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The early initiation of binge-drinking and biological sex are critical risk factors for the development of affective disturbances and cognitive decline, as well as neurodegenerative diseases including Alzheimers disease. Further, a history of excessive alcohol consumption alters normal age-related changes in the pattern of protein expression in the brain, which may relate to an acceleration of cognitive decline. Here, we aimed to disentangle the interrelation between a history of binge-drinking during adolescence, biological sex and normal aging on the manifestation of negative affect, cognitive decline and associated biochemical pathology. To this end, adolescent male and female C57BL/6J mice (PND 28-29) underwent 30 days of alcohol binge-drinking using a modified drinking-in-the-dark (DID) paradigm. Then, mice were assayed for negative affect, sensorimotor gating and cognition at three developmental stages during adulthood-mature adulthood (6 months), pre-middle age (9 months) and middle age (12 months). Behavioural testing was then followed by immunoblotting to index the protein expression of glutamate receptors, neuropathological markers [Tau, p (Thr217)-Tau, p (Ser396)-Tau, BACE, APP, Aβ], as well as ERK activation within the entorhinal cortex, prefrontal cortex and amygdala. Across this age span, we detected only a few age-related changes in our measures of negative affect or spatial learning/memory in the Morris water maze and all of these changes were sex-specific. Prior adolescent binge-drinking impaired behaviour only during reversal learning in 9-month-old females and during radial arm maze testing in 12-month-old females. In contrast to behaviour, we detected a large number of protein changes related to prior binge-drinking history, several of which manifested as early as 6 months of age, with the prefrontal cortex particularly affected at this earlier age. While 6-month-old mice exhibited relatively few alcohol-related protein changes within the entorhinal cortex and amygdala, the number of alcohol-related protein changes within the entorhinal cortex increased with age, while the 12-month-old mice exhibited the largest number of protein changes within the amygdala. Approximately a third of the alcohol-related protein changes were sex-selective. Taken together, the results of our longitudinal study using a murine model of binge-drinking indicate that a prior history of heavy alcohol consumption, beginning in adolescence, is sufficient to induce what we presume to be latent changes in protein indices of cellular activity, glutamate transmission and neuropathology within key brain regions governing cognition, executive function and emotion that appear to precede the onset of robust behavioural signs of dysregulated affect and cognitive impairment.

Amyloid β (Aβ) peptides accumulating in the brain are proposed to trigger Alzheimers disease (AD). However, molecular cascades underlying their toxicity are poorly defined. Here, we explored a novel hypothesis for Aβ42 toxicity that arises from its proven affinity for γ-secretases. We hypothesized that the reported increases in Aβ42, particularly in the endolysosomal compartment, promote the establishment of a product feedback inhibitory mechanism on γ-secretases, and thereby impair downstream signaling events. We conducted kinetic analyses of γ-secretase activity in cell-free systems in the presence of Aβ, as well as cell-based and ex vivo assays in neuronal cell lines, neurons, and brain synaptosomes to assess the impact of Aβ on γ-secretases. We show that human Aβ42 peptides, but neither murine Aβ42 nor human Aβ17-42 (p3), inhibit γ-secretases and trigger accumulation of unprocessed substrates in neurons, including C-terminal fragments (CTFs) of APP, p75, and pan-cadherin. Moreover, Aβ42 treatment dysregulated cellular homeostasis, as shown by the induction of p75-dependent neuronal death in two distinct cellular systems. Our findings raise the possibility that pathological elevations in Aβ42 contribute to cellular toxicity via the γ-secretase inhibition, and provide a novel conceptual framework to address Aβ toxicity in the context of γ-secretase-dependent homeostatic signaling.

Autonomic dysfunction has been observed in Alzheimer's disease (AD); however, the effects of genes involved in AD on the peripheral nervous system are not well understood. Previous studies have shown that presenilin-1 (PSEN1), the catalytic subunit of the gamma secretase (γ-secretase) complex, mutations in which are associated with familial AD function, regulates dendritic growth in hippocampal neurons. In this study, we examined whether the γ-secretase pathway also influences dendritic growth in primary sympathetic neurons. Using immunoblotting and immunocytochemistry, molecules of the γ-secretase complex, PSEN1, PSEN2, PEN2, nicastrin and APH1a, were detected in sympathetic neurons dissociated from embryonic (E20/21) rat sympathetic ganglia. Addition of bone morphogenetic protein-7 (BMP-7), which induces dendrites in these neurons, did not alter expression or localization of γ-secretase complex proteins. BMP-7-induced dendritic growth was inhibited by siRNA knockdown of PSEN1 and by three γ-secretase inhibitors, γ-secretase inhibitor IX (DAPT), LY-411575 and BMS-299897. These effects were specific to dendrites and concentration-dependent and did not alter early downstream pathways of BMP signaling. In summary, our results indicate that γ-secretase activity enhances BMP-7 induced dendritic growth in sympathetic neurons. These findings provide insight into the normal cellular role of the γ-secretase complex in sympathetic neurons.

BackgroundDesmoid tumors are rare, locally aggressive, highly recurrent soft-tissue tumors without approved treatments.MethodsWe conducted a phase 3, international, double-blind, randomized, placebo-controlled trial of nirogacestat in adults with progressing desmoid tumors according to the Response Evaluation Criteria in Solid Tumors, version 1.1. Patients were assigned in a 1:1 ratio to receive the oral γ-secretase inhibitor nirogacestat (150 mg) or placebo twice daily. The primary end point was progression-free survival.ResultsFrom May 2019 through August 2020, a total of 70 patients were assigned to receive nirogacestat and 72 to receive placebo. Nirogacestat had a significant progression-free survival benefit over placebo (hazard ratio for disease progression or death, 0.29; 95% confidence interval, 0.15 to 0.55; P

Mutations or triplication of the alpha synuclein (ASYN) gene contribute to synucleinopathies including Parkinsons disease (PD), Dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). Recent evidence suggests that ASYN also plays an important role in amyloid-induced neurotoxicity, although the mechanism(s) remains unknown. One hypothesis is that accumulation of ASYN alters endolysosomal pathways to impact axonal trafficking and processing of the amyloid precursor protein (APP). To define an axonal function for ASYN, we used a transgenic mouse model of synucleinopathy that expresses a GFP-human ASYN (GFP-hASYN) transgene and an ASYN knockout (ASYN-/-) mouse model. Our results demonstrate that expression of GFP-hASYN in primary neurons derived from a transgenic mouse impaired axonal trafficking and processing of APP. In addition, axonal transport of BACE1, Rab5, Rab7, lysosomes and mitochondria were also reduced in these neurons. Interestingly, axonal transport of these organelles was also affected in ASYN-/- neurons, suggesting that ASYN plays an important role in maintaining normal axonal transport function. Therefore, selective impairment of trafficking and processing of APP by ASYN may act as a potential mechanism to induce pathological features of Alzheimers disease (AD) in PD patients.

Adherens junctions (AJs) create spatially, chemically and mechanically discrete microdomains at cellular interfaces. Here, using a mechanogenetic platform that generates artificial AJs with controlled protein localization, clustering and mechanical loading, we find that AJs also organize proteolytic hotspots for γ-secretase with a spatially regulated substrate selectivity that is critical in the processing of Notch and other transmembrane proteins. Membrane microdomains outside of AJs exclusively organize Notch ligand-receptor engagement (LRE microdomains) to initiate receptor activation. Conversely, membrane microdomains within AJs exclusively serve to coordinate regulated intramembrane proteolysis (RIP microdomains). They do so by concentrating γ-secretase and primed receptors while excluding full-length Notch. AJs induce these functionally distinct microdomains by means of lipid-dependent γ-secretase recruitment and size-dependent protein segregation. By excluding full-length Notch from RIP microdomains, AJs prevent inappropriate enzyme-substrate interactions and suppress spurious Notch activation. Ligand-induced ectodomain shedding eliminates size-dependent segregation, releasing Notch to translocate into AJs for processing by γ-secretase. This mechanism directs radial differentiation of ventricular zone-neural progenitor cells in vivo and more broadly regulates the proteolysis of other large cell-surface receptors such as amyloid precursor protein. These findings suggest an unprecedented role of AJs in creating size-selective spatial switches that choreograph γ-secretase processing of multiple transmembrane proteins regulating development, homeostasis and disease.

Stroke is ranked as the fifth leading cause of death and the leading cause of adult disability in the USA. The progression of neuronal damage after stroke is recognized to be a complex integration of glia, neurons, and the surrounding extracellular matrix, therefore potential treatments must target the detrimental effects created by these interactions. In this study, we examined the spatial cellular and neuroinflammatory mechanisms occurring early after ischemic stroke utilizing Nanostring Digital Spatial Profiling (DSP) technology. Male C57bl/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO) and sacrificed at 3 days post-ischemia. Spatial distinction of the ipsilateral hemisphere was studied according to the regions of interest: the ischemic core, peri-infarct tissues, and peri-infarct normal tissue (PiNT) in comparison to the contralateral hemisphere. We demonstrated that the ipsilateral hemisphere initiates distinct spatial regulatory proteomic profiles with DSP technology that can be identified consistently with the immunohistochemical markers, FJB, GFAP, and Iba-1. The core border profile demonstrated an induction of neuronal death, apoptosis, autophagy, immunoreactivity, and early degenerative proteins. Most notably, the core border resulted in a decrease of the neuronal proteins Map2 and NeuN; an increase in the autophagy proteins BAG3 and CTSD; an increase in the microglial and peripheral immune invasion proteins Iba1, CD45, CD11b, and CD39; and an increase in the neurodegenerative proteins BACE1, APP, amyloid β 1-42, ApoE, and hyperphosphorylated tau protein S-199. The peri-infarct region demonstrated increased astrocytic, immunoreactivity, apoptotic, and neurodegenerative proteomic profiles, with an increase in BAG3, GFAP, and hyperphosphorylated tau protein S-199. The PiNT region displayed minimal changes compared to the contralateral cortex with only an increase in GFAP. In this study, we showed that mechanisms known to be associated with stroke, such as apoptosis and inflammation, occur in distinct spatial domains of the injured brain following ischemia. We also demonstrated the dysregulation of specific autophagic pathways that may lead to neurodegeneration in peri-infarct brain tissues. Taken together, these data suggest that identifying post-ischemic mechanisms occurring in a spatiotemporal manner may lead to more precise targets for successful therapeutic interventions to treat stroke.

While amyloid-β (Aβ) plaques are considered a hallmark of Alzheimer's disease, clinical trials focused on targeting gamma secretase, an enzyme involved in aberrant Aβ peptide production, have not led to amelioration of AD symptoms or synaptic dysregulation. Screening strategies based on mechanistic, multi-omics approaches that go beyond pathological readouts can aid in the evaluation of therapeutics. Using early-onset Alzheimer's (EOFAD) disease patient lineage PSEN1A246E iPSC-derived neurons, we performed RNA-seq to characterize AD-associated endotypes, which are in turn used as a screening evaluation metric for two gamma secretase drugs, the inhibitor Semagacestat and the modulator BPN-15606. We demonstrate that drug treatment partially restores the neuronal state while concomitantly inhibiting cell cycle re-entry and dedifferentiation endotypes to different degrees depending on the mechanism of gamma secretase engagement. Our endotype-centric screening approach offers a new paradigm by which candidate AD therapeutics can be evaluated for their overall ability to reverse disease endotypes.

We determined the effectiveness of absolute binding free energy (ABFE) calculations to refine the selection of active compounds in virtual compound screening, a setting where the more commonly used relative binding free energy approach is not readily applicable. To do this, we conducted baseline docking calculations of structurally diverse compounds in the DUD-E database for three targets, BACE1, CDK2 and thrombin, followed by ABFE calculations for compounds with high docking scores. The docking calculations alone achieved solid enrichment of active compounds over decoys. Encouragingly, the ABFE calculations then improved on this baseline. Analysis of the results emphasizes the importance of establishing high quality ligand poses as starting points for ABFE calculations, a nontrivial goal when processing a library of diverse compounds without informative co-crystal structures. Overall, our results suggest that ABFE calculations can play a valuable role in the drug discovery process.

IntroductionThere is increasing interest in plasma amyloid beta (Aβ) as an endophenotype of Alzheimer's disease (AD). Identifying the genetic determinants of plasma Aβ levels may elucidate important biological processes that determine plasma Aβ measures.MethodsWe included 12,369 non-demented participants from eight population-based studies. Imputed genetic data and measured plasma Aβ1-40, Aβ1-42 levels and Aβ1-42/Aβ1-40 ratio were used to perform genome-wide association studies, and gene-based and pathway analyses. Significant variants and genes were followed up for their association with brain positron emission tomography Aβ deposition and AD risk.ResultsSingle-variant analysis identified associations with apolipoprotein E (APOE) for Aβ1-42 and Aβ1-42/Aβ1-40 ratio, and BACE1 for Aβ1-40. Gene-based analysis of Aβ1-40 additionally identified associations for APP, PSEN2, CCK, and ZNF397. There was suggestive evidence for interaction between a BACE1 variant and APOE ε4 on brain Aβ deposition.DiscussionIdentification of variants near/in known major Aβ-processing genes strengthens the relevance of plasma-Aβ levels as an endophenotype of AD.

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.

Amyloid disorders such as Alzheimer's disease (AD) involve the aggregation of secreted proteins. However, it is largely unclear how secretory-pathway proteins contribute to amyloid formation. We developed a systems biology framework integrating expression data with protein-protein interaction networks to estimate a tissue's fitness for producing specific secreted proteins and analyzed the fitness of the secretory pathway of various brain regions and cell types for synthesizing the AD-associated amyloid precursor protein (APP). While key amyloidogenic pathway components were not differentially expressed in AD brains, we found Aβ deposition correlates with systemic down- and upregulation of the secretory-pathway components proximal to APP and amyloidogenic secretases, respectively, in AD. Our analyses suggest that perturbations from three AD risk loci cascade through the APP secretory-support network and into the endocytosis pathway, connecting amyloidogenesis to dysregulation of secretory-pathway components supporting APP and suggesting novel therapeutic targets for AD. A record of this paper's transparent peer review process is included in the supplemental information.

Hidradenitis suppurativa (HS) is a chronic, relapsing, and remitting inflammatory disease of the skin with significant heritability and racial disposition. The pathogenesis of HS remains enigmatic, but occlusion of the terminal hair follicle and dysregulation of the local innate immune response may contribute to pathogenesis. Genetic predisposition might also contribute to disease susceptibility and phenotypic heterogeneity because mutations in γ-secretase have been found to underlie a minor but characteristic subset of patients with HS. In this review, we synthesized the current data on γ-secretase in HS, evaluated its importance in the context of disease pathobiology, and discussed avenues of future studies.

A potent γ-secretase modulator (GSM) has been developed to circumvent problems associated with γ-secretase inhibitors (GSIs) and to potentially enable use in primary prevention of early-onset familial Alzheimer's disease (EOFAD). Unlike GSIs, GSMs do not inhibit γ-secretase activity but rather allosterically modulate γ-secretase, reducing the net production of Aβ42 and to a lesser extent Aβ40, while concomitantly augmenting production of Aβ38 and Aβ37. This GSM demonstrated robust time- and dose-dependent efficacy in acute, subchronic, and chronic studies across multiple species, including primary and secondary prevention studies in a transgenic mouse model. The GSM displayed a >40-fold safety margin in rats based on a comparison of the systemic exposure (AUC) at the no observed adverse effect level (NOAEL) to the 50% effective AUC or AUCeffective, the systemic exposure required for reducing levels of Aβ42 in rat brain by 50%.

BACE1 (beta-site amyloid precursor protein cleaving enzyme 1) was initially cloned and characterized in 1999. It is required for the generation of all monomeric forms of amyloid-β (Aβ), including Aβ42, which aggregates into bioactive conformational species and likely initiates toxicity in Alzheimer's disease (AD). BACE1 concentrations and rates of activity are increased in AD brains and body fluids, thereby supporting the hypothesis that BACE1 plays a critical role in AD pathophysiology. Therefore, BACE1 is a prime drug target for slowing down Aβ production in early AD. Besides the amyloidogenic pathway, BACE1 has other substrates that may be important for synaptic plasticity and synaptic homeostasis. Indeed, germline and adult conditional BACE1 knockout mice display complex neurological phenotypes. Despite BACE1 inhibitor clinical trials conducted so far being discontinued for futility or safety reasons, BACE1 remains a well-validated therapeutic target for AD. A safe and efficacious compound with high substrate selectivity as well as a more accurate dose regimen, patient population, and disease stage may yet be found. Further research should focus on the role of Aβ and BACE1 in physiological processes and key pathophysiological mechanisms of AD. The functions of BACE1 and the homologue BACE2, as well as the biology of Aβ in neurons and glia, deserve further investigation. Cellular and molecular studies of BACE1 and BACE2 knockout mice coupled with biomarker-based human research will help elucidate the biological functions of these important enzymes and identify their substrates and downstream effects. Such studies will have critical implications for BACE1 inhibition as a therapeutic approach for AD.

BackgroundAlzheimer's disease (AD) is the most frequent and costly neurodegenerative disorder. Although diverse lines of evidence suggest that the amyloid precursor protein (APP) is involved in its causation, the precise mechanisms remain unknown and no treatments are available to prevent or halt the disease. A favorite hypothesis has been that APP contributes to AD pathogenesis through the cerebral accumulation of the amyloid-β peptide (Aβ), which is derived from APP through sequential proteolytic cleavage by BACE1 and γ-secretase. However, inhibitors of these enzymes have failed in clinical trials despite clear evidence for target engagement.MethodsTo further elucidate the roles of APP and its metabolites in AD pathogenesis, we analyzed transgenic mice overexpressing wildtype human APP (hAPP) or hAPP carrying mutations that cause autosomal dominant familial AD (FAD), as well as App knock-in mice that do not overexpress hAPP but have two mouse App alleles with FAD mutations and a humanized Aβ sequence.ResultsAlthough these lines of mice had marked differences in cortical and hippocampal levels of APP, APP C-terminal fragments, soluble Aβ, Aβ oligomers and age-dependent amyloid deposition, they all developed cognitive deficits as well as non-convulsive epileptiform activity, a type of network dysfunction that also occurs in a substantive proportion of humans with AD. Pharmacological inhibition of BACE1 effectively reduced levels of amyloidogenic APP C-terminal fragments (C99), soluble Aβ, Aβ oligomers, and amyloid deposits in transgenic mice expressing FAD-mutant hAPP, but did not improve their network dysfunction and behavioral abnormalities, even when initiated at early stages before amyloid deposits were detectable.ConclusionshAPP transgenic and App knock-in mice develop similar pathophysiological alterations. APP and its metabolites contribute to AD-related functional alterations through complex combinatorial mechanisms that may be difficult to block with BACE inhibitors and, possibly, also with other anti-Aβ treatments.

β-Secretase1 (BACE1) protein concentrations and rates of enzyme activity, analyzed in human bodily fluids, are promising candidate biological markers for guidance in clinical trials investigating BACE1 inhibitors to halt or delay the dysregulation of the amyloid-β pathway in Alzheimer's disease (AD). A robust body of evidence demonstrates an association between cerebrospinal fluid/blood BACE1 biomarkers and core pathophysiological mechanisms of AD, such as brain protein misfolding and aggregration, neurodegeneration, and synaptic dysfunction.In pharmacological trials, BACE1 candidate biomarkers may be applied to a wide set of contexts of use (CoU), including proof of mechanism, dose-finding, response and toxicity dose estimation. For clinical CoU, BACE1 biomarkers show good performance for prognosis and disease prediction.The roadmap toward validation and qualification of BACE1 biomarkers requires standardized pre-analytical and analytical protocols to reduce inter-site variance that may have contributed to inconsistent results.BACE1 biomarker-drug co-development programs, including biomarker-guided outcomes and endpoints, may support the identification of sub-populations with a higher probability to benefit from BACE1 inhibitors with a reduced risk of adverse effects, in line with the evolving precision medicine paradigm.

The evolution of gamma-secretase modulators (GSMs) through the introduction of novel heterocycles with the goal of aligning activity for reducing the levels of Aβ42 and properties consistent with a drug-like molecule are described. The insertion of a methoxypyridine motif within the tetracyclic scaffold provided compounds with improved activity for arresting Aβ42 production as well as improved properties, including solubility. In vivo pharmacokinetic analysis demonstrated that several compounds within the novel series were capable of crossing the BBB and accessing the therapeutic target. Treatment with methoxypyridine-derived compound 64 reduced Aβ42 levels in the plasma of J20 mice, in addition to reducing Aβ42 levels in the plasma and brain of Tg2576 mice.

Intrahepatic cholangiocarcinoma (iCCA) is a deadly disease with rising incidence and few treatment options. An altered expression and/or activation of NOTCH1-3 receptors has been shown to play a role in iCCA development and progression. In this study, we established a new CCA patient-derived xenograft model, which was validated by immunohistochemistry and transcriptomic analysis. The effects of Notch pathway suppression by the Crenigacestat (LY3039478)-specific inhibitor were evaluated in human iCCA cell lines and the PDX model. In vitro, LY3039478 significantly reduced Notch pathway components, including NICD1 and HES1, but not the other Notch receptors, in a panel of five different iCCA cell lines. In the PDX model, LY3039478 significantly inhibited the Notch pathway and tumor growth to the same extent as gemcitabine. Furthermore, gene expression analysis of iCCA mouse tissues treated with LY3039478 revealed a downregulation of VEGFA, HES1, and MMP13 genes. In the same tissues, DLL4 and CD31 co-localized, and their expression was significantly inhibited in the treated mice, as it happened in the case of MMP13. In an in vitro angiogenesis model, LY3039478 inhibited vessel formation, which was restored by the addition of MMP13. Finally, RNA-sequencing expression data of iCCA patients and matched surrounding normal liver tissues downloaded from the GEO database demonstrated that NOTCH1, HES1, MMP13, DLL4, and VEGFA genes were significantly upregulated in tumors compared with adjacent nontumorous tissues. These data were confirmed by our group, using an independent cohort of iCCA specimens. Conclusion: We have developed and validated a new iCCA PDX model to test in vivo the activity of LY3039478, demonstrating its inhibitory role in Notch-dependent angiogenesis. Thus, the present data provide new knowledge on Notch signaling in iCCA, and support the inhibition of the Notch cascade as a promising strategy for the treatment of this disease.

Background: Alzheimer's disease (AD) is one of the most common forms of dementia, is characterized by memory loss and cognitive impairment that affects more than 30 million people worldwide. The pathogenesis of Alzheimer's disease is primary driven by brain accumulation of the amyloid ß peptide generated from the amyloid-ß precursor protein (APP) via cleavages by ß- and -secretase. In this study, we propose an approach by molecular docking to select compounds as -secretase inhibitors for decreasing the APP generation. Methods: We selected potential -secretase inhibitors by molecular docking in the potential site between Asp257, Lue268, Asp385, Ile387, Phe388, and Leu432 amino acids in presenilin-1 (PS-1), using a chemical library of over 500,000 compounds. Results: Eight compounds (AZ1 - AZ8) were selected by molecular docking to develop -secretase inhibitors for decreasing the APP generation. Conclusion: AZ1 - AZ8 compounds could be interacting in the potential site between Asp257, Lue268, Asp385, Ile387, Phe388, and Leu432 amino acids in PS-1. These compounds could specifically interact in the binding pocket in PS-1 to prevent/decrease the APP generation, to develop a new drug against Alzheimer's disease. [ABSTRACT FROM AUTHOR]

The Drug Design Data Resource (D3R) Grand Challenges present an opportunity to assess, in the context of a blind predictive challenge, the accuracy and the limits of tools and methodologies designed to help guide pharmaceutical drug discovery projects. Here, we report the results of our participation in the D3R Grand Challenge 4 (GC4), which focused on predicting the binding poses and affinity ranking for compounds targeting the [Formula: see text]-amyloid precursor protein (BACE-1). Our ligand similarity-based protocol using HYBRID (OpenEye Scientific Software) successfully identified poses close to the native binding mode for most of the ligands with less than 2 Å RMSD accuracy. Furthermore, we compared the performance of our HYBRID-based approach to that of AutoDock Vina and DOCK 6 and found that using a reference ligand to guide the docking process is a better strategy for pose prediction and helped HYBRID to perform better here. We also conducted end-point free energy estimates on molecules dynamics based ensembles of protein-ligand complexes using molecular mechanics combined with generalized Born surface area method (MM-GBSA). We found that the binding affinity ranking based on MM-GBSA scores have poor correlation with the experimental values. Finally, the main lessons from our participation in D3R GC4 are: (i) the generation of the macrocyclic conformers is a key step for successful pose prediction, (ii) the protonation states of the BACE-1 binding site should be treated carefully, (iii) the MM-GBSA method could not discriminate well between different predicted binding poses, and (iv) the MM-GBSA method does not perform well at predicting protein-ligand binding affinities here.

The Drug Design Data Resource (D3R) aims to identify best practice methods for computer aided drug design through blinded ligand pose prediction and affinity challenges. Herein, we report on the results of Grand Challenge 4 (GC4). GC4 focused on proteins beta secretase 1 and Cathepsin S, and was run in an analogous manner to prior challenges. In Stage 1, participant ability to predict the pose and affinity of BACE1 ligands were assessed. Following the completion of Stage 1, all BACE1 co-crystal structures were released, and Stage 2 tested affinity rankings with co-crystal structures. We provide an analysis of the results and discuss insights into determined best practice methods.

We describe a new template-based method for docking flexible ligands such as macrocycles to proteins. It combines Monte-Carlo energy minimization on the manifold, a fast manifold search method, with BRIKARD for complex flexible ligand searching, and with the MELD accelerator of Replica-Exchange Molecular Dynamics simulations for atomistic degrees of freedom. Here we test the method in the Drug Design Data Resource blind Grand Challenge competition. This method was among the best performers in the competition, giving sub-angstrom prediction quality for the majority of the targets.

X-ray crystallography often requires non-native constructs involving mutations or truncations, and is challenged by membrane proteins and large multicomponent complexes. We present here a bottom-up endogenous structural proteomics approach whereby near-atomic-resolution cryo electron microscopy (cryoEM) maps are reconstructed ab initio from unidentified protein complexes enriched directly from the endogenous cellular milieu, followed by identification and atomic modeling of the proteins. The proteins in each complex are identified using cryoID, a program we developed to identify proteins in ab initio cryoEM maps. As a proof of principle, we applied this approach to the malaria-causing parasite Plasmodium falciparum, an organism that has resisted conventional structural-biology approaches, to obtain atomic models of multiple protein complexes implicated in intraerythrocytic survival of the parasite. Our approach is broadly applicable for determining structures of undiscovered protein complexes enriched directly from endogenous sources.

Molecular docking has been successfully used in computer-aided molecular design projects for the identification of ligand poses within protein binding sites. However, relying on docking scores to rank different ligands with respect to their experimental affinities might not be sufficient. It is believed that the binding scores calculated using molecular mechanics combined with the Poisson-Boltzman surface area (MM-PBSA) or generalized Born surface area (MM-GBSA) can predict binding affinities more accurately. In this perspective, we decided to take part in Stage 2 of the Drug Design Data Resource (D3R) Grand Challenge 4 (GC4) to compare the performance of a quick scoring function, AutoDock4, to that of MM-GBSA in predicting the binding affinities of a set of [Formula: see text]-Amyloid Cleaving Enzyme 1 (BACE-1) ligands. Our results show that re-scoring docking poses using MM-GBSA did not improve the correlation with experimental affinities. We further did a retrospective analysis of the results and found that our MM-GBSA protocol is sensitive to details in the protein-ligand system: (i) neutral ligands are more adapted to MM-GBSA calculations than charged ligands, (ii) predicted binding affinities depend on the initial conformation of the BACE-1 receptor, (iii) protonating the aspartyl dyad of BACE-1 correctly results in more accurate binding affinity predictions.

Alchemical free energy calculations have made a dramatic impact upon the field of structure-based drug design by allowing functional group modifications to be explored computationally prior to experimental synthesis and assay evaluation, thereby informing and directing synthetic strategies. In furthering the advancement of this area, a series of 21 β-secretase 1 (BACE1) inhibitors developed by Janssen Pharmaceuticals were examined to evaluate the ability to explore large substituent perturbations, some of which contain scaffold modifications, with multisite λ-dynamics (MSλD), an innovative alchemical free energy framework. Our findings indicate that MSλD is able to efficiently explore all structurally diverse ligand end-states simultaneously within a single MD simulation with a high degree of precision and with reduced computational costs compared to the widely used approach TI/MBAR. Furthermore, computational predictions were shown to be accurate to within 0.5-0.8 kcal/mol when CM1A partial atomic charges were combined with CHARMM or OPLS-AA-based force fields, demonstrating that MSλD is force field independent and a viable alternative to FEP or TI approaches for drug design.

Beta-secretase 1 (BACE-1) is an aspartyl protease implicated in the overproduction of β-amyloid fibrils responsible for Alzheimer disease. The process of β-amyloid genesis is known to be pH dependent, with an activity peak between solution pH of 3.5 and 5.5. We have studied the pH-dependent dynamics of BACE-1 to better understand the pH dependent mechanism. We have implemented support for graphics processor unit (GPU) accelerated constant pH molecular dynamics within the AMBER molecular dynamics software package and employed this to determine the relative population of different aspartyl dyad protonation states in the pH range of greatest β-amyloid production, followed by conventional molecular dynamics to explore the differences among the various aspartyl dyad protonation states. We observed a difference in dynamics between double-protonated, mono-protonated, and double-deprotonated states over the known pH range of higher activity. These differences include Tyr 71-aspartyl dyad proximity and active water lifetime. This work indicates that Tyr 71 stabilizes catalytic water in the aspartyl dyad active site, enabling BACE-1 activity.

Despite growing evidence linking Drosophila melanogaster tweety-homologue 1 (Ttyh1) to normal mammalian brain development and cell proliferation, its exact role has not yet been determined. Here, we show that Ttyh1 is required for the maintenance of neural stem cell (NSC) properties as assessed by neurosphere formation and in vivo analyses of cell localization after in utero electroporation. We find that enhanced Ttyh1-dependent stemness of NSCs is caused by enhanced γ-secretase activity resulting in increased levels of Notch intracellular domain (NICD) production and activation of Notch targets. This is a unique function of Ttyh1 among all other Ttyh family members. Molecular analyses revealed that Ttyh1 binds to the regulator of γ-secretase activity Rer1 in the endoplasmic reticulum and thereby destabilizes Rer1 protein levels. This is the key step for Ttyh1-dependent enhancement of γ-secretase activity, as Rer1 overexpression completely abolishes the effects of Ttyh1 on NSC maintenance. Taken together, these findings indicate that Ttyh1 plays an important role during mammalian brain development by positively regulating the Notch signaling pathway through the downregulation of Rer1.

Developing effective therapeutics for complex diseases such as late-onset, sporadic Alzheimer's disease (SAD) is difficult due to genetic and environmental heterogeneity in the human population and the limitations of existing animal models. Here, we used hiPSC-derived neurons to test a compound that stabilizes the retromer, a highly conserved multiprotein assembly that plays a pivotal role in trafficking molecules through the endosomal network. Using this human-specific system, we have confirmed previous data generated in murine models and show that retromer stabilization has a potentially beneficial effect on amyloid beta generation from human stem cell-derived neurons. We further demonstrate that manipulation of retromer complex levels within neurons affects pathogenic TAU phosphorylation in an amyloid-independent manner. Taken together, our work demonstrates that retromer stabilization is a promising candidate for therapeutic development in AD and highlights the advantages of testing novel compounds in a human-specific, neuronal system.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disorder. The major pathological changes in AD progression are the generation and accumulation of amyloid-beta (Aβ) peptides as well as the presence of abnormally hyperphosphorylated tau proteins in the brain. Autophagy is a conserved degradation pathway that eliminates abnormal protein aggregates and damaged organelles. Previous studies have suggested that autophagy plays a key role in the production and clearance of Aβ peptides to maintain a steady-state of Aβ peptides levels. However, a growing body of evidence suggests that autophagy is significantly impaired in the pathogenesis of AD, especially in Aβ metabolism. Therefore, this article reviews the latest studies concerning the mechanisms of autophagy, the metabolism of Aβ peptides, and the defective autophagy in the production and clearance of Aβ peptides. Here, we also summarize the established and new strategies for targeting autophagy in vivo and through clinical AD trials to identify gaps in our knowledge and to generate further questions. [ABSTRACT FROM AUTHOR]

Pericytes regulate cerebral blood flow (CBF) and excess amyloid in the brain. Pericyte dysfunction may contribute to the pathology of Alzheimer's disease (AD). Acorus tatarinowii (AT), a Chinese medicine commonly used to treat AD, protects the central nervous system. However, whether AT can regulate pericyte function and ameliorate cognitive dysfunction remains unclear. We employed a novel target recognition assay, quantitative measurement of CBF, hematoxylin and eosin staining, immunofluorescence staining, and Western blot to investigate the role of AT in improving cognitive function in patients with AD. Additionally, we investigated the therapeutic potential of β-Asarone, the primary active compound in AT, for treating AD by modulating pericyte function using transmission electron microscopy, silver staining, electrical impedance, and other methodologies. The results revealed that administration of AT effectively alleviated the cognitive impairments induced by D-galactose in mice, as evidenced by enhanced CBF, improved histological characteristics of damaged brain tissue cells, increased expression of platelet-derived growth factor-β (PDGF-β), decreased Aβ accumulation via enhanced lipoprotein receptor-related protein 1 (LRP1), and reduced beta-site APP-cleaving enzyme 1 (BACE1). β-Asarone treatment mitigated ROS release and BACE1 expression while elevating the cell index in Aβ1-40 injured mouse brain vascular pericytes (MBVP). These findings suggest that AT has the potential to enhance CBF and mitigate pericellular dysfunction, thereby ameliorating Aβ deposition in the brain and improving cognitive impairment in patients with AD., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

An in-situ nanozyme signal tag combined with a DNA-mediated universal antibody-oriented strategy was proposed to establish a high-performance immunosensing platform for Alzheimer's disease (AD)-related biomarker detection. Briefly, a Zr-based metal-organic framework (MOF) with peroxidase (POD)-like activity was synthesized to encapsulating the electroactive molecule methylene blue (MB), and subsequently modified with a layer of gold nanoparticles on its surface. This led to the creation of double POD-like activity nanozymes surrounding the MB molecule to form a nanozyme signal tag. A large number of hydroxyl radicals were generated by the nanozyme signal tag with the help of H 2 O 2 , which catalyzed MB molecules in situ to achieve efficient signal amplification. Subsequently, a DNA-aptamer-mediated universal antibody-oriented strategy was proposed to enhance the binding efficiency for the antigen (target). Meanwhile, a poly adenine was incorporated at the end of the aptamer, facilitating binding to the gold electrode and providing anti-fouling properties due to the hydrophilicity of the phosphate group. Under optimal conditions, this platform was successfully employed for highly sensitive detection of AD-associated tau protein and BACE1, achieving limits of detection with concentrations of 3.34 fg/mL and 1.67 fg/mL, respectively. It is worth mentioning that in the tau immunosensing mode, 20 clinical samples from volunteers of varying ages were analyzed, revealing significantly higher tau expression levels in the blood samples of elderly volunteers compared to young volunteers. This suggests that the developed strategy holds great promise for early AD diagnosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Alzheimer's disease (AD) is a neurodegenerative disease that leads to development of cognition and memory dysfunctions. Currently, there is no known cure for AD, although limited medications are approved for the management of disease condition. Various plant-based leads give new hope for considering phytoconstituents as anti-AD drugs. The Piper nigrum L. fruit extract was reported to have anti-Alzheimer's activity. It creates an interest in the finding of active moieties that may be accountable for anti-AD activity., Hypothesis/purpose: Identification of multitarget directed ligands isolated from Piper nigrum fruits through AI based studies., Study Design: The phytochemical analysis of alkaloid fraction was carried out by LCMS, followed by the evaluation of constituents through in silico studies., Methods: The fruits methanolic extract was prepared by cold maceration technique. The chemical profiling of the alkaloidal fraction was carried out using LCMS/MS analysis. The obtained compound's target hit genes were identified through network pharmacology studies using String, Metascape, and Cytoscape tools. Further, docking studies and MD simulations were carried out using AutoDock4 and Desmond-Maestro software. Then, electrochemical properties of hit compound P4 were determined using Gaussview6 software., Results: From LCMS/MS analysis data, 29 compounds were considered based on compound intensity and accuracy (>95 %). Then, 41 common gene targets were identified from AD genes and compound-targeted genes. The 41 common genes in the PPI network suggested that AChE and BACE1 were the most abundant proteins. Further, docking studies revealed the hit compound P4 binding interaction and energies when compared to other 28 ligands. The molecular dynamics studies showed that P4-AChE and P4-BACE1 complexes were stable, and there were no RMSD and RMSF fluctuations were observed up to 100 ns. Further, PCA and MM-GBSA analysis data supported that complexes (P4-AChE and P4-BACE1) were stable. The DFT and surface properties indicated that compound P4 was ideal candidate for AD treatment and must be considered for further biological activity studies., Conclusion: The study identified compound P4 (dehydropipernonaline) from alkaloidal fraction of Piper nigrum fruits, suggesting it may be hit candidate for AD treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier GmbH. All rights reserved.)

Abstract Objective NGFR/p75NTR and NRADD/NRH proteins are closely related structurally and are encoded by genes that arose from a duplication event early in vertebrate evolution. The transmembrane domain (TMD) of NGFR is cleaved by γ-secretase but there is conflicting data around the susceptibility to γ-secretase cleavage of NRADD proteins. If NGFR and NRADD show differential susceptibility to γ-secretase, then they can be used to dissect the structural constraints determining substrate susceptibility. We sought to test this differential susceptibility. Results We developed labelled, lumenally-truncated forms of zebrafish Ngfrb and Nradd and a chimeric protein in which the TMD of Nradd was replaced with the TMD of Ngfrb. We expressed these in zebrafish embryos to test their susceptibility to γ-secretase cleavage by monitoring their stability using western immunoblotting. Inhibition of γ-secretase activity using DAPT increased the stability of only the Ngfrb construct. Our results support that only NGFR is cleaved by γ-secretase. Either NGFR evolved γ-secretase-susceptibility since its creation by gene duplication, or NRADD evolved to be refractory to γ-secretase. Protein structure outside of the TMD of NGFR is likely required for susceptibility to γ-secretase.

Generation of amyloid-β peptides (Aβs) by proteolytic cleavage of the amyloid-β protein precursor (AβPP), especially increased production of Aβ42/Aβ43 over Aβ40, and their aggregation as oligomers and plaques, represent a characteristic feature of Alzheimer's disease (AD). In familial AD (FAD), altered Aβ production originates from specific mutations of AβPP or presenilins 1/2 (PS1/PS2), the catalytic subunits of γ-secretase. In sporadic AD, the origin of altered production of Aβs remains unknown. We hypothesize that the 'human chemical exposome' contains products able to favor the production of Aβ42/Aβ43 over Aβ40 and shorter Aβs. To detect such products, we screened a library of 3500 + compounds in a cell-based assay for enhanced Aβ42/Aβ43 production. Nine pyrazole insecticides were found to induce a β- and γ-secretase-dependent, 3-10-fold increase in the production of extracellular Aβ42 in various cell lines and neurons differentiated from induced pluripotent stem cells derived from healthy and FAD patients. Immunoprecipitation/mass spectrometry analyses showed increased production of Aβs cleaved at positions 42/43, and reduced production of peptides cleaved at positions 38 and shorter. Strongly supporting a direct effect on γ-secretase activity, pyrazoles shifted the cleavage pattern of another γ-secretase substrate, alcadeinα, and shifted the cleavage of AβPP by highly purified γ-secretase toward Aβ42/Aβ43. Focusing on fipronil, we showed that some of its metabolites, in particular the persistent fipronil sulfone, also favor the production of Aβ42/Aβ43 in both cell-based and cell-free systems. Fipronil administered orally to mice and rats is known to be metabolized rapidly, mostly to fipronil sulfone, which stably accumulates in adipose tissue and brain. In conclusion, several widely used pyrazole insecticides enhance the production of toxic, aggregation prone Aβ42/Aβ43 peptides, suggesting the possible existence of environmental "Alzheimerogens" which may contribute to the initiation and propagation of the amyloidogenic process in sporadic AD.

In the amyloidogenic pathway associated with Alzheimer disease (AD), the amyloid precursor protein (APP) is cleaved by β-secretase to generate a 99-aa C-terminal fragment (C99) that is then cleaved by γ-secretase to generate the β-amyloid (Aβ) found in senile plaques. In previous reports, we and others have shown that γ-secretase activity is enriched in mitochondria-associated endoplasmic reticulum (ER) membranes (MAM) and that ER-mitochondrial connectivity and MAM function are upregulated in AD We now show that C99, in addition to its localization in endosomes, can also be found in MAM, where it is normally processed rapidly by γ-secretase. In cell models of AD, however, the concentration of unprocessed C99 increases in MAM regions, resulting in elevated sphingolipid turnover and an altered lipid composition of both MAM and mitochondrial membranes. In turn, this change in mitochondrial membrane composition interferes with the proper assembly and activity of mitochondrial respiratory supercomplexes, thereby likely contributing to the bioenergetic defects characteristic of AD.

A central pathogenic event of Alzheimer's disease (AD) is the accumulation of the Aβ42 peptide, which is generated from amyloid-β precursor protein (APP) via cleavages by β- and γ-secretase. We have developed a class of soluble 2-aminothiazole γ-secretase modulators (SGSMs) that preferentially decreases Aβ42 levels. However, the effects of SGSMs in AD animals and cells expressing familial AD mutations, as well as the mechanism of γ-secretase modulation remain largely unknown. Here, a representative of this SGSM scaffold, SGSM-36, was investigated using animals and cells expressing FAD mutations. SGSM-36 preferentially reduced Aβ42 levels without affecting either α- and β-secretase processing of APP nor Notch processing. Furthermore, an allosteric site was identified within the γ-secretase complex that allowed access of SGSM-36 using cell-based, fluorescence lifetime imaging microscopy analysis. Collectively, these studies provide mechanistic insights regarding SGSMs of this class and reinforce their therapeutic potential in AD.

Alzheimer's disease (AD) is characterized neuropathologically by an abundance of 1) neuritic plaques, which are primarily composed of a fibrillar 42-amino-acid amyloid-β peptide (Aβ), as well as 2) neurofibrillary tangles composed of aggregates of hyperphosporylated tau. Elevations in the concentrations of the Aβ42 peptide in the brain, as a result of either increased production or decreased clearance, are postulated to initiate and drive the AD pathologic process. We initially introduced a novel class of bridged aromatics referred tγ-secretase modulatoro as γ-secretase modulators that inhibited the production of the Aβ42 peptide and to a lesser degree the Aβ40 peptide while concomitantly increasing the production of the carboxyl-truncated Aβ38 and Aβ37 peptides. These modulators potently lower Aβ42 levels without inhibiting the γ-secretase-mediated proteolysis of Notch or causing accumulation of carboxyl-terminal fragments of APP. In this study, we report a large number of pharmacological studies and early assessment of toxicology characterizing a highly potent γ-secretase modulator (GSM), (S)-N-(1-(4-fluorophenyl)ethyl)-6-(6-methoxy-5-(4-methyl-1H-imidazol-1-yl)pyridin-2-yl)-4-methylpyridazin-3-amine (BPN-15606). BPN-15606 displayed the ability to significantly lower Aβ42 levels in the central nervous system of rats and mice at doses as low as 5-10 mg/kg, significantly reduce Aβ neuritic plaque load in an AD transgenic mouse model, and significantly reduce levels of insoluble Aβ42 and pThr181 tau in a three-dimensional human neural cell culture model. Results from repeat-dose toxicity studies in rats and dose escalation/repeat-dose toxicity studies in nonhuman primates have designated this GSM for 28-day Investigational New Drug-enabling good laboratory practice studies and positioned it as a candidate for human clinical trials.

Proteolytic processing of amyloid precursor protein (APP) C-terminal fragments (CTFs) by γ-secretase underlies the pathogenesis of Alzheimer's disease (AD). An RNA interference screen using APP-CTF [99-residue CTF (C99)]- and Notch-specific γ-secretase interaction assays identified a unique ErbB2-centered signaling network that was predicted to preferentially govern the proteostasis of APP-C99. Consistently, significantly elevated levels of ErbB2 were confirmed in the hippocampus of human AD brains. We then found that ErbB2 effectively suppressed autophagic flux by physically dissociating Beclin-1 from the Vps34-Vps15 complex independent of its kinase activity. Down-regulation of ErbB2 by CL-387,785 decreased the levels of C99 and secreted amyloid-β in cellular, zebrafish, and mouse models of AD, through the activation of autophagy. Oral administration of an ErbB2-targeted CL-387,785 for 3 wk significantly improves the cognitive functions of APP/presenilin-1 (PS1) transgenic mice. This work unveils a noncanonical function of ErbB2 in modulating autophagy and establishes ErbB2 as a therapeutic target for AD.

Metastatic castration-resistant prostate cancer (CRPC) is the primary cause of prostate cancer-specific mortality. Defining new mechanisms that can predict recurrence and drive lethal CRPC is critical. Here, we demonstrate that localized high-risk prostate cancer and metastatic CRPC, but not benign prostate tissues or low/intermediate-risk prostate cancer, express high levels of nuclear Notch homolog 1, translocation-associated (Notch1) receptor intracellular domain. Chronic activation of Notch1 synergizes with multiple oncogenic pathways altered in early disease to promote the development of prostate adenocarcinoma. These tumors display features of epithelial-to-mesenchymal transition, a cellular state associated with increased tumor aggressiveness. Consistent with its activation in clinical CRPC, tumors driven by Notch1 intracellular domain in combination with multiple pathways altered in prostate cancer are metastatic and resistant to androgen deprivation. Our study provides functional evidence that the Notch1 signaling axis synergizes with alternative pathways in promoting metastatic CRPC and may represent a new therapeutic target for advanced prostate cancer.

Citrobacter rodentium uses a type III secretion system (T3SS) to induce colonic crypt hyperplasia in mice, thereby gaining an edge during its competition with the gut microbiota through an unknown mechanism. Here, we show that by triggering colonic crypt hyperplasia, the C. rodentium T3SS induced an excessive expansion of undifferentiated Ki67-positive epithelial cells, which increased oxygenation of the mucosal surface and drove an aerobic C. rodentium expansion in the colon. Treatment of mice with the γ-secretase inhibitor dibenzazepine to diminish Notch-driven colonic crypt hyperplasia curtailed the fitness advantage conferred by aerobic respiration during C. rodentium infection. We conclude that C. rodentium uses its T3SS to induce histopathological lesions that generate an intestinal microenvironment in which growth of the pathogen is fueled by aerobic respiration.

Objective: NGFR/p75NTR and NRADD/NRH proteins are closely related structurally and are encoded by genes that arose from a duplication event early in vertebrate evolution. The transmembrane domain (TMD) of NGFR is cleaved by γ-secretase but there is conflicting data around the susceptibility to γ-secretase cleavage of NRADD proteins. If NGFR and NRADD show differential susceptibility to γ-secretase, then they can be used to dissect the structural constraints determining substrate susceptibility. We sought to test this differential susceptibility. Results: We developed labelled, lumenally-truncated forms of zebrafish Ngfrb and Nradd and a chimeric protein in which the TMD of Nradd was replaced with the TMD of Ngfrb. We expressed these in zebrafish embryos to test their susceptibility to γ-secretase cleavage by monitoring their stability using western immunoblotting. Inhibition of γ-secretase activity using DAPT increased the stability of only the Ngfrb construct. Our results support that only NGFR is cleaved by γ-secretase. Either NGFR evolved γ-secretase-susceptibility since its creation by gene duplication, or NRADD evolved to be refractory to γ-secretase. Protein structure outside of the TMD of NGFR is likely required for susceptibility to γ-secretase. [ABSTRACT FROM AUTHOR]

BackgroundAmyloid-β (Aβ) 42 has been implicated as the initiating molecule in the pathogenesis of Alzheimer's disease (AD); thus, therapeutic strategies that target Aβ42 are of great interest. γ-Secretase modulators (GSMs) are small molecules that selectively decrease Aβ42. We have previously reported that many acidic steroids are GSMs with potencies ranging in the low to mid micromolar concentration with 5β-cholanic acid being the most potent steroid identified GSM with half maximal effective concentration (EC50) of 5.7 μM.ResultsWe find that the endogenous cholesterol metabolite, 3β-hydroxy-5-cholestenoic acid (CA), is a steroid GSM with enhanced potency (EC50 of 250 nM) relative to 5β-cholanic acid. CA i) is found in human plasma at ~100-300 nM concentrations ii) has the typical acidic GSM signature of decreasing Aβ42 and increasing Aβ38 levels iii) is active in in vitro γ-secretase assay iv) is made in the brain. To test if CA acts as an endogenous GSM, we used Cyp27a1 knockout (Cyp27a1-/-) and Cyp7b1 knockout (Cyp7b1-/-) mice to investigate if manipulation of cholesterol metabolism pathways relevant to CA formation would affect brain Aβ42 levels. Our data show that Cyp27a1-/- had increased brain Aβ42, whereas Cyp7b1-/- mice had decreased brain Aβ42 levels; however, peripheral dosing of up to 100 mg/kg CA did not affect brain Aβ levels. Structure-activity relationship (SAR) studies with multiple known and novel CA analogs studies failed to reveal CA analogs with increased potency.ConclusionThese data suggest that CA may act as an endogenous GSM within the brain. Although it is conceptually attractive to try and increase the levels of CA in the brain for prevention of AD, our data suggest that this will not be easily accomplished.

BACE-1 is the β-secretase responsible for the initial amyloidogenesis in Alzheimer's disease, catalyzing hydrolytic cleavage of substrate in a pH-sensitive manner. The catalytic mechanism of BACE-1 requires water-mediated proton transfer from aspartyl dyad to the substrate, as well as structural flexibility in the flap region. Thus, the coupling of protonation and conformational equilibria is essential to a full in silico characterization of BACE-1. In this work, we perform constant pH replica exchange molecular dynamics simulations on both apo BACE-1 and five BACE-1-inhibitor complexes to examine the effect of pH on dynamics and inhibitor binding properties of BACE-1. In our simulations, we find that solution pH controls the conformational flexibility of apo BACE-1, whereas bound inhibitors largely limit the motions of the holo enzyme at all levels of pH. The microscopic pKa values of titratable residues in BACE-1 including its aspartyl dyad are computed and compared between apo and inhibitor-bound states. Changes in protonation between the apo and holo forms suggest a thermodynamic linkage between binding of inhibitors and protons localized at the dyad. Utilizing our recently developed computational protocol applying the binding polynomial formalism to the constant pH molecular dynamics (CpHMD) framework, we are able to obtain the pH-dependent binding free energy profiles for various BACE-1-inhibitor complexes. Our results highlight the importance of correctly addressing the binding-induced protonation changes in protein-ligand systems where binding accompanies a net proton transfer. This work comprises the first application of our CpHMD-based free energy computational method to protein-ligand complexes and illustrates the value of CpHMD as an all-purpose tool for obtaining pH-dependent dynamics and binding free energies of biological systems.

The biological underpinnings linking stress to Alzheimer's disease (AD) risk are poorly understood. We investigated how corticotrophin releasing factor (CRF), a critical stress response mediator, influences amyloid-β (Aβ) production. In cells, CRF treatment increases Aβ production and triggers CRF receptor 1 (CRFR1) and γ-secretase internalization. Co-immunoprecipitation studies establish that γ-secretase associates with CRFR1; this is mediated by β-arrestin binding motifs. Additionally, CRFR1 and γ-secretase co-localize in lipid raft fractions, with increased γ-secretase accumulation upon CRF treatment. CRF treatment also increases γ-secretase activity in vitro, revealing a second, receptor-independent mechanism of action. CRF is the first endogenous neuropeptide that can be shown to directly modulate γ-secretase activity. Unexpectedly, CRFR1 antagonists also increased Aβ. These data collectively link CRF to increased Aβ through γ-secretase and provide mechanistic insight into how stress may increase AD risk. They also suggest that direct targeting of CRF might be necessary to effectively modulate this pathway for therapeutic benefit in AD, as CRFR1 antagonists increase Aβ and in some cases preferentially increase Aβ42 via complex effects on γ-secretase.

Free energy calculations are used to study how strongly potential drug molecules interact with their target receptors. The accuracy of these calculations depends on the accuracy of the molecular dynamics (MD) force field as well as proper sampling of the major conformations of each molecule. However, proper sampling of ligand conformations can be difficult when there are large barriers separating the major ligand conformations. An example of this is for ligands with an asymmetrically substituted phenyl ring, where the presence of protein loops hinders the proper sampling of the different ring conformations. These ring conformations become more difficult to sample when the size of the functional groups attached to the ring increases. The Adaptive Integration Method (AIM) has been developed, which adaptively changes the alchemical coupling parameter λ during the MD simulation so that conformations sampled at one λ can aid sampling at the other λ values. The Accelerated Adaptive Integration Method (AcclAIM) builds on AIM by lowering potential barriers for specific degrees of freedom at intermediate λ values. However, these methods may not work when there are very large barriers separating the major ligand conformations. In this work, we describe a modification to AIM that improves sampling of the different ring conformations, even when there is a very large barrier between them. This method combines AIM with conformational Monte Carlo sampling, giving improved convergence of ring populations and the resulting free energy. This method, called AIM/MC, is applied to study the relative binding free energy for a pair of ligands that bind to thrombin and a different pair of ligands that bind to aspartyl protease β-APP cleaving enzyme 1 (BACE1). These protein-ligand binding free energy calculations illustrate the improvements in conformational sampling and the convergence of the free energy compared to both AIM and AcclAIM.

UnlabelledChronic HIV infection results in a loss of HIV-specific CD8(+) T cell effector function, termed "exhaustion," which is mediated, in part, by the membrane coinhibitory receptor T cell immunoglobulin mucin domain-3 (Tim-3). Like many other receptors, a soluble form of this protein has been described in human blood plasma. However, soluble Tim-3 (sTim-3) is poorly characterized, and its role in HIV disease is unknown. Here, we show that Tim-3 is shed from the surface of responding CD8(+) T cells by the matrix metalloproteinase ADAM10, producing a soluble form of the coinhibitory receptor. Despite previous reports in the mouse model, no alternatively spliced, soluble form of Tim-3 was observed in humans. Shed sTim-3 was found in human plasma and was significantly elevated during early and chronic untreated HIV infection, but it was not found differentially modulated in highly active antiretroviral therapy (HAART)-treated HIV-infected subjects or in elite controllers compared to HIV-uninfected subjects. Plasma sTim-3 levels were positively correlated with HIV load and negatively correlated with CD4 counts. Thus, plasma sTim-3 shedding correlated with HIV disease progression. Despite these correlations, we found that shedding Tim-3 did not improve the function of CD8(+) T cells in terms of gamma interferon production or prevent their apoptosis through galectin-9. Further characterization studies of sTim-3 function are needed to understand the contribution of sTim-3 in HIV disease pathogenesis, with implications for novel therapeutic interventions.ImportanceDespite the overall success of HAART in slowing the progression to AIDS in HIV-infected subjects, chronic immune activation and T cell exhaustion contribute to the eventual deterioration of the immune system. Understanding these processes will aid in the development of interventions and therapeutics to be used in combination with HAART to slow or reverse this deterioration. Here, we show that a soluble form of T cell exhaustion associated coinhibitory molecule 3, sTim-3, is shed from the surface of T cells. Furthermore, sTim-3 is elevated in the plasma of treatment-naive subjects with acute or chronic HIV infection and is associated with markers of disease progression. This is the first study to characterize sTim-3 in human plasma, its source, and mechanism of production. While it is still unclear whether sTim-3 contributes to HIV pathogenesis, sTim-3 may represent a new correlate of HIV disease progression.

BackgroundAberrant Notch activation confers a proliferative advantage to many human tumors, including melanoma. This phase 2 trial assessed the antitumor activity of RO4929097, a gamma-secretase inhibitor of Notch signaling, with respect to the progression-free and overall survival of patients with advanced melanoma.MethodsChemotherapy-naive patients with metastatic melanoma of cutaneous or unknown origin were treated orally with RO4929097 at a dose of 20 mg daily 3 consecutive days per week. A 2-step accrual design was used with an interim analysis of the first 32 patients and with continuation of enrollment if 4 or more of the 32 patients responded.ResultsThirty-six patients from 23 institutions were enrolled; 32 patients were evaluable. RO4929097 was well tolerated, and most toxicities were grade 1 or 2. The most common toxicities were nausea (53%), fatigue (41%), and anemia (22%). There was 1 confirmed partial response lasting 7 months, and there were 8 patients with stable disease lasting at least through week 12, with 1 of these continuing for 31 months. The 6-month progression-free survival rate was 9% (95% confidence interval [CI], 2%-22%), and the 1-year overall survival rate was 50% (95% CI, 32%-66%). Peripheral blood T-cell assays showed no significant inhibition of the production of interleukin-2, a surrogate pharmacodynamic marker of Notch inhibition, and this suggested that the drug levels were insufficient to achieve Notch target inhibition.ConclusionsRO4929097 showed minimal clinical activity against metastatic melanoma in this phase 2 trial, possibly because of inadequate exposure to therapeutic drug levels. Although Notch inhibition remains a compelling target in melanoma, the results do not support further investigation of RO4929097 with this dose and schedule.