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.

BACE1 Inhibition Utilizing Organic Compounds Holds Promise as a Potential Treatment for Alzheimer's and Parkinson's Diseases.

Background: Neurological disorders like Alzheimer's disease (AD) and Parkinson's disease (PD) manifest through gradually deteriorating cognitive functions. An encouraging strategy for addressing these disorders involves the inhibition of precursor-cleaving enzyme 1 (BACE1). Objectives: In the current research, a virtual screening technique was employed to identify potential BACE1 inhibitors among selected herbal isolates. Methods: This study evaluated 79 flavonoids, anthraquinones (AQs), and cinnamic acid derivatives for their potential blood-brain barrier (BBB) permeability. Using the AutoDock 4.0 tool, molecular docking analysis was conducted to determine the binding affinity of BBB permeable compounds to the BACE1 active site. Molecular dynamics (MD) simulations were performed to assess the stability of the docked poses of the most potent inhibitors. The interactions between the most effective plant-based inhibitors and the residues within the BACE1 catalytic site were examined before and after MD simulations. Results: Ponciretin, danthron, chrysophanol, and N-p-coumaroyltyramine were among the highest-ranking BACE1 inhibitors, with inhibition constant values calculated in the nanomolar range. Furthermore, during 10 ns simulations, the docked poses of these ligands were observed to be stable. Conclusion: The findings propose that ponciretin, danthron, chrysophanol, and N-p-coumaroyltyramine might serve as potential choices for the treatment of AD and PD, laying the groundwork for the creation of innovative BACE1 inhibitors.

Evaluating a Retro-Inverso Type Inhibitor of HTLV-1 Protease as a Competitive Inhibitor.

The inhibition mode of a retro-inverso (RI) inhibitor containing a hydroxyethylamine dipeptide isostere against the human T-cell leukemia virus type-1 (HTLV-1) protease was examined. Enzymatic evaluation of the RI-modified inhibitor containing a D-allo-Ile residue revealed that HTLV-1 was competitively inhibited. IC50 values of the RI-modified inhibitor and pepstatin A, a standard inhibitor of aspartic proteases, were nearly equivalent.

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.

Structure and function of SPP/SPPL proteases: insights from biochemical evidence and predictive modeling.

More than 20 years ago, signal peptide peptidase (SPP) and its homologues, the signal peptide peptidase-like (SPPL) proteases have been identified based on their sequence similarity to presenilins, a related family of intramembrane aspartyl proteases. Other than those for the presenilins, no high-resolution structures for the SPP/SPPL proteases are available. Despite this limitation, over the years bioinformatical and biochemical data have accumulated, which altogether have provided a picture of the overall structure and topology of these proteases, their localization in the cell, the process of substrate recognition, their cleavage mechanism, and their function. Recently, the artificial intelligence-based structure prediction tool AlphaFold has added high-confidence models of the expected fold of SPP/SPPL proteases. In this review, we summarize known structural aspects of the SPP/SPPL family as well as their substrates. Of particular interest are the emerging substrate recognition and catalytic mechanisms that might lead to the prediction and identification of more potential substrates and deeper insight into physiological and pathophysiological roles of proteolysis.

Exploring the Binding Pathway of Novel Nonpeptidomimetic Plasmepsin V Inhibitors.

Predicting the interaction modes and binding affinities of virtual compound libraries is of great interest in drug development. It reduces the cost and time of lead compound identification and selection. Here we apply path-based metadynamics simulations to characterize the binding of potential inhibitors to the Plasmodium falciparum aspartic protease plasmepsin V (plm V), a validated antimalarial drug target that has a highly mobile binding site. The potential plm V binders were identified in a high-throughput virtual screening (HTVS) campaign and were experimentally verified in a fluorescence resonance energy transfer (FRET) assay. Our simulations allowed us to estimate compound binding energies and revealed relevant states along binding/unbinding pathways in atomistic resolution. We believe that the method described allows the prioritization of compounds for synthesis and enables rational structure-based drug design for targets that undergo considerable conformational changes upon inhibitor binding.

Pepstatin-Based Probes for Photoaffinity Labeling of Aspartic Proteases and Application to Target Identification.

Aspartic proteases are a small class of proteases implicated in a wide variety of human diseases. Covalent chemical probes for photoaffinity labeling (PAL) of these proteases are underdeveloped. We here report a full on-resin synthesis of clickable PAL probes based on the natural product inhibitor pepstatin incorporating a minimal diazirine reactive group. The position of this group in the inhibitor determines the labeling efficiency. The most effective probes sensitively detect cathepsin D, a biomarker for breast cancer, in cell lysates. Moreover, through chemical proteomics experiments and deep learning algorithms, we identified sequestosome-1, an important player in autophagy, as a direct interaction partner and substrate of cathepsin D.

Synthesis of beta-site amyloid precursor protein-cleaving enzyme 1 inhibitors BI 1147560 and BI 1181181 labeled with carbon-14 and deuterium.

The generation of amyloid beta peptides that aggregate in the brain is believed to play a major role in Alzheimer's disease. In theory, the inhibition of beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1), which catalyzes the initial rate-limiting step in amyloid beta production, may slow or stop Alzheimer's disease. Herein, we report the preparation of two potent BACE1 inhibitors, BI 1147560 (1) and BI 1181181 (2), labeled with carbon-14 and with deuterium. The use of advanced key chiral intermediates like 3 and 5 shortened the carbon-14 syntheses of these two compounds to five and six steps, respectively, and helped in preparing them with very high chemical purity and enantiomeric excess without deviating from the process chemistry route. For the deuterium synthesis, oxetan-3-ylmethanamine [2 H6 ]-7 and 2-fluoro-2-methylpropan-1-amine [2 H6 ]-9 were prepared then used with the chiral intermediate 5 to furnish deuterium labeled 1 and 2, respectively.

Repurposing antiviral drugs against HTLV-1 protease by molecular docking and molecular dynamics simulation.

Human T-cell leukemia virus type I (HTLV-1) belongs to the delta retrovirus family and the etiological agent of adult T-cell leukemia (ATL(. While the current HTLV-1 therapy, relies on using Zidovudine plus IFN-γ, there is no FDA approved drugs against it. In silico drug repurposing is a fast and accurate way for screening US-FDA approved drugs to find a therapeutic option for the HTLV-1 infection. So that, this research aims to analyze a dataset of approved antiviral drugs as a potential prospect for an anti-viral drug against HTLV-1 infection. Molecular docking simulation was performed to identify interactions of the antiviral drugs with the key residues in the HTLV-1 protease binding site. Then, molecular dynamics simulation was also performed for the potential protein-ligand complexes to confirm the stable behavior of the ligands inside the binding pocket. The best docking scores with the target was found to be Simeprevir, Atazanavir, and Saquinavir compounds which indicate that these drugs can firmly bind to the HTLV-1 protease. The MD simulation confirmed the stability of Simeprevir-protease, Atazanavir-Protease, and Saquinavir-Protease interactions. Clearly, these compounds should be further evaluated in experimental assays and clinical trials to confirm their actual activity against HTLV-1 infection.Communicated by Ramaswamy H. Sarma.

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

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

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.

In Silico and In Vitro Antimalarial Screening and Validation Targeting Plasmodium falciparum Plasmepsin V.

Malaria chemotherapy is greatly threatened by the recent emergence and spread of resistance in the Plasmodium falciparum parasite against artemisinins and their partner drugs. Therefore, it is an urgent priority to develop new antimalarials. Plasmepsin V (PMV) is regarded as a superior drug target for its essential role in protein export. In this study, we performed virtual screening based on homology modeling of PMV structure, molecular docking and pharmacophore model analysis against a library with 1,535,478 compounds, which yielded 233 hits. Their antimalarial activities were assessed amongst four non-peptidomimetic compounds that demonstrated the promising inhibition of parasite growth, with mean IC50 values of 6.67 µM, 5.10 µM, 12.55 µM and 8.31 µM. No significant affection to the viability of L929 cells was detected in these candidates. These four compounds displayed strong binding activities with the PfPMV model through H-bond, hydrophobic, halogen bond or π-π interactions in molecular docking, with binding scores under -9.0 kcal/mol. The experimental validation of molecule-protein interaction identified the binding of four compounds with multiple plasmepsins; however, only compound 47 showed interaction with plasmepsin V, which exhibited the potential to be developed as an active PfPMV inhibitor.

Basis for drug selectivity of plasmepsin IX and X inhibition in Plasmodium falciparum and vivax.

Plasmepsins IX (PMIX) and X (PMX) are essential aspartyl proteases for Plasmodium spp. egress, invasion, and development. WM4 and WM382 inhibit PMIX and PMX in Plasmodium falciparum and P. vivax. WM4 inhibits PMX, while WM382 is a dual inhibitor of PMIX and PMX. To understand their function, we identified protein substrates. Enzyme kinetic and structural analyses identified interactions responsible for drug specificity. PMIX and PMX have similar substrate specificity; however, there are distinct differences for peptide and protein substrates. Differences in WM4 and WM382 binding for PMIX and PMX map to variations in the S' region and engagement of the active site S3 pocket. Structures of PMX reveal interactions and mechanistic detail of drug binding important for development of clinical candidates against these targets.

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

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

Flap Dynamics in Pepsin-Like Aspartic Proteases: A Computational Perspective Using Plasmepsin-II and BACE-1 as Model Systems.

The flexibility of ß hairpin structure known as the flap plays a key role in catalytic activity and substrate intake in pepsin-like aspartic proteases. Most of these enzymes share structural and sequential similarity. In this study, we have used apo Plm-II and BACE-1 as model systems. In the apo form of the proteases, a conserved tyrosine residue in the flap region remains in a dynamic equilibrium between the normal and flipped states through rotation of the χ1 and χ2 angles. Independent MD simulations of Plm-II and BACE-1 remained stuck either in the normal or flipped state. Metadynamics simulations using side-chain torsion angles (χ1 and χ2 of tyrosine) as collective variables sampled the transition between the normal and flipped states. Qualitatively, the two states were predicted to be equally populated. The normal and flipped states were stabilized by H-bond interactions to a tryptophan residue and to the catalytic aspartate, respectively. Further, mutation of tyrosine to an amino-acid with smaller side-chain, such as alanine, reduced the flexibility of the flap and resulted in a flap collapse (flap loses flexibility and remains stuck in a particular state). This is in accordance with previous experimental studies, which showed that mutation to alanine resulted in loss of activity in pepsin-like aspartic proteases. Our results suggest that the ring flipping associated with the tyrosine side-chain is the key order parameter that governs flap dynamics and opening of the binding pocket in most pepsin-like aspartic proteases.

Structures of plasmepsin X from Plasmodium falciparum reveal a novel inactivation mechanism of the zymogen and molecular basis for binding of inhibitors in mature enzyme.

Plasmodium falciparum plasmepsin X (PfPMX), involved in the invasion and egress of this deadliest malarial parasite, is essential for its survival and hence considered as an important drug target. We report the first crystal structure of PfPMX zymogen containing a novel fold of its prosegment. A unique twisted loop from the prosegment and arginine 244 from the mature enzyme is involved in zymogen inactivation; such mechanism, not previously reported, might be common for apicomplexan proteases similar to PfPMX. The maturation of PfPMX zymogen occurs through cleavage of its prosegment at multiple sites. Our data provide thorough insights into the mode of binding of a substrate and a potent inhibitor 49c to PfPMX. We present molecular details of inactivation, maturation, and inhibition of PfPMX that should aid in the development of potent inhibitors against pepsin-like aspartic proteases from apicomplexan parasites.

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

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

Signal peptide peptidase-like 2 proteases: Regulatory switches or proteasome of the membrane?

Signal peptide peptidase-like 2 (SPPL) proteases constitute a subfamily of SPP/SPPL intramembrane proteases which are homologues of the presenilins, the catalytic core of the γ-secretase complex. The three SPPL2 proteases SPPL2a, SPPL2b and SPPL2c proteolyse single-span, type II-oriented transmembrane proteins and/or tail-anchored proteins within their hydrophobic transmembrane segments. We review recent progress in defining substrate spectra and in vivo functions of these proteases. Characterisation of the respective knockout mice has implicated SPPL2 proteases in immune cell differentiation and function, prevention of atherosclerotic plaque development and spermatogenesis. Mechanisms how substrates are selected by these enzymes are still incompletely understood. We will discuss current views on how selective SPPL2-mediated cleavage is or whether these proteases may exhibit a generalised role in the turnover of membrane proteins. This has been suggested previously for the mechanistically related γ-secretase for which the term "proteasome of the membrane" has been coined based on its broad substrate spectrum. With regard to individual substrates, potential signalling functions of the resulting cytosolic cleavage fragments remain a controversial aspect. However, it has been clearly shown that SPPL2 proteases can influence cellular signalling and membrane trafficking by controlling levels of their membrane-bound substrate proteins which highlights these enzymes as regulatory switches. Based on this, regulatory mechanisms controlling activity of SPPL2 proteases would need to be postulated, which are just beginning to emerge. These different questions, which are relevant for other families of intramembrane proteases in a similar way, will be critically discussed based on the current state of knowledge.

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

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

Alzheimer's Disease and ß-secretase Inhibition: An Update with a Focus on Computer-aided Inhibitor Design.

INTRODUCTION: Alzheimer's disease (AD) is an intensifying neurodegenerative illness due to its irreversible nature. Identification of ß-site Amyloid Precursor Protein (APP) cleaving en-zyme1 (BACE1) has been a significant medicinal focus towards AD treatment, and this has opened ground for several investigations. Despite the numerous works in this direction, no BACE1 inhibitor has made it to the final approval stage as an anti-AD drug. METHODS: We provide an introductory background of the subject with a general overview of the pathogenesis of AD. The review features BACE1 inhibitor design and development with a focus on some clinical trials and discontinued drugs. Using the topical keywords BACE1, inhibitor design, and computational/theoretical study in the Web of Science and Scopus database, we retrieved over 49 relevant articles. The search years are from 2010 and 2020, with analysis conducted from May 2020 to March 2021. RESULTS AND DISCUSSION: Researchers have employed computational methodologies to unravel po-tential BACE1 inhibitors with a significant outcome. The most used computer-aided approach in BACE1 inhibitor design and binding/interaction studies are pharmacophore development, quantita-tive structure-activity relationship (QSAR), virtual screening, docking, and molecular dynamics (MD) simulations. These methods, plus more advanced ones including quantum mechan-ics/molecular mechanics (QM/MM) and QM, have proven substantial in the computational frame-work for BACE1 inhibitor design. Computational chemists have embraced the incorporation of in vitro assay to provide insight into the inhibition performance of identified molecules with potential inhibition towards BACE1. Significant IC50 values up to 50 nM, better than clinical trial com-pounds, are available in the literature. CONCLUSION: The continuous failure of potent BACE1 inhibitors at clinical trials is attracting many queries prompting researchers to investigate newer concepts necessary for effective inhibitor de-sign. The considered properties for efficient BACE1 inhibitor design seem enormous and require thorough scrutiny. Lately, researchers noticed that besides appreciable binding affinity and Blood-Brain Barrier (BBB) permeation, BACE1 inhibitor must show low or no affinity for permeability-glycoprotein. Computational modeling methods have profound applications in drug discovery strat-egies. With the volume of recent in silico studies on BACE1 inhibition, the prospect of identifying potent molecules that would reach the approved level is feasible. Investigators should try pushing many of the identified BACE1 compounds with significant anti-AD properties to preclinical and clinical trial stages. We also advise computational research on allosteric inhibitor design, exosite modeling, and multisite inhibition of BACE1. These alternatives might be a solution to BACE1 drug discovery in AD therapy.