Discovery of Natural Inhibitors of Cholinesterases from Hydrangea: In Vitro and In Silico Approaches.

Alzheimer's disease (AD) is a neurodegenerative disease conceptualized as a clinical-biological neurodegenerative construct where amyloid-beta pathophysiology is supposed to play a role. The loss of cognitive functions is mostly characterized by the rapid hydrolysis of acetylcholine by cholinesterases including acetylcholinesterase (AChE) and butyrylcholinesterase (BChE). Moreover, both enzymes are responsible for non-catalytic actions such as interacting with amyloid ß peptide (Aß) which further leads to promote senile plaque formation. In searching for a natural cholinesterase inhibitor, the present study focused on two isocoumarines from hydrangea, thunberginol C (TC) and hydrangenol 8-O-glucoside pentaacetate (HGP). Hydrangea-derived compounds were demonstrated to act as dual inhibitors of both AChE and BChE. Furthermore, the compounds exerted selective and non-competitive mode of inhibition via hydrophobic interaction with peripheral anionic site (PAS) of the enzymes. Overall results demonstrated that these natural hydrangea-derived compounds acted as selective dual inhibitors of AChE and BChE, which provides the possibility of potential source of new type of anti-cholinesterases with non-competitive binding property with PAS.

Rapid identification of isoprenylated flavonoids constituents with inhibitory activity on bacterial neuraminidase from root barks of paper mulberry (Broussonetia papyrifera).

This study was to assess the possibility of using competitive and slow binding experiments with affinity-based ultrafiltration UPLC-QTof-MS analysis to identify potent bacterial neuraminidase (bNA) inhibitors from the Broussonetia papyrifera roots extract. To isolate unbound compounds from the enzyme-binding complex, the root bark extracts were either incubated in the absence of bNA, in the presence of bNA, or with the time-dependent bNA before the ultrafiltration was performed. Thirteen flavonoids were separated from the target extract, and their inhibitory activities were tested against bNA. The isolated flavonoids exhibited potent inhibition against NA (IC50 = 0.7-54.0 µM). Our kinetic analysis of representative active flavonoids (1, 2, and 6) showed slow and time-dependent reversible inhibition. Additionally, chalcones exhibited noncompetitive inhibition characteristics, whereas flavonols and flavans showed mixed-type behavior. The computational results supported the experimental behaviors of flavonoids 2, 6, 10, and 12, indicating that bounded to the active site, but flavonoids 6 and 10 binds near but not accurately at the active site. Although this is mixed-type inhibition, their binding can be considered competitive.

ReSimNet: drug response similarity prediction using Siamese neural networks.

MOTIVATION: Traditional drug discovery approaches identify a target for a disease and find a compound that binds to the target. In this approach, structures of compounds are considered as the most important features because it is assumed that similar structures will bind to the same target. Therefore, structural analogs of the drugs that bind to the target are selected as drug candidates. However, even though compounds are not structural analogs, they may achieve the desired response. A new drug discovery method based on drug response, which can complement the structure-based methods, is needed. RESULTS: We implemented Siamese neural networks called ReSimNet that take as input two chemical compounds and predicts the CMap score of the two compounds, which we use to measure the transcriptional response similarity of the two compounds. ReSimNet learns the embedding vector of a chemical compound in a transcriptional response space. ReSimNet is trained to minimize the difference between the cosine similarity of the embedding vectors of the two compounds and the CMap score of the two compounds. ReSimNet can find pairs of compounds that are similar in response even though they may have dissimilar structures. In our quantitative evaluation, ReSimNet outperformed the baseline machine learning models. The ReSimNet ensemble model achieves a Pearson correlation of 0.518 and a precision@1% of 0.989. In addition, in the qualitative analysis, we tested ReSimNet on the ZINC15 database and showed that ReSimNet successfully identifies chemical compounds that are relevant to a prototype drug whose mechanism of action is known. AVAILABILITY AND IMPLEMENTATION: The source code and the pre-trained weights of ReSimNet are available at https://github.com/dmis-lab/ReSimNet. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Piscroside C inhibits TNF-α/NF-κB pathway by the suppression of PKCδ activity for TNF-RSC formation in human airway epithelial cells.

BACKGROUND: Piscroside C, isolated from Pseudolysimachion rotundum var. subintegrum, is a novel iridoid glycoside with therapeutic efficacy in a mouse model of chronic obstructive pulmonary disease (COPD). Piscroside C has been reported as a constituent of YPL-001 (under Phase 2a study, ClinicalTrials.gov identifier NCT02272634). PURPOSE: To investigate the mechanisms behind piscroside C therapeutic effects on COPD in human airway epithelial NCI-H292 cells. METHODS: We tested if piscroside C effectively suppresses MUC5AC gene expression and TNF-RSC/IKK/NF-κB cascades in TNF-α-stimulated NCI-H292 cells by employing, reverse transcription-polymerase chain reaction, enzyme-linked immunosorbent assay, luciferase reporter assays, chromatin immunoprecipitation assays and immunoprecipitation. RESULTS: Piscroside C markedly suppressed the expression of TNF-α-induced MUC5AC mucus protein by inhibiting the transcriptional activity of NF-κB in NCI-H292 cells. Indeed, piscroside C negatively regulated the function of TNF receptor 1 signaling complex (TNF-RSC, an upstream regulator of the NF-κB pathway) without affecting its extracellular interaction with the TNF-α ligand. This inhibitory effect by piscroside C is mediated by the inactivation of protein kinase C (PKC), an essential regulator of TNF-RSC. PKC inactivation by piscroside C results in decreased PKCδ binding to a TRAF2 subunit of TNF-RSC and subsequent reduced IKK phosphorylation, resulting in NF-κB inactivation. CONCLUSION: We propose that piscroside C is a promising therapeutic constituent of YPL-001 through its inhibition of PKCδ activity in the TNF-RSC/IKK/NF-κB/MUC5AC signaling cascade.

BACE1 Inhibition by Genistein: Biological Evaluation, Kinetic Analysis, and Molecular Docking Simulation.

ß-site amyloid precursor protein cleaving enzyme 1 (BACE1) plays a role in generating amyloid ß (Aß), thus playing a major part early in the pathogenesis of Alzheimer's disease (AD). BACE1 has emerged as a crucial therapeutic target for decreasing the Aß concentration in the AD brain. To explore natural BACE1 inhibitors, the present study concentrated on isoflavones, including genistein, formononetin, glycitein, daidzein, and puerarin. In this study, in vitro anti-AD activities were assessed using BACE1 inhibition assays, as well as enzyme kinetic predictions. Molecular docking analysis was applied to design potential BACE1 inhibitors. Among the major isoflavones, genistein exerted a notable BACE1 inhibition through reversible noncompetitive mechanism, while other compounds were less potent against BACE1. The docking study revealed that genistein had negative binding energy (-8.5 kcal/mol) and was stably positioned in the allosteric domains of BACE1 residues. It interacted with important amino acid residues in BACE1, such as ASN37, GLN73, and TRP76, through hydrogen bonding. The results suggested that genistein may be beneficial for preventing and/or treating AD. Furthermore, it may provide potential guidelines for the design of new BACE1 inhibitors.

Molecular dynamics simulation integrating the inhibition kinetics of hydroxysafflor yellow A on α-glucosidase.

Inhibition of α-glucosidase has attracted the attention of researchers due to its connection to type-2 diabetes. Hydroxysafflor yellow A (HSYA) extracted from Carthamus tinctorius L. is a natural antioxidant used in traditional Chinese medicine. In this study, the effect of HSYA on α-glucosidase was evaluated using inhibitory kinetics based on the antioxidant properties of HSYA and by performing computational simulation integration methods. HSYA reversibly inhibited α-glucosidase in a competitive inhibition manner and the evaluated kinetic parameters were IC50 = 1.1 ± 0.22 mM and Ki = 1.04 ± 0.23 mM, respectively. The results of spectrofluorimetry showed that the inner hydrophobic regions of α-glucosidase, which are mostly in the active site, were exposed to the surface with increasing HSYA concentrations, indicating that the inactivation of α-glucosidase by HSYA was accompanied by regional unfolding. The molecular dynamics simulations indicated that the four rings of HSYA interact with four residues such as G217, A278, H279, and G280 at the entrance of the active site. Our study provides insight into the inhibition of α-glucosidase and the accompanying structural changes by HSYA. Based on its α-glucosidase-inhibiting effect and its potential as a natural antioxidant, HSYA is a potential agent for treating α-glucosidase-associated type-2 diabetes.

The Identification of Biochanin A as a Potent and Selective ß-Site App-Cleaving Enzyme 1 (Bace1) Inhibitor.

Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the enzyme involved in the abnormal production of the amyloidogenic peptide Aß, one of the major causes of histological hallmarks of Alzheimer's disease (AD). Thus, BACE1 represents a key target protein in the development of new potential target for the prevention and treatment of AD. In this study, in vitro anti-AD activity of biochanin A, a dietary isoflavone found in legumes and most notably red clover, were evaluated via human recombinant BACE1 inhibition assay, as well as enzyme kinetic and molecular docking predictions. Enzyme-based assays revealed that biochanin A exhibited a non-competitive inhibitory effect on BACE1 with an IC50 value of 28 µM and a Ki of 43 µM. In addition, docking simulation results demonstrated that ASN37, SER35, SER36, TRP76, and ARG128 residues of BACE1 interacted with biochanin A. Moreover, the binding energy of biochanin A was negative (-8.4 kcal/mol), indicating that it might potentiate a strong binding between the compound and the allosteric site of BACE1, resulting in further effective BACE1 inhibition. The present novel findings raise the possibility that biochanin A may be used as a preventative, developed into a therapeutic agent for AD, or both.

Mg2+ effect on argonaute and RNA duplex by molecular dynamics and bioinformatics implications.

RNA interference (RNAi), mediated by small non-coding RNAs (e.g., miRNAs, siRNAs), influences diverse cellular functions. Highly complementary miRNA-target RNA (or siRNA-target RNA) duplexes are recognized by an Argonaute family protein (Ago2), and recent observations indicate that the concentration of Mg2+ ions influences miRNA targeting of specific mRNAs, thereby modulating miRNA-mRNA networks. In the present report, we studied the thermodynamic effects of differential [Mg2+] on slicing (RNA silencing cycle) through molecular dynamics simulation analysis, and its subsequent statistical analysis. Those analyses revealed different structural conformations of the RNA duplex in Ago2, depending on Mg2+ concentration. We also demonstrate that cation effects on Ago2 structural flexibility are critical to its catalytic/functional activity, with low [Mg2+] favoring greater Ago2 flexibility (e.g., greater entropy) and less miRNA/mRNA duplex stability, thus favoring slicing. The latter finding was supported by a negative correlation between expression of an Mg2+ influx channel, TRPM7, and one miRNA's (miR-378) ability to downregulate its mRNA target, TMEM245. These results imply that thermodynamics could be applied to siRNA-based therapeutic strategies, using highly complementary binding targets, because Ago2 is also involved in RNAi slicing by exogenous siRNAs. However, the efficacy of a siRNA-based approach will differ, to some extent, based on the Mg2+ concentration even within the same disease type; therefore, different siRNA-based approaches might be considered for patient-to-patient needs.

Oleic acid and linoleic acid from Tenebrio molitor larvae inhibit BACE1 activity in vitro: molecular docking studies.

In our ongoing research to find therapeutic compounds for Alzheimer's disease (AD) from natural resources, the inhibitory activity of the BACE1 enzyme by Tenebrio molitor larvae and its major compounds were evaluated. The T. molitor larvae extract and its fractions exhibited strong BACE1 suppression. The major components of hexane fraction possessing both high yield and strong BACE1 inhibition were determined by thin layer chromatography, gas chromatography, and nuclear magnetic resonance analysis. A remarkable composition of unsaturated long chain fatty acids, including oleic acid and linoleic acid, were identified. Oleic acid, in particular, noncompetitively attenuated BACE1 activity with a half-maximal inhibitory concentration (IC50) value of 61.31 µM and Ki value of 34.3 µM. Furthermore, the fatty acids were stably interacted with BACE1 at different allosteric sites of the enzyme bound with the OH of CYS319 and the NH3 of TYR320 for oleic acid and with the C=O group of GLN304 for linoleic acid. Here, we first revealed novel pharmacophore features of oleic acids and linoleic acid to BACE1 by in silico docking studies. The present findings would clearly suggest potential guidelines for designing novel BACE1 selective inhibitors.

The effect of Zn(2+) on Pelodiscus sinensis creatine kinase: unfolding and aggregation studies.

We studied the effects of Zn(2+) on creatine kinase from the Chinese soft-shelled turtle, Pelodiscus sinensis (PSCK). Zn(2+) inactivated the activity of PSCK (IC(50) = .079 ± .004 mM) following first-order kinetics consistent with multiple phases. The spectrofluorimetry results showed that Zn(2+) induced significant tertiary structural changes of PSCK with exposure to hydrophobic surfaces and that Zn(2+) directly induced PSCK aggregation. The addition of osmolytes such as glycine, proline, and liquaemin successfully blocked PSCK aggregation, recovering the conformation and activity of PSCK. We measured the ORF gene sequence of PSCK by rapid amplification of cDNA end and simulated the 3D structure of PSCK. The results of molecular dynamics simulations showed that eight Zn(2+) bind to PSCK and one Zn(2+) is predicted to bind in a plausible active site of creatine and ATP. The interaction of Zn(2+) with the active site could mostly block the activity of PSCK. Our study provides important insight into the action of Zn(2+) on PSCK as well as more insights into the PSCK folding and ligand-binding mechanisms, which could provide important insight into the metabolic enzymes of P. sinensis.

Caspase and mitogen activated protein kinase pathways are involved in Solanum lyratum herba induced apoptosis.

AIM OF STUDY: Solanum lyratum herba (SLH) has been traditionally used for the treatment of febrifuge, diarrhea, eye disease and cancer with little scientific evidences. Thus, in the present study, to elucidate the antitumor mechanism of SLH: in vitro and in vivo experiments were performed with hexane fraction of Solanum lyratum herba (HSLH). MATERIALS AND METHODS: Cytotoxicity assay, 4'-6-diamidino-2-phenylindole (DAPI) staining, flow cytometric analysis for sub-G1 peaks, Western blot analysis were used with the antibodies of apoptosis related proteins in vitro. In addition, the effect of HSLH on in vivo tumor growth was evaluated in Lewis lung carcinoma (LLC) tumor model and immunohistochemistry also was performed with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining in tumor section. RESULTS: HSLH exhibited cytotoxicity against LLC cells most effectively among its solvent fractions. Ladder like DNA fragmentation and apoptotic features such as chromatin condensation and apoptotic bodies were observed in HSLH treated LLC cells by 4'-6-diamidino-2-phenylindole staining. HSLH also significantly increased sub-G(1) peaks, activated caspase-8, -9 and -3 proteins and cleaved poly(ADP-ribose) polymerase (PARP). Furthermore, HSLH increased the phosphorylation of extracellular signal-regulated kinase (ERK), transiently activated phospho-JNK (c-jun N-terminal kinase) and downregulated phospho-p38 MAPK. In addition, we have found for the first time HSLH treatment effectively suppressed the in vivo growth of LLC to up to approximately 30% of untreated control at 50mg/kg and significantly increased apoptotic expression in tumor section by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining. Taken together, these findings strongly demonstrate that hexane fraction of Solanum lyratum herba exerts antitumor activity via caspase activation and MAPK regulation and can be effectively applied to lung cancer as a cancer chemopreventive agent.

[Clinical factors associated with response to biofeedback therapy for patients with chronic constipation].

BACKGROUND/AIMS: Biofeedback therapy has been widely used for the treatment of constipated patients. However, there are only a few reports about the clinical factors that can predict the effectiveness of biofeedback therapy. The aim of this study was to evaluate prognostic factors before the initiation of biofeedback treatment in constipated patients. METHODS: Biofeedback treatment was performed in 114 patients with constipation. After classifying the patients into two groups, responder and non-responder by subjective and objective parameters, univariate and multivariate analysis were performed to evaluate the factors associated with effectiveness of biofeedback therapy. RESULTS: Eighty-five patients (74.6%) responded to biofeedback therapy. Pre-treatment balloon expulsion test, paradoxical contraction on manometry, defecation index and anal residual pressure during straining were the factors that influenced the results of biofeedback treatment. On multivariate analysis, defecation index (odds ratio=67.5, p<0.05) and paradoxical contraction on manometry (odds ratio=0.053, p<0.05) were the factors that showed significant difference between the responders and non-responders. CONCLUSIONS: This study suggests that several pre-treatment prognostic factors are associated with response to biofeedback for the constipated patients. Using prognostic factors, we may be able to evaluate the patterns of pelvic floor dysfunction and responsiveness of biofeedback therapy for the patients with constipation.