Proposed Mechanism for Emodin as Agent for Methicillin Resistant Staphylococcus Aureus: In Vitro Testing and In Silico Study.

In the search for a new anti-MRSA lead compound, emodin was identified as a good lead against methicillin-resistant Staphylococcus aureus (MRSA). Emodin serves as a new scaffold to design novel and effective anti-MRSA agents. Because rational drug discovery is limited by the knowledge of the drug target, α-hemolysin of Staphylococcus aureus was used in this study because it has an essential role in Staphylococcus infections and because emodin shares structural features with compounds that target this enzyme. In order to explore emodin's interactions with α-hemolysin, all possible ligand binding pockets were identified and investigated. Two ligand pockets were detected based on bound ligands and other reports. The third pocket was identified as a cryptic site after molecular dynamics (MD) simulations. MD simulations were conducted for emodin in each pocket to identify the most plausible ligand site and to aid in the design of potent anti-MRSA agents. Binding of emodin to site 1 was most stable (RMSD changes within 1 Å), while in site 2, the binding pose of emodin fluctuated, and it left after 20 ns. In site 3, it was stable during the first 50 ns, and then it started to move out of the binding site. Site 1 is a possible ligand binding pocket, and this study sheds more light on interaction types, binding mode, and key amino acids involved in ligand binding essential for better lead design. Emodin showed an IC50 value of 6.3 µg/mL, while 1, 6, and 8 triacetyl emodin showed no activity against MRSA. A molecular modeling study was pursued to better understand effective binding requirements for a lead.

Discovery of novel class of histone deacetylase inhibitors as potential anticancer agents.

Selective inhibition of histone deacetylases (HDACs) is an important strategy in the field of anticancer drug discovery. However, lack of inhibitors that possess high selectivity toward certain HDACs isozymes is associated with adverse side effects that limits their clinical applications. We have initiated a collaborative initiatives between multi-institutions aimed at the discovery of novel and selective HDACs inhibitors. To this end, a phenotypic screening of an in-house pilot library of about 70 small molecules against various HDAC isozymes led to the discovery of five compounds that displayed varying degrees of HDAC isozyme selectivity. The anticancer activities of these molecules were validated using various biological assays including transcriptomic studies. Compounds 15, 14, and 19 possessed selective inhibitory activity against HDAC5, while 28 displayed selective inhibition of HDAC1 and HDAC2. Compound 22 was found to be a selective inhibitor for HDAC3 and HDAC9. Importantly, we discovered a none-hydroxamate based HDAC inhibitor, compound 28, representing a distinct chemical probe of HDAC inhibitors. It contains a trifluoromethyloxadiazolyl moiety (TFMO) as a non-chelating metal-binding group. The new compounds showed potent anti-proliferative activity when tested against MCF7 breast cancer cell line, as well as increased acetylation of histones and induce cells apoptosis. The new compounds apoptotic effects were validated through the upregulation of proapoptotic proteins caspases3 and 7 and downregulation of the antiapoptotic biomarkers C-MYC, BCL2, BCL3 and NFĸB genes. Furthermore, the new compounds arrested cell cycle at different phases, which was confirmed through downregulation of the CDK1, 2, 4, 6, E2F1 and RB1 proteins. Taken together, our findings provide the foundation for the development of new chemical probes as potential lead drug candidates for the treatment of cancer.

Proposed Mechanism for the Antitrypanosomal Activity of Quercetin and Myricetin Isolated from Hypericum afrum Lam.: Phytochemistry, In Vitro Testing and Modeling Studies.

The in vitro activity of L. donovani (promastigotes, axenic amastigotes and intracellular amastigotes in THP1 cells) and T. brucei, from the fractions obtained from the hydroalcoholic extract of the aerial part of Hypericum afrum and the isolated compounds, has been evaluated. The chloroform, ethyl acetate and n-butanol extracts showed significant antitrypanosomal activity towards T. brucei, with IC50 values of 12.35, 13.53 and 12.93 µg/mL and with IC90 values of 14.94, 19.31 and 18.67 µg/mL, respectively. The phytochemical investigation of the fractions led to the isolation and identification of quercetin (1), myricitrin (2), biapigenin (3), myricetin (4), hyperoside (5), myricetin-3-O-ß-d-galactopyranoside (6) and myricetin-3'-O-ß-d-glucopyranoside (7). Myricetin-3'-O-ß-d-glucopyranoside (7) has been isolated for the first time from this genus. The chemical structures were elucidated by using comprehensive one- and two-dimensional nuclear magnetic resonance (1D and 2D NMR) spectroscopic data, as well as high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). These compounds have also been evaluated for their antiprotozoal activity. Quercetin (1) and myricetin (4) showed noteworthy activity against T. brucei, with IC50 and IC90 values of 7.52 and 5.71 µM, and 9.76 and 7.97 µM, respectively. The T. brucei hexokinase (TbHK1) enzyme was further explored as a potential target of quercetin and myricetin, using molecular modeling studies. This proposed mechanism assists in the exploration of new candidates for novel antitrypanosomal drugs.

Rational design and synthesis of new tetralin-sulfonamide derivatives as potent anti-diabetics and DPP-4 inhibitors: 2D & 3D QSAR, in vivo radiolabeling and bio distribution studies.

Type 2 diabetes (T2D) is a severe disease and it is one of the most raising problems worldwide. This study deals with design, synthesis and in vivo determination of a new set of tetralin-sulfonamide derivatives as anti-diabetic and dipeptidyl peptidase-IV (DPP-4) inhibiting agents. Most of the new compounds exhibited significant hypoglycemic effect alongside with DPP-4 suppression potency considering sitagliptin as a reference drug. The most promising compounds 4, 15 showed 2.80 nM DPP-4 IC50 with 20-40 folds selectivity over DPP-8 and DPP-9. 2D and 3D QSAR models were performed using auto QSAR of Schrödinger, QuaSAR of MOE and 3D Field-based QSAR of Schrödinger, respectively. The experimental results revealed that the alignment-independent descriptors, electrostatic and steric field descriptors were significantly correlated with the antidiabetic activity of the new derivatives. In addition, the new compounds were docked in the active site of DPP-4 in reference to sitagliptin to rationalize the binding modes of the compounds with the amino acid residues of the enzyme. Furthermore, 131I-compound 4 complex was selected to evaluate the pharmacokinetic behavioral profile of compound 4 and its body organs uptakes alongside its elimination pathway as a representative example for the rest of the analogues. The bio distribution pattern of the tracer proved the selective accumulation of 131I-substrate in the pancreas and rapid clearance from most of the body organs.

Damage Resistance of Kevlar® Fabric, UHMWPE, PVB Multilayers Subjected to Concentrated Drop-Weight Impact.

The impact resistance of layered polymer structures using polyvinyl butyral (PVB) in combination with Kevlar® fabric and ultra-high molecular weight polyethylene (UHMWPE) were fabricated and tested. Methods of wet impregnation and hot-press impregnation and consolidation of fabric with PVB and UHMWPE were used to manufacture multilayer constructs. All sandwich constructs were fixed to the surface of ballistic clay and subject to a free drop-weight test with a conical impactor having a small contact area. All tests were made at the same impact energy of 9.3 J and velocity of 2.85 m/s. The change in the resistance force was recorded using a piezoelectric force sensor at the time intervals of 40 µs. Using experimental force-time history, the change in the impactor's velocity, the depth of impactor penetration, the energy transformation at various stages of impactor interaction with the sample, and other parameters were obtained. Three indicators were considered as the main criteria for the effectiveness of a sample's resistance to impact: (1) minimum deformation, bulging, of the panel backside at the moment of impact, (2) minimum absorption of impact energy per areal density, and (3) minimal or, better yet, no destruction of structural integrity. Under the tested conditions, the rigid Kevlar-PVB-Kevlar sandwich at the frontside and relatively soft but flexible UHMWPE-Kevlar-UHMWPE layers in the middle helped to localize and absorb impact energy, while the backside Kevlar-PVB-Kevlar sandwich minimized local bulging providing the best overall performance. The front layer damage area was very shallow and less than two impactor tip diameters. The backside bulging was also less than in any other tested configurations.

ACE2 Activation by Tripeptide IRW (Ile-Arg-Trp) Depends on the G Protein-Coupled Receptor 30 Signaling Cascade.

This study aimed to understand how specific cell-bound receptors influence ACE2 activation by IRW. Our results showed that G protein-coupled receptor 30 (GPR30), a 7-transmembrane domain protein, was involved in IRW-mediated ACE2 increase. IRW treatment (50 µM) significantly increased the GPR30 pool levels (3.2 ± 0.5 folds) (p < 0.001). IRW treatment also boosted the consecutive GEF (guanine nucleotide exchange factor) activity (2.2 ± 0.2 folds) (p < 0.001), and GNB1 levels (2.0 ± 0.5 folds) (p < 0.05), associated with the functional subunits of G proteins, in cells. These results were translated in hypertensive animal studies as well (p < 0.05), indicated by an increase in the aortal levels of GPR30 (p < 0.01); further experiments showed an increase in downstream PIP3/PI3K/Akt pathway activation following IRW treatment. The blockade of GPR30 by an antagonist and siRNA in cells abolished the ACE2-activating ability of IRW, as shown by the depleted levels of ACE2 mRNA (p < 0.001), protein levels in whole cells and membrane, angiotensin (1-7) (p < 0.01), and ACE2 promoter HNF1α (p < 0.05). Finally, the GPR30 blockade in ACE2-overexpressing cells using the antagonist (p < 0.01) and siRNA (p < 0.05) significantly depleted the innate cellular pool of ACE2, thus confirming the relationship between the membrane-bound GPR30 and ACE2. Overall, these results showed that the vasodilatory peptide IRW could activate ACE2 via the membrane-bound receptor GPR30.

Structurally Modified Bioactive Peptide Inhibits SARS-CoV-2 Lentiviral Particles Expression.

Coronavirus disease 2019 (COVID-19), the current global pandemic is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Various pharmaceuticals are being developed to counter the spread of the virus. The strategy of repurposing known drugs and bioactive molecules is a rational approach. A previously described molecule, Ile-Arg-Trp (IRW), is a bioactive tripeptide that exhibits an ability to boost angiotensin converting enzyme-2 (ACE2) expression in animals and cells. Given the importance of SARS-CoV-2 S receptor binding domain (RBD)-ACE2 interaction in SARS-CoV-2 pathophysiology, we synthesized various IRW analogs intending to mitigate the RBD-ACE-2 interaction. Herein, we describe two analogs of IRW, A9 (Acetyl-Ile-Arg-Trp-Amide) and A14 (Formyl-Ile-Arg-Trp-Amide) which lowered the SARS-CoV-2 S RBD-ACE2 (at 50 µM) in vitro. The free energy of binding suggested that A9 and A14 interacted with the SARS-CoV-2 S RBD more favorably than ACE2. The calculated MMGBSA ΔG of spike binding for A9 was -57.22 kcal/mol, while that of A14 was -52.44 kcal/mol. A14 also inhibited furin enzymatic activity at various tested concentrations (25, 50, and 100 µM). We confirmed the effect of the two potent analogs using SARS-CoV-2 spike protein overexpressing cells. Both peptides lowered the protein expression of SARS-CoV-2 spike protein at the tested concentration (50 µM). Similarly, both peptides, A9 and A14 (50 µM), also inhibited pseudotyped lentiviral particles with SARS-CoV-2 Spike in ACE2 overexpressing cells. Further, the molecular dynamics (MD) calculations showed the interaction of A9 and A14 with multiple residues in spike S1 RBD. In conclusion, novel peptide analogs of ACE2 boosting IRW were prepared and confirmed through in vitro, cellular, and computational evaluations to be potential seed candidates for SARS-CoV-2 host cell binding inhibition.

Computationally Assisted Lead Optimization of Novel Potent and Selective MAO-B Inhibitors.

A series of dietary flavonoid acacetin 7-O-methyl ether derivatives were computationally designed aiming to improve the selectivity and potency profiles against monoamine oxidase (MAO) B. The designed compounds were evaluated for their potential to inhibit human MAO-A and -B. Compounds 1c, 2c, 3c, and 4c were the most potent with a Ki of 37 to 68 nM against MAO-B. Compounds 1c-4c displayed more than a thousand-fold selectivity index towards MAO-B compared with MAO-A. Moreover, compounds 1c and 2c showed reversible inhibition of MAO-B. These results provide a basis for further studies on the potential application of these modified flavonoids for the treatment of Parkinson's Disease and other neurological disorders.

Purification, Characterization, Identification, and Anticancer Activity of a Circular Bacteriocin From Enterococcus thailandicus.

New anticancer agents are continually needed because cancerous cells continue to evolve resistance to the currently available chemotherapeutic agents. The aim of the present study was to screen, purify and characterize a hepatotoxic bacteriocin from Enterococcus species. The production of bacteriocin from the Enterococcus isolates was achieved based on their antibacterial activity against indicator reference strains. Enterococcus isolates showed a broad spectrum of antibacterial activity by forming inhibition zones with diameters ranged between 12 and 29 mm. The most potent bacteriocin producing strain was molecularly identified as Enterococcus thailandicus. The crude extracted bacteriocin was purified by cation exchange and size exclusion chromatography that resulted in 83 fractions. Among them, 18 factions were considered as bacteriocins based on their positive antibacterial effects. The anticancer effects of the purified bacteriocins were tested against HepG2 cell line. The most promising enterocin (LNS18) showed the highest anticancer effects against HepG2 cells (with 75.24% cellular inhibition percentages), with IC50 value 15.643 µM and without any significant cytotoxic effects on normal fibroblast cells (BJ ATCC® CRL-2522™). The mode of anticancer action of enterocin LNS18 against HepG2 cells could be explained by its efficacy to induce cellular ROS, decrease HepG2 CD markers and arrest cells in G0 phase. Amino acid sequence of enterocin LNS18 was determined and the deduced peptide of the structural gene showed 86 amino acids that shared 94.7% identity with enterocin NKR-5-3B from E. faecium. Enterocin LNS18 consisted of 6 α-helices; 5 circular and one linear. Model-template alignment constructed between enterocin LNS18 and NKR-5-3B revealed 95.31% identity. The predicted 3D homology model of LNS18, after circularization and release of 22 amino acids, showed the formation of a bond between Leu23 and Trp86 amino acid residues at the site of circularization. Furthermore, areas of positive charges were due to the presence of 6 lysine residues resulting in a net positive charge of +4 on the bacteriocin surface. Based on the above mentioned results, our characterized bacteriocin is a promising agent to target liver cancer without any significant toxic effects on normal cell lines.

Computationally aided stereochemical assignment of undescribed bisabolenes from Calea urticifolia.

Calea urticifolia (Mill.) DC. (Compositae) is a medicinal plant found in El Salvador. Calea is used in folkloric medicine as a psychoactive principle with calming effect, as well as in the treatment of diarrhea and fever. Three undescribed bisabolenes, named caleanolenes A-C, as well as, three known sesquiterpene lactones 2,3-epoxyjuanislamin, calealactone B, calein C, and the flavonoid acacetin, were isolated from the chloroform extract of the leaves of C. urticifolia. The chemical structures of the isolated compounds were determined on the basis of HRMS, IR, CD, and from 1D and 2D NMR spectroscopic studies. The absolute configurations of the caleanolenes have been partly established using GIAO NMR and ECD calculations. The isolated compounds were evaluated for cytotoxicity against the CA46 and Raji lymphoma, and the MCF7 breast cancer cell lines, with 2,3-epoxyjuanislamin showing the best activity in all cell lines (IC50 value range 2.9-12.3 µM).

Secondary metabolites from the aerial parts of Cytisus villosus Pourr.

Phytochemical investigation of the aerial parts of Cytisus villosus Pourr. resulted in the isolation and characterization of a new isoflavan, (3S, 4S)-2',4'-dihydroxy-3'-methoxy-6,7-methylenedioxyisoflavan- 4-ol (1), and a new monoterpene, (4R,6S)-4-hydroxy-2,2,6-trimethyl-9-oxabicyclo [4.2.1] non-1(8)-en-7-one (2), together with four known flavonoids: geinstein (3), chrysin (4), chrysin -7-O-ß-D-glucopyranoside (5) and 2″-O-α-L-rhamnosylorientin (6). The structures of the new compounds were elucidated on the basis of extensive spectroscopic analysis, including 1D, 2D NMR (1H, 13C, COSY, TOCSY, HMBC and HSQC) and HRESIMS. The absolute configurations of 1 and 2 were established by the comparison of experimental and calculated electronic circular dichroism (ECD) spectra.

Inhibition of human monoamine oxidase A and B by flavonoids isolated from two Algerian medicinal plants.

BACKGROUND: Monoamine oxidases (MAOs) are outer mitochondrial membrane flavoenzymes. They catalyze the oxidative deamination of a variety of neurotransmitters. MAO-A and MAO-B may be considered as targets for inhibitors to treat neurodegenerative diseases and depression and for managing symptoms associated with Parkinson's and Alzheimer's diseases. PURPOSE: The objective was to evaluate the inhibitory effect of Hypericum afrum and Cytisus villosus against MAO-A and B and to isolate the compounds responsible for the MAO-inhibitory activity. METHODS: The inhibitory effect of extracts and purified constituents of H. afrum and C. villosus were investigated in vitro using recombinant human MAO-A and B, and through bioassay-guided fractionation of ethyl acetate fractions of areal parts of the two plants collected in northeastern Algeria. In addition, computational protein-ligand docking and molecular dynamics simulations were carried out to explain the MAO binding at the molecular level. RESULTS: The ethyl acetate (EtOAc) fractions of H. afrum and C. villosus showed the highest MAO inhibition activity against MAO A and B with IC50 values of 3.37 µg/ml and 13.50 µg/ml as well as 5.62 and 1.87 µg/ml, respectively. Bioassay-guided fractionation of the EtOAc fractions resulted in the purification and identification of the known flavonoids quercetin, myricetin, genistein and chrysin as the principal MAO-inhibitory constituents. Their structures were established by extensive 1 and 2D NMR studies and mass spectrometry. Quercetin, myricetin and chrysin showed potent inhibitory activity towards MAO-A with IC50 values of 1.52, 9.93 and 0.25 µM, respectively, while genistein more efficiently inhibited MAO-B (IC50 value: 0.65 µM). The kinetics of the inhibition and the study of dialysis dissociation of the complex of quercetin and myricetin and the isoenzyme MAO-A showed competitive and mixed inhibition, respectively. Both compounds showed reversible binding. Molecular docking experiments and molecular dynamics simulations allowed to estimate the binding poses and to identify the most important residues involved in the selective recognition of molecules in the MAOs enzymatic clefts. CONCLUSION: Quercetin and myricetin isolated from H. afrum together with genistein and chrysin isolated from C. villosus have been identified as potent MAO-A and -B inhibitors. H. afrum and C. villosus have properties indicative of potential neuroprotective ability and may be new candidates for selective MAO-A and B inhibitors.

Nitroreductase-triggered activation of a novel caged fluorescent probe obtained from methylene blue.

A near-infrared fluorescent probe based on methylene blue (p-NBMB) was developed for the detection of nitroreductase. Conjugating methylene blue with a p-nitrobenzyl moiety enables it to be activated by nitroreductase-catalyzed 1,6-elimination, resulting in the release of an active methylene blue fluorophore.