition states for endoproteolysis, TMD helix unwinding, and lateral gating of the substrate, identifying potent inhibitors for each class. These TMD mimetics exhibited non-competitive inhibition and occupy both the exosite and the active site, as demonstrated by inhibitor cross-competition experiments and photoaffinity probe binding assays. The new probes should be important structural tools for trapping different stages of substrate recognition and processing via ongoing cryo-electron microscopy with γ-secretase, ultimately aiding rational drug design.

The Role of Cholesterol in Amyloidogenic Substrate Binding to the γ-Secretase Complex.

Alzheimer's disease is the most common progressive neurodegenerative disorder and is characterized by the presence of amyloid ß (Aß) plaques in the brain. The γ-secretase complex, which produces Aß, is an intramembrane-cleaving protease consisting of four membrane proteins. In this paper we investigated the amyloidogenic fragments of amyloid precursor protein (substrates Aß43 and Aß45, leading to less amyloidogenic Aß40 and more amyloidogenic Aß42, respectively) docked to the binding site of presenilin, the catalytic subunit of γ-secretase. In total, we performed 9 µs of all-atom molecular dynamics simulations of the whole γ-secretase complex with both substrates in low (10%) and high (50%) concentrations of cholesterol in the membrane. We found that, at the high cholesterol level, the Aß45 helix was statistically more flexible in the binding site of presenilin than Aß43. An increase in the cholesterol concentration was also correlated with a higher flexibility of the Aß45 helix, which suggests incompatibility between Aß45 and the binding site of presenilin potentiated by a high cholesterol level. However, at the C-terminal part of Aß45, the active site of presenilin was more compact in the case of a high cholesterol level, which could promote processing of this substrate. We also performed detailed mapping of the cholesterol binding sites at low and high cholesterol concentrations, which were independent of the typical cholesterol binding motifs.

Assembly of γ-secretase occurs through stable dimers after exit from the endoplasmic reticulum.

γ-Secretase affects many physiological processes through targeting >100 substrates; malfunctioning links γ-secretase to cancer and Alzheimer's disease. The spatiotemporal regulation of its stoichiometric assembly remains unresolved. Fractionation, biochemical assays, and imaging support prior formation of stable dimers in the ER, which, after ER exit, assemble into full complexes. In vitro ER budding shows that none of the subunits is required for the exit of others. However, knockout of any subunit leads to the accumulation of incomplete subcomplexes in COPII vesicles. Mutating a DPE motif in presenilin 1 (PSEN1) abrogates ER exit of PSEN1 and PEN-2 but not nicastrin. We explain this by the preferential sorting of PSEN1 and nicastrin through Sec24A and Sec24C/D, respectively, arguing against full assembly before ER exit. Thus, dimeric subcomplexes aided by Sec24 paralog selectivity support a stepwise assembly of γ-secretase, controlling final levels in post-Golgi compartments.

1,3,5-Triazine Nitrogen Mustards with Different Peptide Group as Innovative Candidates for AChE and BACE1 Inhibitors.

A series of new analogs of nitrogen mustards (4a-4h) containing the 1,3,5-triazine ring substituted with dipeptide residue were synthesized and evaluated for the inhibition of both acetylcholinesterase (AChE) and ß-secretase (BACE1) enzymes. The AChE inhibitory activity studies were carried out using Ellman's colorimetric method, and the BACE1 inhibitory activity studies were carried out using fluorescence resonance energy transfer (FRET). All compounds displayed considerable AChE and BACE1 inhibition. The most active against both AChE and BACE1 enzymes were compounds A and 4a, with an inhibitory concentration of AChE IC50 = 0.051 µM; 0.055 µM and BACE1 IC50 = 9.00 µM; 11.09 µM, respectively.