Inhibition Of Binding Activity Research Articles

A structure-based approach to discover a potential isomerase Pin1 inhibitor for cancer therapy using computational simulation and biological studies

Peptidyl-prolyl cis/trans isomerase Pin1 occupies a prominent role in preventing the development of certain malignant tumors. Pin1 is considered a target for the treatment of related malignant tumors, so the identification of novel Pin1 inhibitors is particularly urgent. In this study, we preliminarily predicted eight candidates from FDA-approved drug database as the potential Pin1 inhibitors through virtual screening combined with empirical screening. Therefore, we selected these eight candidates and tested their binding affinity and inhibitory activity against Pin1 using fluorescence titration and PPIase activity assays, respectively. Subsequently, we found that four FDA-approved drugs showed good binding affinities and inhibition effects. In addition, we also observed that bexarotene can reduce cell viability in a dose-dependent and time-dependent manner and induce apoptosis. Finally, we inferred that residues K63, R68 and R69 are important in the binding process between bexarotene and Pin1. All in all, repurposing of FDA-approved drugs to inhibit Pin1 may provide a promising insight into the identification and development of new treatments for certain malignant tumors.

A novel small molecule ZYZ384 targeting SMYD3 for hepatocellular carcinoma via reducing H3K4 trimethylation of the Rac1 promoter.

SMYD3 (SET and MYND domain-containing 3) is a histone lysine methyltransferase highly expressed in different types of cancer(s) and is a promising epigenetic target for developing novel antitumor therapeutics. No selective inhibitors for this protein have been developed for cancer treatment. Therefore, the current study describes developing and characterizing a novel small molecule ZYZ384 screened and synthesized based on SMYD3 structure. Virtual screening was initially used to identify a lead compound and followed up by modification to get the novel molecules. Several technologies were used to facilitate compound screening about these novel molecules' binding affinities and inhibition activities with SMYD3 protein; the antitumor activity has been assessed in vitro using various cancer cell lines. In addition, a tumor-bearing nude mice model was established, and the activity of the selected molecule was determined in vivo. Both RNA-seq and chip-seq were performed to explore the antitumor mechanism. This work identified a novel small molecule ZYZ384 targeting SMYD3 with antitumor activity and impaired hepatocellular carcinoma tumor growth by reducing H3K4 trimethylation of the Rac1 promoter triggering the tumor cell cycle arrest through the AKT pathway.

Novel antipyrine substituted 4-thiazolidinones: Synthesis, DNA binding and topoisomerase inhibition activities, and in-silico studies

In this study, we report the synthesis and characterization of novel 4-thiazolidinones containing antipyrine (3a-e) derived from the starting compounds of Schiff bases (2a-e). The Schiff bases were synthesized using aldehydes of natural origin, such as citronellal, cuminaldehyde, 2-thiazolecarboxaldehyde, 5-methylfurfural, and syringaldehyde. Their FTIR spectra revealed the enol- tautomeric solid-state structures of compounds 3b and 3d. The pUC18 plasmid DNA binding specificity and capacity of the compounds to catalytically generate topoisomerase I activity were also examined using agarose gel electrophoresis. The compounds induced changes in DNA mobility and showed a topoisomerase I inhibitory effect at all doses. 4-Thiazolidinones had a higher affinity in DNA and topoisomerase I than the Schiff bases. DNA binding and topoisomerase I activities were most effective at low concentration (6 μM) for the compounds of Schiff bases 2a, d, and 4-thiazolidinones 3a, d. Furthermore, this study presents computational methods for addressing the molecules’ drug-like properties. 2a, d, and 3a, d Compounds were used as representative of the most bioactive among the newly synthesized molecules in in-silico studies. The web tools of ADMETlab, SwissADME, OSIRIS, and the BOILED-Egg method were used to conduct in-silico biological studies involving ADME prediction, BBB penetration, gastrointestinal absorption, and toxicity studies. The ADME profile of the query compounds gets within the range of applicability and is at an appropriate level. In-silico studies revealed that compounds 2a, d, and 3a, d are neither hERG blockers nor AMES toxic. However, compounds 2a and 3a have hepatotoxicity.

Spike-Specific Memory B Cell Response in Hematopoietic Cell Transplantation Recipients following Multiple mRNA-1273 Vaccinations: A Longitudinal Observational Study.

Preventing SARS-CoV-2 infection is of utmost importance in allogeneic hematopoietic cell transplantation patients (allo-HCT), given their heightened susceptibility to adverse outcomes associated with SARS-CoV-2 infection. However, limited data are available regarding the immune response to COVID-19 vaccines in these subjects, particularly concerning the generation and persistence of spike-specific memory response. Here, we analyzed the spike-specific memory B cells in a cohort of allo-HCT recipients vaccinated with multiple doses of the mRNA-1273 vaccine and monitored the spike-specific antibody response from baseline up to one month after the fourth dose. After the primary vaccine series, the frequency of spike-specific B cells, detected within the pool of Ig-switched CD19+ cells, significantly increased. The booster dose further induced a significant expansion, reaching up to 0.28% of spike-specific B cells. The kinetics of this expansion were slower in the allo-HCT recipients compared to healthy controls. Spike-specific IgG and ACE2/RBD binding inhibition activity were observed in 80% of the allo-HCT recipients after the first two doses, with a significant increase after the third and fourth booster doses, including in the subjects who did not respond to the primary vaccine series. Additionally, 87% of the allo-HCT recipients exhibited positive cross-inhibition activity against the BA.1 variant. Our findings provide evidence that allo-HCT recipients need repeated doses of the mRNA-1273 vaccine to induceSARS-CoV-2 specific immune response similar to that observed in healthy individuals. This is particularly crucial for vulnerable individuals who may exhibit a limited response to the primary series of SARS-CoV-2 vaccination.

Mudanpioside C Discovered from Paeonia suffruticosa Andr. Acts as a Protein Disulfide Isomerase Inhibitor with Antithrombotic Activities.

Paeonia suffruticosa Andr. is a well-known landscape plant worldwide and also holds significant importance in China due to its medicinal and dietary properties. Previous studies have found that Cortex Moutan (CM), the dried root bark of P. suffruticosa, showed antiplatelet and cardioprotective effects, although the underlying mechanism and active compounds remain to be revealed. In this study, protein disulfide isomerase (PDI) inhibitors in CM were identified using a ligand-fishing method combined with the UHPLC-Q-TOF-MS assay. Further, their binding sites and inhibitory activities toward PDI were validated. The antiplatelet aggregation and antithrombotic activity were investigated. The results showed that two structurally similar compounds in CM were identified as the inhibitor for PDI with IC50 at 3.22 μM and 16.73 μM; among them Mudanpioside C (MC) is the most effective PDI inhibitor. Molecular docking, site-directed mutagenesis, and MST assay unequivocally demonstrated the specific binding of MC to the b'-x domain of PDI (Kd = 3.9 μM), acting as a potent PDI inhibitor by interacting with key amino acids K263, D292, and N298 within the b'-x domain. Meanwhile, MC could dose-dependently suppress collagen-induced platelet aggregation and interfere with platelet activation, adhesion, and spreading. Administration of MC can significantly inhibit thrombosis formation without disturbing hemostasis in mice. These findings present a promising perspective on the antithrombotic properties of CM and highlight the potential application of MC as lead compounds for targeting PDI in thrombosis therapy.

Monocarboxylate transporter-1 (MCT-1) inhibitors screened from autodisplayed FV-antibody library

Monocarboxylate transporter-1 (MCT-1) inhibitors were screened from the Fv-antibody library, which contained complementary determining region 3 with randomized amino acid sequences (11 residues) through site-directed mutagenesis. Fv-antibodies against MCT-1 were screened from the autodisplayed Fv-antibody library. Two clones were screened, and the binding affinity (KD) against MCT-1 was estimated using flow cytometry. The screened Fv-antibodies were expressed as soluble fusion proteins (Fv-1 and Fv-2) and the KD for MCT-1 was estimated using the SPR biosensor. The inhibitory activity of the expressed Fv-antibodies was observed in HEK293T and Jurkat cell lines by measuring intracellular pH and lactate accumulation. The level of cell viability in HEK293T and Jurkat cell lines was decreased by the inhibitory activity of the expressed Fv-antibodies. The binding properties of the Fv-antibodies to MCT-1 were analyzed using molecular docking simulations. Overall, the results showed that the screened Fv-antibodies against MCT-1 from the Fv-antibody library had high binding affinity and inhibitory activity against MCT-1, which could be used as potential therapeutic drug candidates for the MCT-1 inhibitor.

Mechanistic and thermodynamic characterization of antiviral inhibitors targeting nucleocapsid N-terminal domain of SARS-CoV-2

The nucleocapsid (N) protein of SARS-CoV-2 plays a pivotal role in encapsulating the viral genome. Developing antiviral treatments for SARS-CoV-2 is imperative due to the diminishing immunity of the available vaccines. This study targets the RNA-binding site located in the N-terminal domain (NTD) of the N-protein to identify the potential antiviral molecules against SARS-CoV-2. A structure-based repurposing approach identified the twelve high-affinity molecules from FDA-approved drugs, natural products, and the LOPAC1280 compound libraries that precisely bind to the RNA binding site within the NTD. The interaction of these potential antiviral agents with the purified NTD protein was thermodynamically characterized using isothermal titration calorimetry (ITC). A fluorescence-based plate assay to assess the RNA binding inhibitory activity of small molecules against the NTD has been employed, and the selected compounds exhibited significant RNA binding inhibition with calculated IC50 values ranging from 8.8 μM to 15.7 μM. Furthermore, the antiviral efficacy of these compounds was evaluated using in vitro cell-based assays targeting the replication of SARS-CoV-2. Remarkably, two compounds, Telmisartan and BMS-189453, displayed potential antiviral activity against SARS-CoV-2, with EC50 values of approximately 1.02 μM and 0.98 μM, and a notable selective index of >98 and > 102, respectively. This study gives valuable insight into developing therapeutic interventions against SARS-CoV-2 by targeting the N-protein, a significant effort given the global public health concern posed due to the virus re-emergence and long COVID-19 disease.

Exploring the Potential of Furofuran Lignans Isolated from Beilschmiedia pulverulenta for Drug Development: A Computational Approach

Natural products have played a significant role in drug discovery and continue to be an important source of lead for new drugs. In recent years, computer-based drug discovery methods have emerged as an effective approach for identifying small molecule leads with desirable pharmacokinetic and toxicity profiles. This study investigated the pharmacological and bioactivity of five furofuran lignans, namely, epiexcelsin, sesamin, sesartemin, syringaresinol, and yangambin, isolated from the plant Beilschmiedia pulverulenta. In silico studies were conducted to predict the pharmacological activities, toxicity, and drug likeliness properties of the lead compounds. The results showed that all compounds had promising pharmacokinetic activities, with epiexcelsin exhibiting strong binding affinity (-8.13 kcal mol-1) and inhibitory activity (1.1 µM) against estrogen receptor-α, and predicted to be bioavailable and effective lead. The findings of this study provide important insights into the potential therapeutic uses of natural medicinal plants and emphasize the potential of combining traditional medicinal knowledge with modern scientific approaches in drug discovery. Overall, the furofuran lignans isolated from Beilschmiedia pulverulenta represent a promising source of natural compounds for the development of effective drugs.

Clinical Utility of SARS-CoV-2 Antibody Titer Multiplied by Binding Avidity of Receptor-Binding Domain (RBD) in Monitoring Protective Immunity and Clinical Severity.

Conventional serum antibody titer, which expresses antibody level, does not provide antigen binding avidity of the variable region of the antibody, which is essential for the defense response to infection. Here, we quantified anti-SARS-CoV-2 antibody binding avidity to the receptor-binding domain (RBD) by competitive binding-inhibition activity (IC50) between SARS-CoV-2 S1 antigen immobilized on the DCP microarray and various RBD doses added to serum and expressed as 1/IC50 nM. The binding avidity analyzed under equilibrium conditions of antigen-antibody binding reaction is different from the avidity index measured with the chaotropic agent, such as urea, under nonequilibrium and short-time conditions. Quantitative determination of the infection-protection potential of antibodies was assessed by ABAT (antigen binding avidity antibody titer), which was calculated by the quantity (level) × quality (binding avidity) of antibodies. The binding avidity correlated strongly (r = 0.811) with cell-based virus-neutralizing activity. Maturation of the protective antibody induced by repeated vaccinations or SARS-CoV-2 infection was classified into three categories of ABAT, such as an initial, low, and high ABAT. Antibody maturity correlated with the clinical severity of COVID-19. Once a mature high binding avidity was achieved, it was maintained for at least 6-8 months regardless of the subsequent change in the antibody levels.

Application of Ligand- and Structure-Based Prediction Models for the Design of Alkylhydrazide-Based HDAC3 Inhibitors as Novel Anti-Cancer Compounds.

Histone deacetylases (HDAC) represent promising epigenetic targets for several diseases including different cancer types. The HDAC inhibitors approved to date are pan-HDAC inhibitors and most show a poor selectivity profile, side effects, and in particular hydroxamic-acid-based inhibitors lack good pharmacokinetic profiles. Therefore, the development of isoform-selective non-hydroxamic acid HDAC inhibitors is a highly regarded field in medicinal chemistry. In this study, we analyzed different ligand-based and structure-based drug design techniques to predict the binding mode and inhibitory activity of recently developed alkylhydrazide HDAC inhibitors. Alkylhydrazides have recently attracted more attention as they have shown promising effects in various cancer cell lines. In this work, pharmacophore models and atom-based quantitative structure-activity relationship (QSAR) models were generated and evaluated. The binding mode of the studied compounds was determined using molecular docking as well as molecular dynamics simulations and compared with known crystal structures. Calculated free energies of binding were also considered to generate QSAR models. The created models show a good explanation of in vitro data and were used to develop novel HDAC3 inhibitors.

Six-month immune responses to mRNA-1273 vaccine in combination antiretroviral therapy treated late presenter people with HIV according to previous SARS-CoV-2 infection.

Immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) mRNA vaccines in people with HIV (PWH) with a history of late presentation (LP) and their durability have not been fully characterized. In this prospective, longitudinal study, we sought to assess T-cell and humoral responses to SARS-CoV-2 mRNA vaccination up to 6 months in LP-PWH on effective combination antiretroviral therapy (cART) as compared to HIV-negative healthcare workers (HCWs), and to evaluate whether previous SARS-CoV-2 infection modulates immune responses to vaccine. SARS-CoV-2 spike (S)-specific T-cell responses were determined by two complementary flow cytometry methodologies, namely activation-induced marker (AIM) assay and intracellular cytokine staining (ICS), whereas humoral responses were measured by ELISA [anti-receptor binding domain (RBD) antibodies) and receptor-binding inhibition assay (spike-ACE2 binding inhibition activity), before vaccination (T0), 1 month (T1) and 5 months (T2) after the second dose. LP-PWH showed at T1 and T2 significant increase of: S-specific memory and circulating T follicular helper (cTfh) CD4 + T cells; polyfunctional Th1-cytokine (IFN-γ, TNF-α, IL-2)- and Th2-cytokine (IL-4)-producing S-specific CD4 + T cells; anti-RBD antibodies and spike-ACE2 binding inhibition activity. Immune responses to vaccine in LP-PWH were not inferior to HCWs overall, yet S-specific CD8 + T cells and spike-ACE2 binding inhibition activity correlated negatively with markers of immune recovery on cART. Interestingly, natural SARS-CoV-2 infection, while able to sustain S-specific antibody response, seems less efficacious in inducing a T-cell memory and in boosting immune responses to vaccine, possibly reflecting an enduring partial immunodeficiency. Altogether, these findings support the need for additional vaccine doses in PWH with a history of advanced immune depression and poor immune recovery on effective cART.

Structure-based drug design, molecular dynamics simulation, ADMET, and quantum chemical studies of some thiazolinones targeting influenza neuraminidase

The genetic mutability of the influenza virus leads to the existence of drug-resistant strains which is dangerous, particularly with the lingering coronavirus disease (COVID-19). This necessitated the need for the search and discovery of more potential anti-influenza agents to avert future outbreaks. In furtherance of our previous in-silico studies on 5-benzyl-4-thiazolinones as anti-influenza neuraminidase (NA) inhibitors, molecule 11 was selected as the template scaffold for the structure-based drug design due to its good binding, pharmacokinetic profiling, and better NA inhibitory activity. As such, eighteen (18) new molecules (11a-r) were designed with better MolDock scores as compared with the template scaffold and the zanamivir reference drug. However, the dynamic stability of molecule 11a in the binding cavity of the NA target (3TI5) showed water-mediated hydrogen and hydrophobic bondings with the active residues such as Arg118, Ile149, Arg152, Ile222, Trp403, and Ile427 after the MD simulation for 100 ns. The drug-likeness and ADMET assessment of all designed molecules predicted non-violation of the stipulated thresholds of Lipinski’s rule and good pharmacokinetic properties respectively. In addition, the quantum chemical calculations also suggested the significant chemical reactivity of molecules with their smaller band energy gap, high electrophilicity, high softness, and low hardness. The results obtained in this study proposed a reliable in-silico viewpoint for anti-influenza drug discovery and development.Communicated by Ramaswamy H. Sarma

Screening of anti‐thrombin active components from Ligusticum chuanxiong by affinity‐ultrafiltration coupled with HPLC‐Q‐Orbitrap‐MSn

Ligusticum chuanxiong ('chuanxiong') is a traditional Chinese medicine for promoting blood circulation and removing blood stasis, which is often used to treat thrombotic diseases. However, its potential anticoagulant active ingredients have been unexplored. The study aims to establish an affinity ultrafiltration mass spectrometry (AUF-MS) method for rapid screening of anti-thrombin active components of chuanxiong and to verify it in vitro. In this study, the chemical constituents of different parts of chuanxiong were determined. A method for rapid screening of anticoagulant active ingredients by AUF-MS was established using thrombin as an affinity receptor target. Subsequently, the anticoagulant effect of such ligands was verified by in vitro anticoagulation experiments such as chromogenic substrate method and in vitro coagulation assay. Then the possible interaction mechanism between these ligands and thrombin was further studied by molecular docking. Twenty-one components were detected from different parts of chuanxiong. And three potential anti-thrombin active components were screened: ferulic acid, chlorogenic acid, isochlorogenic acid A by AUF coupled with high-performance liquid chromatography-quadrupole-Orbitrap mass spectrometry (HPLC-Q-Orbitrap-MSn ). The in vitro activity experiments and molecular docking revealed that these potential ligands exhibited strong binding ability and inhibitory activities on thrombin. The present study revealed that chuanxiong is a traditional Chinese medicine with excellent anticoagulation effects. Meanwhile, the integrated strategy based on AUF-MS, in vitro experiments and molecular docking also provided a powerful tool for further exploration of active ingredients responsible for the anticoagulant activity in chuanxiong.

Skullcapflavone II, a novel NQO1 inhibitor, alleviates aristolochic acid I-induced liver and kidney injury in mice.

Aristolochic acid I (AAI) is a well established nephrotoxin and human carcinogen. Cytosolic NAD(P)H quinone oxidoreductase 1 (NQO1) plays an important role in the nitro reduction of aristolochic acids, leading to production of aristoloactam and AA-DNA adduct. Application of a potent NQO1 inhibitor dicoumarol is limited by its life-threatening side effect as an anticoagulant and the subsequent hemorrhagic complications. As traditional medicines containing AAI remain available in the market, novel NQO1 inhibitors are urgently needed to attenuate the toxicity of AAI exposure. In this study, we employed comprehensive 2D NQO1 biochromatography to screen candidate compounds that could bind with NQO1 protein. Four compounds, i.e., skullcapflavone II (SFII), oroxylin A, wogonin and tectochrysin were screened out from Scutellaria baicalensis. Among them, SFII was the most promising NQO1 inhibitor with a binding affinity (KD = 4.198 μmol/L) and inhibitory activity (IC50 = 2.87 μmol/L). In human normal liver cell line (L02) and human renal proximal tubular epithelial cell line (HK-2), SFII significantly alleviated AAI-induced DNA damage and apoptosis. In adult mice, oral administration of SFII dose-dependently ameliorated AAI-induced renal fibrosis and dysfunction. In infant mice, oral administration of SFII suppressed AAI-induced hepatocellular carcinoma initiation. Moreover, administration of SFII did not affect the coagulation function in short term in adult mice. In conclusion, SFII has been identified as a novel NQO1 inhibitor that might impede the risk of AAI to kidney and liver without obvious side effect.

Lead Optimization and Avoidance of Metabolic-perturbing Motif Developing Novel Diarylpyrimidines as Potent HIV-1 NNRTIs.

Non-nucleoside reverse transcriptase inhibitors (NNRTIs) represent an indispensable part of anti-HIV-1 therapy. To discover novel HIV-1 NNRTIs with increased drug resistance profiles and improved pharmacokinetic (PK) properties, a series of novel diarylpyrimidine derivatives were generated via the cocrystal structure-based drug design strategy. Among them, 36a exhibited outstanding antiviral activity against HIV-1 IIIB and a panel of mutant strains (L100I, K103N, Y181C, Y188L, E138K, F227L + V106A, and RES056), with EC50 ranging from 2.22 to 53.3 nM. Besides, 36a was identified with higher binding affinity (KD = 2.50 μM) and inhibitory activity (IC50 = 0.03 μM) to HIV-1 RT. Molecular docking and molecular dynamics simulation were performed to rationalize the design and the improved drug resistance of these novel inhibitors. Additionally, 36a·HCl exhibited favorable PK (T1/2 = 5.12 h, F = 12.1%) and safety properties (LD50 > 2000 mg/kg). All these suggested that 36a·HCl may serve as a novel drug candidate anti-HIV-1 therapy.

Effect of booster vaccination against Delta and Omicron SARS-CoV-2 variants in Iceland

By the end of July 2021, the majority of the Icelandic population had received vaccination against COVID-19. In mid-July a wave of SARS-CoV-2 infections, dominated by the Delta variant, spread through the population, followed by an Omicron wave in December. A booster vaccination campaign was initiated to curb the spread of the virus. We estimate the risk of infection for different vaccine combinations using vaccination data from 276,028 persons and 963,557 qPCR tests for 277,687 persons. We measure anti-Spike-RBD antibody levels and ACE2-Spike binding inhibitory activity in 371 persons who received one of four recommended vaccination schedules with or without an mRNA vaccine booster. Overall, we find different antibody levels and inhibitory activity in recommended vaccination schedules, reflected in the observed risk of SARS-CoV-2 infections. We observe an increased protection following mRNA boosters, against both Omicron and Delta variant infections, although BNT162b2 boosters provide greater protection against Omicron than mRNA-1273 boosters.

Glycan-Adhering Lectins and Experimental Evaluation of a Lectin FimH Inhibitor in Enterohemorrhagic Escherichia coli (EHEC) O157:H7 Strain EDL933.

In this study, we tried to develop a FimH inhibitor that inhibits adhesion of enterohemorrhagic Escherichia coli (EHEC) on the epithelium of human intestine during the initial stage of infections. Using a T7 phage display method with a reference strain, EHEC EDL933, FimH was selected as an adherent lectin to GM1a and Gb3 glycans. In order to detect the ligand binding domain (LBD) of FimH, we used a docking simulation and found three binding site sequences of FimH, i.e., P1, P2, and P3. Among Gb3 mimic peptides, P2 was found to have the strongest binding strength. Moreover, in vitro treatment with peptide P2 inhibited binding activity in a concentration-dependent manner. Furthermore, we conducted confirmation experiments through several strains isolated from patients in Korea, EHEC NCCP15736, NCCP15737, and NCCP15739. In addition, we analyzed the evolutionary characteristics of the predicted FimH lectin-like adhesins to construct a lectin-glycan interaction (LGI). We selected 70 recently differentiated strains from the phylogenetic tree of 2240 strains with Shiga toxin in their genome. We can infer EHEC strains dynamically evolved but FimH was conserved during the evolution time according to the phylogenetic tree. Furthermore, FimH could be a reliable candidate of drug target in terms of evolution. We examined how pathogen lectins interact with host glycans early in infection in EDL933 as well as several field strains and confirmed that glycan-like peptides worked as an initial infection inhibitor.

Antioxidant, Anti-Proliferative Activity and Chemical Fingerprinting of Centaurea calcitrapa against Breast Cancer Cells and Molecular Docking of Caspase-3.

Centaurea calcitrapa has been intensively utilized in ethnomedicinal practices as a natural therapeutic recipe to cure various ailments. The current study aimed to chemically characterize ethanolic extract of C. calcitrapa (EECC) aerial parts (leaves and shoots) by use of gas chromatography-mass spectrometry analyses (GC-MS) and investigate its antioxidant and in vitro anticancer activities, elucidating the underlying molecular mechanism by use of flow cytometry-based fluorescence-activated cell sorting (FACS) and conducting in silico assessment of binding inhibitory activities of EECC major compounds docked to caspase-3. CG-MS profiling of EECC identified a total of 26 major flavonoids and polyphenolic compounds. DPPH and ABTS assays revealed that EECC exhibits potent antioxidant activity comparable to standard reducing agents. Results of the proliferation assay revealed that EECC exhibit potent, dose-dependent cytotoxic activities against triple-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cell models, with IC50 values of 1.3 × 102 and 8.7 × 101 µg/mL, respectively. The observed cytotoxic effect was specific to studied cancer cells since EECC exhibited minimal (~<10%) cytotoxicity against MCF-12, a normal breast cell line. FACS analysis employing annexin V-FITC/propidium iodide double labeling demonstrated that the observed anti-proliferative activity against MCF-7 and MDA-MB-231 was mediated via apoptotic as well as necrotic signaling transduction processes. The increase in fluorescence intensity associated with DCFH oxidation to DCF, as reported by FACS, indicated that apoptosis is caused by generation of ROS. The use of caspase-3-specific fluorogenic substrate revealed a dose-dependent elevation in caspase-3 substrate-cleavage activity, which further supports EECC-mediated apoptosis in MCF-7 cells. The major EECC compounds were examined for their inhibitory activity against caspase-3 receptor (1HD2) using molecular docking. Three compounds exhibited the highest glide score energy of −5.156, −4.691 and −4.551 kcal/mol, respectively. Phenol, 2,6-dimethoxy established strong binding in caspase-3 receptor of hydrogenic type, with residue ARG 207 and of PI-PI stacking type with residue HIS 121. By contract, hexadecenoic acid showed 3 H-bond with the following residues: ASN 615, ASN 616a and THR 646. Taken together, the current findings reveal that EECC exhibits significant and specific cytotoxicity against breast cancer cells mediated by the generation of ROS and culminating into necrosis and apoptosis. Further investigations of the phytoconstituents-rich C. calcitrapa are therefore warranted against breast as well as other human cancer cell models.

Anticholinesterase Inhibition, Drug-Likeness Assessment, and Molecular Docking Evaluation of Milk Protein-Derived Opioid Peptides for the Control of Alzheimer’s Disease

The drug-likeness and pharmacokinetic properties of 23 dairy-protein-derived opioid peptides were studied using SwissADME and ADMETlab in silico tools. All the opioid peptides had poor drug-like properties based on violations of Lipinski’s rule-of-five. Moreover, prediction of their pharmacokinetic properties showed that the peptides had poor intestinal absorption and bioavailability. Following this, two well-known opioid peptides (βb-casomorphin-5, βb-casomorphin-7) from A1 bovine milk and caffeine (positive control) were selected for in silico molecular docking and in vitro inhibition study with two cholinesterase enzyme receptors important for the pathogenesis of Alzheimer’s disease. Both peptides showed higher binding free energies and inhibitory activities to butyrylcholinesterase (BChE) than caffeine, but in vitro binding energy values were lower than those from the docking model. Moreover, the two casomorphins had lower inhibitory properties against acetylcholinesterase (AChE) than caffeine, although the docking model predicted the opposite. At 1 mg/mL concentrations, βb-casomorphin-5 and βb-casomorphin-7 showed promising results in inhibiting both cholinesterases (i.e., respectively 34% and 43% inhibition of AChE, and 67% and 81% inhibition of BChE). These dairy-derived opioid peptides have the potential to treat Alzheimer’s disease via cholinesterase inhibition. However, appropriate derivatization may be required to improve their poor predicted intestinal absorption and bioavailability.

New acylglycosides from the roots of Rheum palmatum L.

A phytochemical investigation of the EtOH extract of the roots of Rheum palmatum L. was performed, leading to the isolation of 3 new acylglycosides ( 1 – 3 ) and 19 previously reported compounds, including 4 cinnamoyl glucosides ( 4 – 7 ) and 15 anthraquinones ( 8 – 22 ). Their structures were elucidated using nuclear magnetic resonance, infrared, ultraviolet and high-resolution electrospray ionization mass spectra and X-ray diffraction data. The biological activities of compounds 2 , 4 – 9 , and 11 – 13 , including their lymphocyte activation gene 3 (LAG3)/major histocompatibility complex class II (MHC II) and LAG3/fibrinogen-like protein 1 (FGL1) binding inhibition activities, were evaluated. Among the isolates, compound 7 exhibited clear inhibitory activity against the LAG3/FGL1 interaction with an IC 50 of 7.89 μ M, meanwhile compound 6 inhibited the LAG3/MHC II interaction with an IC 50 of 6.00 μ M. • Three undescribed acylglycosides were isolated from roots of Rheum palmatum L. • The absolute configuration of 1 was confirmed by single crystal X-ray diffraction. • This is the first report of the crystal structure of a cinnamoyl glucoside. • Their activities against the interaction of LAG3 with MHC II or FGL1 were evaluated.