Searching for potential acetylcholinesterase inhibitors: a combined approach of multi-step similarity search, machine learning and molecular dynamics simulations.

Targeting acetylcholinesterase is one of the most important strategies for developing therapeutics against Alzheimer's disease. In this work, we have employed a new approach that combines machine learning models, a multi-step similarity search of the PubChem library and molecular dynamics simulations to investigate potential inhibitors for acetylcholinesterase. Our search strategy has been shown to significantly enrich the set of compounds with strong predicted binding affinity to acetylcholinesterase. Both machine learning prediction and binding free energy calculation, based on linear interaction energy, suggest that the compound CID54414454 would bind strongly to acetylcholinesterase and hence is a promising inhibitor.

Design, synthesis and biological evaluation of novel quinazoline derivatives as immune checkpoint inhibitors.

In this work, we report 14 novel quinazoline derivatives as immune checkpoint inhibitors, IDO1 and PD-L1. The antitumor screening of synthesized compounds on ovarian cancer cells indicated that compound V-d and V-l showed the most activity with IC50 values of about 5 µM. Intriguingly, compound V-d emerges as a stand out, triggering cell death through caspase-dependent and caspase-independent manners. More importantly, V-d presents its ability to hinder tumor sphere formation and re-sensitized cisplatin-resistant A2780 cells to cisplatin treatment. These findings suggest that compound V-d emerges as a promising lead candidate for the future development of immuno anticancer agents.

Silver nanoparticles alter the dimerization of Aß42 studied by REMD simulations.

The aggregation of amyloid beta (Aß) peptides is associated with the development of Alzheimer's disease (AD). However, there has been a growing belief that the oligomerization of Aß species in different environments has a neurotoxic effect on the patient's brain, causing damage. It is necessary to comprehend the compositions of Aß oligomers in order to develop medications that may effectively inhibit these neurotoxic forms that affect the nervous system of AD patients. Thus, dissociation or inhibition of Aß aggregation may be able to prevent AD. To date, the search for traditional agents and biomolecules has largely been unsuccessful. In this context, nanoparticles have emerged as potential candidates to directly inhibit the formation of Aß oligomers. The oligomerization of the dimeric Aß peptides with or without the influence of a silver nanoparticle was thus investigated using temperature replica-exchange molecular dynamics (REMD) simulations. The physical insights into the dimeric Aß oligomerization were clarified by analyzing intermolecular contact maps, the free energy landscape of the dimeric oligomer, secondary structure terms, etc. The difference in obtained metrics between Aß with or without a silver nanoparticle provides a picture of the influence of silver nanoparticles on the oligomerization process. The underlying mechanisms that are involved in altering Aß oligomerization will be discussed. The obtained results may play an important role in searching for Aß inhibitor pathways.

Triptan use in elderly over 65 years and the risk of hospitalization for serious vascular events.

BACKGROUND: Several studies have focused on the use of triptan and the risk of acute vascular events but the existence of such association is still debated and has never been quantified in patients over 65 years. To assess whether triptan use among older is associated with an increased risk of hospitalization for acute vascular events. METHODS: A propensity score-matched cohort study was designed using the French national health insurance database linked to hospital stays. Patients aged ≥ 65 years, newly treated by triptans between 2011 and 2014, were included… The primary event was hospitalization for an acute ischemic vascular event within de 90 days following triptan initiation. Association with triptan exposure was investigated through cox regression model, considering exposure at inclusion, and with exposure as a time-varying variable A case-crossover (CCO) and a self-controlled case series (SCCS) analyses were also conducted to address potential residual confounding. RESULTS: The cohort included 24, 774 triptan users and 99 096 propensity matched controls (mean (SD) age: 71 years (5.9), 74% of women). Within 90 days after cohort entry, 163 events were observed in the triptan group, and 523 in the control group (0.66% vs. 0.53%, adjusted hazard ratio (aHR) exposed/not exposed 1.25 95%CI [1.05-1.49]; aHR time-varying 8.74 [5.21-14.66]). The association was significant (CCO) for all events (adjusted odds ratio (aOR1.63 [1.22-2.19]) with a more consistent association with cerebral events (aOR 2.14 [1.26-3.63]). The relative incidence (RI) for all events was 2.13 [1.76-2.58] in the SCCS, for cardiac (RI: 1.67 [1.23-2.27]) and for cerebral events (RI: 3.20, [2.30-4.45]). CONCLUSION: The incidence of acute vascular events was low among triptan users. We found that triptan use among older may be associated with a low increased risk for acute vascular events, which may be more marked for cerebral events such as stroke, than for cardiac events.

Natural compounds inhibit Monkeypox virus methyltransferase VP39 in silico studies.

VP39, an essential 2'-O-RNA methyltransferase enzyme discovered in Monkeypox virus (MPXV), plays a vital role in viral RNA replication and transcription. Inhibition of the enzyme may prevent viral replication. In this context, using a combination of molecular docking and molecular dynamics (MDs) simulations, the inhibitory ability of NCI Diversity Set VII natural compounds to VP39 protein was investigated. It should be noted that the computed binding free energy of ligand via molecular docking and linear interaction energy (LIE) approaches are in good agreement with the corresponding experiments with coefficients of R=0.72 and 0.75, respectively. NSC 319990, NSC 196515 and NSC 376254 compounds were demonstrated that can inhibit MPVX methyltransferase VP39 protein with the similar affinity compared to available inhibitor sinefungin. Moreover, nine residues involving Gln39, Gly68, Gly72, Asp95, Arg97, Val116, Asp138, Arg140 and Asn156 may be argued that they play an important role in binding process of inhibitors to VP39.Communicated by Ramaswamy H. Sarma.

Evidence of the protective effect of anti-pollution products against oxidative stress in skin ex vivo using EPR spectroscopy and autofluorescence measurements.

The concentration of air pollution is gradually increasing every year so that daily skin exposure is unavoidable. Dietary supplements and topical formulations currently represent the protective strategies to guard against the effects of air pollution on the body and the skin. Unfortunately, there are not yet enough methods available to measure the effectiveness of anti-pollution products on skin. Here, we present two ex vivo methods for measuring the protective effect against air pollution of different cream formulations on the skin: Electron paramagnetic resonance (EPR) spectroscopy and autofluorescence excited by 785 nm using a confocal Raman microspectrometer (CRM). Smoke from one cigarette was used as a model pollutant. EPR spectroscopy enables the direct measurement of free radicals in excised porcine skin after smoke exposure. The autofluorescence in the skin was measured ex vivo, which is an indicator of oxidative stress. Two antioxidants and a chelating agent in a base formulation and a commercial product containing an antioxidant mixture were investigated. The ex vivo studies show that the antioxidant epigallocatechin-3-gallate (EGCG) in the base cream formulation provided the best protection against oxidative stress from smoke exposure for both methods.

RAS­stimulated release of exosomal miR­494­3p promotes the osteolytic bone metastasis of breast cancer cells.

RAS activation is a key determinant of breast cancer progression and metastasis. However, the role of the interaction among exosomes, RAS and microRNAs (miRNAs/miRs) in the osteolytic bone metastasis of breast cancer remains unclear. Therefore, the present study aimed to examine the role of activated RAS (KRAS, HRAS and NRAS) in the release of exosome­mediated osteoclastogenic miRNAs and to elucidate their functional role in bone microenvironment remodeling in vitro and in vivo. Exosomes derived from RAS­activated breast cancer cells promoted RANKL­induced osteoclastogenesis; however, RAS inhibition abolished this effect. miR­494­3p, miR­4508 and miR­6869­5p were identified as osteoclastogenic miRNAs in the exosomes secreted by RAS­activated breast cancer cells. The levels of these osteoclastogenic miRNAs in the sera of patients with human epidermal growth factor receptor 2­positive luminal breast cancer were significantly higher than those in the sera of patients with triple­negative breast cancer. miR­494­3p exhibited both osteoclastogenic and anti­osteoblastogenic activity. Treatment with a miR­494­3p inhibitor abolished the exosome­mediated increase in RANKL­induced osteoclastogenesis. Treatment with a miR­494­3p mimic enhanced RANKL­induced osteoclast formation; however, treatment with its inhibitor suppressed this effect by targeting leucine­rich repeat­containing G­protein coupled receptor 4 in osteoclast precursors. Furthermore, miR­494­3p inhibited bone morphogenetic protein 2­induced osteoblast formation by targeting semaphorin 3A. In a mouse model, exosomes derived from breast cancer cells promoted osteolytic bone lesions; however, treatment with a miR­494­3p inhibitor significantly suppressed this effect. On the whole, the present study provides a novel mechanism, demonstrating that the RAS activation of breast cancer cells induces osteolytic bone metastasis by stimulating the exosome­mediated transfer of osteoclastogenic miRNAs, including miR­494­3p to bone cells.

Design, synthesis and evaluation the bioactivities of novel 1,3-dimethyl-6-amino-1H-indazole derivatives as anticancer agents.

Indoleamine 2,3-dioxygenase (IDO1) is a heme-containing enzyme mainly responsible for the metabolism of tryptophan to kynurenine. To date, the IDO1 inhibitors have been developed intensively for the re-activation of the anticancer immune response. In this report, we designed, and synthesized novel 1,3-dimethyl-6-amino indazole derivatives as IDO1 inhibitors based on the structure of IDO1 active site. We further examined their anticancer activity on hypopharyngeal carcinoma cells (FaDu), squamous cell carcinoma of the oral tongue (YD-15), breast cancer cells (MCF7), and human dental pulp stem cells (HDPSC). Of them, compound N-(4-bromobenzyl)-1,3-dimethyl-1H-indazol-6-amine (7) remarkably suppressed IDO1 expression in a concentration - dependent manner. In addition, 7 was the most potential anticancer compound with inducing apoptosis activity as well as selectively activated extracellular signal-regulated kinases (ERK) in mitogen-activated protein kinase (MAPK) pathways on FaDu cells. Finally, compound 7 suppressed cell mobility in wound healing assay with the reduced expression of matrix metalloproteinase MMP9. Taken together, we believe that 7 is the most promising compound, which may be applied to treatment of hypopharyngeal carcinoma.

Red- and Near-Infrared-Excited Autofluorescence as a Marker for Acute Oxidative Stress in Skin Exposed to Cigarette Smoke Ex Vivo and In Vivo.

Air pollution is increasing worldwide and skin is exposed to high levels of pollution daily, causing oxidative stress and other negative consequences. The methods used to determine oxidative stress in the skin are invasive and non-invasive label-free in vivo methods, which are severely limited. Here, a non-invasive and label-free method to determine the effect of cigarette smoke (CS) exposure on skin ex vivo (porcine) and in vivo (human) was established. The method is based on the measurement of significant CS-exposure-induced enhancement in red- and near-infrared (NIR)-excited autofluorescence (AF) intensities in the skin. To understand the origin of red- and NIR-excited skin AF, the skin was exposed to several doses of CS in a smoking chamber. UVA irradiation was used as a positive control of oxidative stress in the skin. The skin was measured with confocal Raman microspectroscopy before CS exposure, immediately after CS exposure, and after skin cleaning. CS exposure significantly increased the intensity of red- and NIR-excited skin AF in a dose-dependent manner in the epidermis, as confirmed by laser scanning microscopy AF imaging and fluorescence spectroscopy measurements. UVA irradiation enhanced the intensity of AF, but to a lower extent than CS exposure. We concluded that the increase in red- and NIR-excited AF intensities of the skin after CS exposure could clearly be related to the induction of oxidative stress in skin, where skin surface lipids are mainly oxidized.

Establishment of a method to expose and measure pollution in excised porcine skin with electron paramagnetic resonance spectroscopy.

Health problems associated with the amount of air pollutants are increasing worldwide. Pollution damages not only the lungs; it also has an impact on skin health and is co-responsible for the development of skin diseases. Anti-pollution products are on the rise in the cosmetic market but so far, there is no established method to directly assess the impact of pollution on the skin and to test the efficacy of anti-pollution products. To address this problem, two different chambers were developed for the reproducible exposure to realistic air pollutant concentrations. One chamber for the exclusive use of excised skin and hair samples, the second chamber for ex vivo and in vivo measurements. Measurements of nicotine next to the investigated skin area allow conclusions to be drawn on the particle concentration to which the skin is exposed. Electron paramagnetic resonance spectroscopy, which enables the detection of free radicals in different systems, was applied to assess the hazard potential of pollution in the skin. A direct proof of the formation of free radicals in the skin by the model pollutant cigarette smoke could be demonstrated. An additional application of UV irradiation even increased the formation of free radicals in the skin seven-fold (sum parameter). Depending on the question of interest, the use of different spin probes allows various assessments of the radical formation in skin: the amount of radicals but also the antioxidant status of the microenvironment can be estimated. Using two exposure chambers, the direct formation of oxidative stress by cigarette smoke on ex vivo skin, with and without additional UV exposure, could be reproducibly examined. This measurement method is promising for the assessment of anti-pollution products and could allow a direct causal connection between pollutant, effect on the skin and the protective function of skin care products.

Smoking trajectory and cancer risk: A population-based cohort study.

INTRODUCTION: Smoking behavior can change with time and lead to different health outcomes. This study explored the trajectory of smoking and its relationship with cancer incidence and mortality among Korean male adults. METHODS: We used 2002-2018 data from the National Health Insurance Service (NHIS). Smoking status was repeatedly measured in four waves of general health examinations provided by the NHIS between 2002 and 2009. Cancer incidence and mortality were tracked from 2010 to 2018. Trajectory analysis was used to identify the patterns of smoking. The hazard ratio was calculated using Cox proportional regression models. RESULTS: For the 2448548 men (≥20 years), 137788 cases of cancers and 41146 cancer deaths were found. We identified six trajectory groups: never smokers, former smokers, new current smokers, decreasing light smokers, steady moderate smokers, and steady heavy smokers. All smoking groups had an increased risk of cancer. The steady heavy smokers showed higher cancer incidence and mortality rate than the steady non-smokers (hazard ratio, HR=1.53; 95% CI: 1.49-1.58 and HR=2.64; 95% CI: 2.50-2.79, respectively). The cancer-specific analysis showed that the larynx and lung cancer incidence and mortality rate of the smoking group were higher than in never smokers. CONCLUSIONS: Smoking, even at low doses, increases the risk of most cancers in men. Quitting or reducing smoking, especially at a young age, can lower cancer incidence and mortality. This study may provide more objective results on the relationship between smoking and cancer, because smoking behavior was examined at multiple time points.

23-Hydroxyursolic acid from Viburnum lutescens inhibits osteoclast differentiation in vitro and lipopolysaccharide-induced bone loss in vivo by suppressing c-Fos and NF-κB signalling.

Bone homeostasis is maintained by a combination of osteoclast-mediated bone resorption and osteoblast-mediated bone formation. Excessive osteoclast activity is linked to several bone-related disorders, including osteoporosis and rheumatoid arthritis. Pharmacological therapy might have a number of adverse effects. Therefore, the development of natural anti-osteoclastogenic drugs with greater efficacy and fewer adverse effects is desirable. In this study, the anti-osteoclastogenic effects of 23-hydroxyursolic acid (HUA), a triterpene isolated from Viburnum lutescens, were investigated in vitro and in vivo. HUA significantly inhibited receptor activator of nuclear factor kappa-B ligand (RANKL)-induced mature osteoclast differentiation by reducing the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts and F-actin ring formation. It also inhibited the expression of osteoclast-specific marker genes such OSCAR, MMP-9, TRAP, DC-STAMP, and CtsK, as well as transcription factors, c-Fos and nuclear factor of activated T cells cytoplasmic 1 (NFATc1) in response to RANKL. Mice orally administered with HUA (25 and 50 mg/kg) exhibited significant protection against bone loss and osteoclast formation induced by lipopolysaccharide (LPS). HUA suppressed RANKL-induced nuclear factor kappa B (NF-κB) activation and phosphorylation of JNK and ERK mitogen-activated protein kinases (MAPKs). These results suggest that HUA attenuates osteoclast formation in vitro and in vivo by suppressing the RANKL-mediated AP1, NF-κB, and NFATc1 pathways. Therefore, HUA may be a lead compound for the prevention or treatment of osteolytic bone disorders.

Mussaendoside O, a N-triterpene cycloartane saponin, attenuates RANKL-induced osteoclastogenesis and inhibits lipopolysaccharide-induced bone loss.

BACKGROUND: Elevated activity of osteoclasts (OCs) is linked to osteolytic bone diseases, such as osteoporosis and rheumatoid arthritis. Developing natural anti-osteoclastogenic compounds with greater efficacy and fewer adverse effects is crucial for preventing or treating osteolytic bone diseases. N-triterpene cycloartane saponins (NTCSs) are rarely found in nature, and their inhibitory effects on OC differentiation in vitro and in vivo have not yet been explored. PURPOSE: This study was aimed to investigate the effect of mussaendoside O, an NTCS isolated from Mussaenda pubescens, on RANKL-induced OC differentiation and its underlying mechanism in vitro, and lipopolysaccharide (LPS)-induced bone resorption in a mouse model. METHODS: The content of mussaendoside O in methanol extract of M. pubescens was determined by HPLC. The inhibitory effects of mussaendoside O on RANKL-induced OC formation were assessed using TRAP staining, western blotting, immunofluorescence staining, and real-time qPCR. Meanwhile, the effects of mussaendoside O on LPS-induced inflammatory responses were assessed using a Griess reagent and qPCR. The effects of mussaendoside O on LPS-induced bone resorption in a mouse model were evaluated using micro-CT and immunohistochemical staining. RESULTS: Mussaendoside O inhibited RANKL-induced TRAP-positive multinucleated OC formation in a concentration-dependent manner without affecting cell viability. However, mussaendoside O did not inhibit LPS-induced mRNA expression of COX-2, iNOS, and TNF-α. Mice orally administrated with mussaendoside O exhibited significant protection from LPS-induced bone resorption and OC formation. At the molecular level, mussaendoside O suppressed RANKL-activated phosphorylation of p38 MAPK and JNK, as well as c-Fos expression. In addition, mussaendoside O suppressed RANKL-induced NFATc1 activation and the expression of its target genes, including OSCAR, DC-STAMP, CtsK, and TRAP. CONCLUSION: Mussaendoside O attenuates OC differentiation in vitro and LPS-induced bone resorption in a mouse model by inhibiting the RANKL-activated c-Fos/NFATc1 signaling pathways. Therefore, mussaendoside O may be a valuable lead compound for preventing or treating of osteolytic bone diseases.

Searching for AChE inhibitors from natural compounds by using machine learning and atomistic simulations.

Acetylcholinesterase (AChE) is one of the most important drug targets for Alzheimer's disease treatment. In this work, a combined approach involving machine-learning (ML) model and atomistic simulations was established to predict the ligand-binding affinity to AChE of the natural compounds from VIETHERB database. The trained ML model was first utilized to rapidly and accurately screen the natural compound database for potential AChE inhibitors. Atomistic simulations including molecular docking and steered-molecular dynamics simulations were then used to confirm the ML outcome. Good agreement between ML and atomistic simulations was observed. Twenty compounds were suggested to be able to inhibit AChE. Especially, four of them including geranylgeranyl diphosphate, 2-phosphoglyceric acid, and 2-carboxy-d-arabinitol 1-phosphate, and farnesyl diphosphate are highly potent inhibitors with sub-nanomolar affinities.

Identifying Possible AChE Inhibitors from Drug-like Molecules via Machine Learning and Experimental Studies.

Acetylcholinesterase (AChE) is one of the most important drug targets for Alzheimer's disease (AD) treatment. In this work, a machine learning model was trained to rapidly and accurately screen large chemical databases for the potential inhibitors of AChE. The obtained results were then validated via in vitro enzyme assay. Moreover, atomistic simulations including molecular docking and molecular dynamics simulations were then used to understand molecular insights into the binding process of ligands to AChE. In particular, two compounds including benzyl trifluoromethyl ketone and trifluoromethylstyryl ketone were indicated as highly potent inhibitors of AChE because they established IC50 values of 0.51 and 0.33 µM, respectively. The obtained IC50 of two compounds is significantly lower than that of galantamine (2.10 µM). The predicted log(BB) suggests that the compounds may be able to traverse the blood-brain barrier. A good agreement between computational and experimental studies was observed, indicating that the hybrid approach can enhance AD therapy.

Syntenin-1-mediated small extracellular vesicles promotes cell growth, migration, and angiogenesis by increasing onco-miRNAs secretion in lung cancer cells.

Small extracellular vesicles (sEVs) play a pivotal role in tumor progression by mediating intercellular communication in the tumor microenvironment (TME). Syntenin-1 induces malignant tumor progression in various types of human cancers, including human lung cancer and regulates biogenesis of sEVs. However, the function of syntenin-1-regulated sEVs and miRNAs in sEVs remains to be elucidated. In the present study, we aimed to demonstrate the role of oncogenic Ras/syntenin-1 axis in the release of sEVs and elucidate the function of syntenin-1-mediated miRNAs in sEVs in lung cancer progression. The results revealed that oncogenic Ras promoted the release of sEVs by inducing syntenin-1 expression; disruption of syntenin-1 expression impaired the release of sEVs as well as sEV-mediated cancer cell migration and angiogenesis. Moreover, we identified three miRNAs, namely miR-181a, miR-425-5p, and miR-494-3p, as onco-miRNAs loaded into syntenin-1-dependent sEVs. Remarkably, miR-494-3p was highly abundant in sEVs and its release was triggered by syntenin-1 expression and oncogenic Ras. Ectopic expression of the miR-494-3p mimic enhanced the migration and proliferation of lung cancer cells as well as tube formation in endothelial cells; however, the miR-494-3p inhibitor blocked sEV-mediated effects by targeting tyrosine-protein phosphatase nonreceptor type 12 (PTPN12), a tumor suppressor. sEVs promoted tumor growth and angiogenesis by downregulating PTPN12 expression; however, the miR-494-3p inhibitor significantly suppressed these effects in vivo, confirming that miR-494-3p acts as a major onco-miRNA loaded into lung cancer cell-derived sEVs. Eventually, the oncogenic Ras/syntenin-1 axis may induce cancer progression by increasing miR-494-3p loading into sEVs in lung cancer cells in the TME.

Anti-osteoclastogenic Effects of Indole Alkaloids Isolated from Barley (Hordeum vulgare Var. Hexastichon) Grass.

As part of our continuous program to identify new potential candidates for controlling osteolytic bone diseases from natural products, the alkaloid fraction of barley (Hordeum vulgare var. hexastichon) grass (HVA) significantly inhibited RANKL-induced osteoclast formation and protected mice from LPS-induced bone loss. A phytochemical investigation of HVA afforded nine indole alkaloids, including one new compound [hordeumin A (1)] and eight known analogues (2-9). Of them, four (1, 2, 4, and 5) were anti-osteoclastogenic compounds. Of these four, compound 5 significantly suppressed RANKL-induced osteoclast formation, actin ring formation, and bone resorption in a concentration-dependent manner. It also suppressed the RANKL-induced NF-κB and MAPK signaling pathways and the activation of c-Fos and NFATc1. Compound 5 also reduced the expression levels of osteoclast-specific marker genes, including TRAP, CtsK, DC-STAMP, OSCAR, and MMP9. Our findings suggest that HVA and its alkaloid constituents could be valuable candidates for the prevention and treatment of osteolytic bone diseases.

Marine derivatives prevent wMUS81 in silico studies.

The winged-helix domain of the methyl methanesulfonate and ultraviolet-sensitive 81 (wMUS81) is a potential cancer drug target. In this context, marine fungi compounds were indicated to be able to prevent wMUS81 structure via atomistic simulations. Eight compounds such as D197 (Tryptoquivaline U), D220 (Epiremisporine B), D67 (Aspergiolide A), D153 (Preussomerin G), D547 (12,13-dihydroxyfumitremorgin C), D152 (Preussomerin K), D20 (Marinopyrrole B) and D559 (Fumuquinazoline K) were indicated that they are able to prevent the conformation of wMUS81 via forming a strong binding affinity to the enzyme via perturbation approach. The electrostatic interaction is the dominant factor in the binding process of ligands to wMUS81. The residues Trp55, Arg59, Leu62, His63 and Arg69 were found to frequently form non-bonded contacts and hydrogen bonds to inhibitors. Moreover, the influence of the ligand D197, which formed the lowest binding free energy to wMUS81, on the structural change of enzyme was investigated using replica exchange molecular dynamics simulations. The obtained results indicated that D197, which forms a strong binding affinity, can modify the structure of wMUS81. Overall, the marine compounds probably inhibit wMUS81 due to forming a strong binding affinity to the enzyme as well as altering the enzymic conformation.

Flavonoids from the peels of Citrus unshiu Markov. and their inhibitory effects on RANKL-induced osteoclastogenesis through the downregulation of c-Fos signaling in vitro.

Phytochemical investigation of Citrus unshiu peels led to the isolation of eight new flavonols (7-9, 11-15) and sixteen known compounds (1-6, 10, 16-24). Their structures were elucidated using spectroscopic analysis (1D, 2D NMR, and HR-MS). Besides, all isolated compounds (1-24) were evaluated for their inhibitory effects on receptor activator of RANKL-induced osteoclastogenesis in BMMs. Among them, dimethylmikanin (1), quercetogetin (2), 3,3',4',5,7,8-hexamethoxyflavone (3), 3-methoxynobiletin (4) showed a significant inhibitory effect on RANKL-induced osteoclast differentiation at a concentration of 10 µM. Moreover, 3-methoxynobiletin (4) suppressed RANKL-induced osteoclastogenesis by decreasing the number of osteoclasts and osteoclast actin-ring formation in a dose-dependent manner without causing any cytotoxic effects on BMMs. At the molecular level, 3-methoxynobiletin (4) inhibited RANKL-induced c-Fos expression and subsequently NFATc1 activation, as well as the expression of osteoclastogenesis-related marker genes c-Src and CtsK. These findings suggested that 3-methoxynobiletin (4) attenuated osteoclast differentiation by inhibiting RANKL-mediated c-Fos signaling and that it may have therapeutic potential for treating or preventing bone resorption-related diseases, such as osteoporosis.

Searching and designing potential inhibitors for SARS-CoV-2 Mpro from natural sources using atomistic and deep-learning calculations.

The spread of severe acute respiratory syndrome coronavirus 2 novel coronavirus (SARS-CoV-2) worldwide has caused the coronavirus disease 2019 (COVID-19) pandemic. A hundred million people were infected, resulting in several millions of death worldwide. In order to prevent viral replication, scientists have been aiming to prevent the biological activity of the SARS-CoV-2 main protease (3CL pro or Mpro). In this work, we demonstrate that using a reasonable combination of deep-learning calculations and atomistic simulations could lead to a new approach for developing SARS-CoV-2 main protease (Mpro) inhibitors. Initially, the binding affinities of the natural compounds to SARS-CoV-2 Mpro were estimated via atomistic simulations. The compound tomatine, thevetine, and tribuloside could bind to SARS-CoV-2 Mpro with nanomolar/high-nanomolar affinities. Secondly, the deep-learning (DL) calculations were performed to chemically alter the top-lead natural compounds to improve ligand-binding affinity. The obtained results were then validated by free energy calculations using atomistic simulations. The outcome of the research will probably boost COVID-19 therapy.