A Strategy for Allowing Earlier Diagnosis and Rigorous Evaluation of BACE1 Inhibitors in Preclinical Alzheimer's Disease.

Given continued failure of BACE1 inhibitor programs at symptomatic and prodromal stages of Alzheimer's disease (AD), clinical trials need to target the earlier preclinical stage. However, trial design is complex in this population with negative diagnosis of classical hippocampal amnesia on standard memory tests. Besides recent advances in brain imaging, electroencephalogram, and fluid-based biomarkers, new cognitive markers should be established for earlier diagnosis that can optimize recruitment to BACE1 inhibitor trials in presymptomatic AD. Notably, accelerated long-term forgetting (ALF) is emerging as a sensitive cognitive measure that can discriminate between asymptomatic individuals with high risks for developing AD and healthy controls. ALF is a form of declarative memory impairment characterized by increased forgetting rates over longer delays (days to months) despite normal storage within the standard delays of testing (20-60 min). Therefore, ALF may represent a harbinger of preclinical dementia and the impairment of systems memory consolidation, during which memory traces temporarily stored in the hippocampus become gradually integrated into cortical networks. This review provides an overview of the utility of ALF in a rational design of next-generation BACE1 inhibitor trials in preclinical AD. I explore potential mechanisms underlying ALF and relevant early-stage biomarkers useful for BACE1 inhibitor evaluation, including synaptic protein alterations, astrocytic dysregulation and neuron hyperactivity in the hippocampal-cortical network. Furthermore, given the physiological role of the isoform BACE2 as an AD-suppressor gene, I also discuss the possible association between the poor selectivity of BACE1 inhibitors and their side effects (e.g., cognitive worsening) in prior clinical trials.

In vitro cleavage of tumor necrosis factor α (TNFα) by Signal-Peptide-Peptidase-like 2b (SPPL2b) resembles mechanistic principles observed in the cellular context.

Members of the Signal Peptide-Peptidase (SPP) and Signal Peptide-Peptidase-like (SPPL) family are intramembrane aspartyl-proteases like their well-studied homologs, the presenilins, which comprise the catalytically active subunit within the γ-secretase complex. The lack of in vitro cleavage assays for SPPL proteases limited their biochemical characterization as well as substrate identification and validation. So far, SPPL proteases have been analyzed exclusively in intact cells or membranes, restricting mechanistic analysis to co-expression of enzyme and substrate variants colocalizing in the same subcellular compartments. We describe the details of developing an in vitro cleavage assay for SPPL2b and its model substrate TNFα and analyzed the influence of phospholipids, detergent supplements, and cholesterol on the SPPL2b in vitro activity. SPPL2b in vitro activity resembles mechanistic principles that have been observed in a cellular context, such as cleavage sites and consecutive turnover of the TNFα transmembrane domain. The novel in vitro cleavage assay is functional with separately isolated protease and substrate and amenable to a high throughput plate-based readout overcoming previous limitations and providing the basis for studying enzyme kinetics, catalytic activity, substrate recognition, and the characteristics of small molecule inhibitors. As a proof of concept, we present the first biochemical in vitro characterization of the SPPL2a and SPPL2b specific small molecule inhibitor SPL-707.

Design, synthesis, and biological evaluation of some 2-(3-oxo-5,6-diphenyl-1,2,4-triazin-2(3H)-yl)-N-phenylacetamide hybrids as MTDLs for Alzheimer's disease therapy.

Inspite of established symptomatic relief drug targets, a multi targeting approach is highly in demand to cure Alzheimer's disease (AD). Simultaneous inhibition of cholinesterase (ChE), ß secretase-1 (BACE-1) and Dyrk1A could be promising in complete cure of AD. A series of 18 diaryl triazine based molecular hybrids were successfully designed, synthesized, and tested for their hChE, hBACE-1, Dyrk1A and Aß aggregation inhibitory potentials. Compounds S-11 and S-12 were the representative molecules amongst the series with multi-targeted inhibitory effects. Compound S-12 showed hAChE inhibition (IC50 value = 0.486 ± 0.047 µM), BACE-1 inhibition (IC50 value = 0.542 ± 0.099 µM) along with good anti-Aß aggregation effects in thioflavin-T assay. Only compound S-02 of the series has shown Dyrk1A inhibition (IC50 value = 2.000 ± 0.360 µM). Compound S-12 has also demonstrated no neurotoxic liabilities against SH-SY5Y as compared to donepezil. The in vivo behavioral studies of the compound S-12 in the scopolamine- and Aß-induced animal models also demonstrated attanuation of learning and memory functions in rats models having AD-like characteristics. The ex vivo studies, on the rat hippocampal brain demonstrated reduction in certain biochemical markers of the AD brain with a significant increase in ACh level. The Western blot and Immunohistochemistry further revealed lower tau, APP and BACE-1 molecular levels. The drosophilla AD model also revealed improved eyephenotype after treatment with compound S-12. The molecular docking studies of the compounds suggested that compound S-12 was interacting with the ChE-PAS & CAS residues and catalytic dyad residues of the BACE-1 enzymes. The 100 ns molecular dynamics simulation studies of the ligand-protein complexed with hAChE and hBACE-1 also suggested stable ligand-protein confirmation throughout the simulation run.

Photoreaction products of extract from the fruiting bodies of Polyozellus multiplex.

Photochemical reactions are powerful tools for synthesizing organic molecules. The input of energy provided by light offers a means to produce strained and unique molecules that cannot be assembled using thermal protocols, allowing for the production of immense molecular complexity in a single chemical step. Furthermore, unlike thermal reactions, photochemical reactions do not require active reagents such as acids, bases, metals, or enzymes. Photochemical reactions play a central role in green chemistry. This article reports the isolation and structure determination of four new compounds (1-4) from the photoreaction products of the Polyozellus multiplex MeOH ext. The structures of the new compounds were elucidated using MS, IR, comprehensive NMR measurements and microED. The four compounds were formed by deacetylation of polyozellin, the main secondary metabolite of P. multiplex, and addition of singlet oxygen generated by sunlight. To develop drugs for treating Alzheimer's disease (AD) on the basis of the amyloid cascade hypothesis, the compounds (1-4) obtained by photoreaction were evaluated for BACE1 inhibitory activity. The hydrolysates (5 and 6) of polyozellin, the main secondary metabolites of P. multiplex, were also evaluated. The photoreaction products (3 and 4) and hydrolysates (5 and 6) of polyozellin showed BACE1 inhibitory activity (IC50: 2.2, 16.4, 23.3, and 5.3 µM, respectively).

Extracting binding energies and binding modes from biomolecular simulations of fragment binding to endothiapepsin.

Fragment-based drug discovery (FBDD) aims to discover a set of small binding fragments that may be subsequently linked together. Therefore, in-depth knowledge of the individual fragments' structural and energetic binding properties is essential. In addition to experimental techniques, the direct simulation of fragment binding by molecular dynamics (MD) simulations became popular to characterize fragment binding. However, former studies showed that long simulation times and high computational demands per fragment are needed, which limits applicability in FBDD. Here, we performed short, unbiased MD simulations of direct fragment binding to endothiapepsin, a well-characterized model system of pepsin-like aspartic proteases. To evaluate the strengths and limitations of short MD simulations for the structural and energetic characterization of fragment binding, we predicted the fragments' absolute free energies and binding poses based on the direct simulations of fragment binding and compared the predictions to experimental data. The predicted absolute free energies are in fair agreement with the experiment. Combining the MD data with binding mode predictions from molecular docking approaches helped to correctly identify the most promising fragments for further chemical optimization. Importantly, all computations and predictions were done within 5 days, suggesting that MD simulations may become a viable tool in FBDD projects.

Rational design of novel diaryl ether-linked benzimidazole derivatives as potent and selective BACE1 inhibitors.

Due to the large size and high flexibility of the catalytic active site of BACE1 enzyme, the development of nonpeptide inhibitors with optimal pharmacological properties is still highly demanding. In this work, we have discovered 2-aminobenzimidazole-containg ether scaffolds having potent and selective inhibitory potentials against BACE1 enzyme. We have synthesized novel 29 compounds and optimization of aryl linker region resulted in highly potent BACE1 inhibitory activities with EC50 values of 0.05-2.71 µM. The aryloxy-phenyl analogs 20j showed the EC50 value as low as 0.07 µM in the enzyme assay, whereas, the benzyloxyphenyl dervative 24b was comparatively less effective in the enzyme assay. But interestingly the latter was more effective in the cell assay (EC50 value 1.2 µM). While comparing synthesized derivatives in the cell assay using PC12-APPSW cell, compound 27f appeared as the most potent BACE1 inhibitor having EC50 value 0.7 µM. This scaffold also showed high selectivity over BACE2 enzyme and cathepsin D. Furthermore, the research findings were bolstered through the incorporation of molecular docking, molecular dynamics, and DFT studies. We firmly believe that these discoveries will pave the way for the development of a novel class of small-molecule selective BACE1 inhibitors.

Exploring Aspartic Protease Inhibitor Binding to Design Selective Antimalarials.

Selectivity is a major issue in the development of drugs targeting pathogen aspartic proteases. Here, we explore the selectivity-determining factors by studying specifically designed malaria aspartic protease (plasmepsin) open-flap inhibitors. Metadynamics simulations are used to uncover the complex binding/unbinding pathways of these inhibitors and describe the critical transition states in atomistic resolution. The simulation results are compared with experimentally determined enzymatic activities. Our findings demonstrate that plasmepsin inhibitor selectivity can be achieved by targeting the flap loop with hydrophobic substituents that enable ligand binding under the flap loop, as such a behavior is not observed for several other aspartic proteases. The ability to estimate the selectivity of compounds before they are synthesized is of considerable importance in drug design; therefore, we expect that our approach will be useful in selective inhibitor designs against not only aspartic proteases but also other enzyme classes.

Finding New Ways How to Control BACE1.

Recently, all applications of BACE1 inhibitors failed as therapeutical targets for Alzheimer´s disease (AD) due to severe side effects. Therefore, alternative ways for treatment development are a hot research topic. The present analysis investigates BACE1 protein-protein interaction networks and attempts to solve the absence of complete knowledge about pathways involving BACE1. A bioinformatics analysis matched the functions of the non-substrate interaction network with Voltage-gated potassium channels, which also appear as top priority protein nodes. Targeting BACE1 interactions with PS1 and GGA-s, blocking of BACE1 access to APP by BRI3 and RTN-s, activation of Wnt signaling and upregulation of ß-catenin, and brain delivery of the extracellular domain of p75NTR, are the main alternatives to the use of BACE 1 inhibitors highlighted by the analysis. The pathway enrichment analysis also emphasized substrates and substrate candidates with essential biological functions, which cleavage must remain controlled. They include ephrin receptors, ROBO1, ROBO2, CNTN-s, CASPR-s, CD147, CypB, TTR, APLP1/APLP2, NRXN-s, and PTPR-s. The analysis of the interaction subnetwork of BACE1 functionally related to inflammation identified a connection to three cardiomyopathies, which supports the hypothesis of the common molecular mechanisms with AD. A lot of potential shows the regulation of BACE1 activity through post-translational modifications. The interaction network of BACE1 and its phosphorylation enzyme CSNK1D functionally match the Circadian clock, p53, and Hedgehog signaling pathways. The regulation of BACE1 glycosylation could be achieved through N-acetylglucosamine transferases, α-(1→6)-fucosyltransferase, ß-galactoside α-(2→6)-sialyltransferases, galactosyltransferases, and mannosidases suggested by the interaction network analysis of BACE1-MGAT3. The present analysis proposes possibilities for the alternative control of AD pathology.

In silico screening of potential ß-secretase (BACE1) inhibitors from VIETHERB database.

For the purpose of discovering potential inhibitors of ß-amyloid (BACE1), which is a crucial element in Alzheimer's disease (AD) pathogenesis, an in silico study of naturally occurring compounds was performed using precise computational approaches. Autodock4 package was preliminary used to predict the binding affinities to BACE1 of more than four thousand compounds presented in the Vietnamese plants (VIETHERB) database. Based on docking results, twenty top-lead compounds having the largest docking energy to BACE1 were rigorously examined using steered molecular dynamics (SMD) simulations. Interestingly, SMD results found that the binding affinity values of three compounds, including myricetin 3-O-(3''-galloylrhamnopyranoside), quercetin 3-O-neohesperidoside, and hydroxysafflor yellow A, are remarkably higher than that of the well-known BACE1 inhibitor, 23I, and these compounds can thus be considered the promising candidates for inhibitors of BACE1.

Synthesis, radiolabeling, and evaluation of a potent ß-site APP cleaving enzyme (BACE1) inhibitor for PET imaging of BACE1 in vivo.

The ß-site APP-cleaving enzyme 1 (BACE1) plays important roles in the proteolytic processing of amyloid precursor protein, and can be regarded as an important target for the diagnosis and treatment of AD. This study aimed to report the synthesis and evaluation of an 18F-labeled 2-amino-3,4-dihydroquinazoline analog as a potential BACE1 radioligand. A fluoropropyl side chain was introduced to the phenyl of this 3,4-dihydroquinazoline scaffold to generate the radioligand. Our preliminary data indicated that although the 2-amino-3,4-dihydroquinazoline scaffold possessed favorable in-vitro properties as a PET ligand, its poor brain uptake hindered the in-vivo imaging of BACE1. Further investigation would be required to optimize the scaffold for the development of a blood-brain-barrier-permeable BACE1-targeted PET ligand.

Tangeretin Inhibits BACE1 Activity and Attenuates Cognitive Impairments in AD Model Mice.

Tangeretin (TAN) exhibits many bioactivities, including neuroprotective effects. However, the efficacy of TAN in Alzheimer's disease (AD) has not been sufficiently investigated. In the present study, we integrated behavioral tests, pathology assessment, and biochemical analyses to elucidate the antidementia activity of TAN in APPswe/PSEN1dE9 transgenic (Tg) mice. At supplementation levels of 100 mg/kg body weight per day, TAN significantly attenuated the cognitive impairment of Tg mice in behavioral tests. These effects were associated with less synaptic impairments and fewer ß-amyloid accumulations after TAN administration. Furthermore, our study revealed that TAN possessed powerful inhibitory activity against ß-secretase both in vitro and in vivo, which played a crucial role in the process of Aß generation. These findings indicate that TAN is a potential drug for preventing AD pathology. The key mechanism underlying the antidementia effect of TAN may include its inhibitory activity against ß-secretase.

The possible effect of microRNA-155 (miR-155) and BACE1 inhibitors in the memory of patients with down syndrome and Alzheimer's disease: Design, synthesis, virtual screening, molecular modeling and biological evaluations.

MiR-155 plays main roles in several physiological and pathological mechanisms, such as Down syndrome (DS), immunity and inflammation and potential anti-AD therapeutic target. The miR-155 is one of the overexpressed miRNAs in DS patients that contribute directly and indirectly to the onset or progression of the DS. Since the miR-155 can simultaneously reduce the translation of several genes at post-transcriptional levels, targeting the miR-155 might set the stage for the treatment of DS. One of the rational strategies in providing therapeutic interventions in this respect is to design and develop novel small molecules inhibiting the miR-155 function or biogenesis or maturation. In the present study, we aim to introduce small molecule compounds with the potential to inhibit the generation of the selectively miR-155 processing by employing computational drug design approaches, as well as in vitro studies. We designed and synthesized a novel series of imidazo[1,2-a]pyridines derivatives as new nonpeptic candidates for the treatment of DS with AD. The designed compounds were investigated for their BACE1 and miR-155 binder inhibitory potential in vitro and in cell. In addition, we present a systematic computational approach that includes 3 D modeling, docking-based virtual screening, and molecular dynamics simulation to identify Small - molecule inhibitors of pre-miR-155 maturation. To confirm the inhibitory potential of compound 8k on miR-155 maturation, qRT- PCR was performed. All our results confirm that compound 8k, in addition to being a good inhibitor of BACE1, can also be a good inhibitor of miR-155.Communicated by Ramaswamy H. Sarma.

A short peptide from sAPPα binding to BACE1-APP action site rescues Alzheimer-like pathology.

Amyloid ß-peptide (Aß) is the driven force of Alzheimer's disease (AD), and reducing Aß production could be a potential therapeutic strategy for AD. sAPPα appears to have the ability to specifically inhibit ß-cleavage of APP without inhibiting BACE1 completely, direct administration of sAPPα may not be clinically applicable due to the low permeability of blood-brain barrier (BBB). In this study, we investigated the neuroprotective effects of a short peptide generated from sAPPα, which could specifically bind to BACE1 at the BACE1-APP action site. We found that this peptide significantly reduced Aß production both in vivo and in vitro, thus further attenuated Aß deposition, Tau hyperphosphorylation, neuroinflammation et al. and rescued behavioral deficits. Therefore, this short peptide may hold promise for the treatment of AD due to its neuroprotective effects, low molecular weight to cross BBB, and less safety concerns. The anti-neurodegenerative capacity of sAPPα may not result solely from direct inhibition of BACE1.

Modulating physicochemical properties of tetrahydropyridine-2-amine BACE1 inhibitors with electron-withdrawing groups: A systematic study.

A common challenge for medicinal chemists is to reduce the pKa of strongly basic groups' conjugate acids into a range that preserves the desired effects, usually potency and/or solubility, but avoids undesired effects like high volume of distribution (Vd), limited membrane permeation, and off-target binding to, notably, the hERG channel and monoamine receptors. We faced this challenge with a 3,4,5,6-tetrahydropyridine-2-amine scaffold harboring an amidine, a key structural component of potential inhibitors of BACE1, the rate-limiting enzyme in the production of Aß species that make up amyloid plaques in Alzheimer's disease. In our endeavor to balance potency with desirable properties to achieve brain penetration, we introduced a diverse set of groups in beta position of the amidine that modulate logD, PSA and pKa. Given the synthetic challenge to prepare these highly functionalized warheads, we first developed a design flow including predicted physicochemical parameters which allowed us to select only the most promising candidates for synthesis. For this we evaluated a set of commercial packages to predict physicochemical properties, which can guide medicinal chemists in their endeavors to modulate pKa values of amidine and amine bases.

Reconstruction of the binding pathway of an anti-HIV drug, Indinavir, in complex with the HTLV-1 protease using unaggregated unbiased molecular dynamics simulation.

Retroviruses are a growing concern for the health of human beings, and one of the dangerous members of this family is the Human T-cell Leukemia Virus 1 (HTLV-1) virus. It has affected more than 20 million people so far, and since there are no registered treatments against it yet, urgent treatment solutions are needed. One of the most promising drug targets to fight this virus is the protease enzyme of the virus's protein machinery. In this study, by utilizing a computational method called Unaggregated Unbiased Molecular Dynamics (UUMD), we reconstructed the binding pathway of a HTLV-1 protease inhibitor, Indinavir, to find the details of the binding pathway, the influential residues, and also the stable states of the binding pathway. We achieved the native conformation of the inhibitor in 6 rounds, 360 replicas by performing over 4 micro-seconds of UMD simulations. We found 3 Intermediate states between the solvated state and the native conformation state in the binding pathway. We also discovered that aromatic residues such as Trp98 and Trp98', catalytic residues Asp32 and Asp32', and the flap region's residues have the most influential roles in the binding pathway and also have the most contribution to the total interaction energies. We believe that the details found in this study would be a great guide for developing new treatment solutions against the HTLV-1 virus by inhibiting the HTLV-1 protease.

Design and discovery of C2-fluoroalkyl iminothiazine dioxides as BACE inhibitors.

This paper describes the structure-activity-relationships of novel fluoroalkyl substituents at the C2 position of iminothiazine dioxide beta secretase inhibitors. Key discoveries include reduced amidine basicity and its effect on Pgp, cell potency, and efficacy in various preclinical in vivo efficacy animal models. Findings from these structure-activity-relationships are discussed.

Inhibition of BACE1 and amyloid ß aggregation by polyketide from Streptomyces sp.

Alzheimer's disease (AD) causes cognitive impairment in the elderly and is a severe problem worldwide. One of the major reasons for the pathogenesis of AD is thought to be due to the accumulation of amyloid beta (Aß) peptides that result in neuronal cell death in the brain. In this study, bioassay-guided fractionation was performed to develop seed compounds for anti-AD drugs that can act as dual inhibitors of BACE1 and Aß aggregation from secondary metabolites produced by Streptomyces sp. To improve the solubility, the crude extracts were methylated with trimethylsilyl (TMS) diazomethane and then purified to yield polyketides 1-5, including the new compound 1. We synthesized the compounds 6 and 7 (original compounds 2 and 3, respectively), and their activities were evaluated. KS-619-1, the demethylated form of 4 and 5, was isolated and evaluated for its inhibitory activity. The IC50 values for BACE1 and Aß aggregation were found to be 0.48 and 1.1 µM, respectively, indicating that KS-619-1 could be a lead compound for the development of therapeutic agents for AD.

Macrocyclic BACE1 inhibitors with hydrophobic cross-linked structures: Optimization of ring size and ring structure.

Based on the X-ray crystallography of recombinant BACE1 and a hydroxyethylamine-type peptidic inhibitor, we introduced a cross-linked structure between the P1 and P3 side chains of the inhibitor to enhance its inhibitory activity. The P1 and P3 fragments bearing terminal alkenes were synthesized, and a ring-closing metathesis of these alkenes was used to construct the cross-linked structure. Evaluation of ring size using P1 and P3 fragments with various side chain lengths revealed that 13-membered rings were optimal, although their activity was reduced compared to that of the parent compound. Furthermore, the optimal ring structure was found to be a macrocycle with a dimethyl branched substituent at the P3 ß-position, which was approximately 100-fold more active than the non-substituted macrocycle. In addition, the introduction of a 4-carboxymethylphenyl group at the P1' position further improved the activity.

Potential of Ramalin and Its Derivatives for the Treatment of Alzheimer's Disease.

The pathogenesis of Alzheimer's disease (AD) is still unclear, and presently there is no cure for the disease that can be used for its treatment or to stop its progression. Here, we investigated the therapeutic potential of ramalin (isolated from the Antarctic lichen, Ramalina terebrata), which exhibits various physiological activities, in AD. Specifically, derivatives were synthesized based on the structure of ramalin, which has a strong antioxidant effect, BACE-1 inhibition activity, and anti-inflammatory effects. Therefore, ramalin and its derivatives exhibit activity against multiple targets associated with AD and can serve as potential therapeutic agents for the disease.

Discovery of Umibecestat (CNP520): A Potent, Selective, and Efficacious ß-Secretase (BACE1) Inhibitor for the Prevention of Alzheimer's Disease.

After identification of lead compound 6, 5-amino-1,4-oxazine BACE1 inhibitors were optimized in order to improve potency, brain penetration, and metabolic stability. Insertion of a methyl and a trifluoromethyl group at the 6-position of the 5-amino-1,4-oxazine led to 8 (NB-360), an inhibitor with a pKa of 7.1, a very low P-glycoprotein efflux ratio, and excellent pharmacological profile, enabling high central nervous system penetration and exposure. Fur color changes observed with NB-360 in efficacy studies in preclinical animal models triggered further optimization of the series. Herein, we describe the steps leading to the discovery of 3-chloro-5-trifluoromethyl-pyridine-2-carboxylic acid [6-((3R,6R)-5-amino-3,6-dimethyl-6-trifluoromethyl-3,6-dihydro-2H-[1,4]oxazin-3-yl)-5-fluoro-pyridin-2-yl]amide 15 (CNP520, umibecestat), an inhibitor with superior BACE1/BACE2 selectivity and pharmacokinetics. CNP520 reduced significantly Aß levels in mice and rats in acute and chronic treatment regimens without any side effects and thus qualified for Alzheimer's disease prevention studies in the clinic.