A New Immunoassay for Detecting All Subtypes of Shiga Toxins Produced by Shiga Toxin-Producing E. coli in Ground Beef

Background

Shiga toxin (Stx) is a common virulence factor of all Shiga toxin producing E. coli (STEC) that cause a wide spectrum of disease, including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Although several commercial kits are available for detection of Stx produced by STEC, none of them are capable of recognizing all subtypes of Stxs, which include three subtypes of Stx1 and seven subtypes of Stx2.

Methods and Findings

New monoclonal and polyclonal antibodies against Stx1 and Stx2 were developed. A universal sandwich ELISA capable of detecting all known subtypes of Stx1 and Stx2 was established using a pool of newly developed antibodies. To precisely monitor the sensitivity of the assay for each subtype of Stxs, recombinant toxoids were created and used as standards in ELISAs. Because of the high affinity of the antibodies incorporated, the ELISA assay is highly sensitive with a limit of detection for the different subtypes of Stx1a and Stx2a between 10 and 50 pg/mL in phosphate buffered saline (PBS). The assay was also able to identify STEC based on the production of Stxs using the supernatants of culture fluids or even single colonies on agar plates without lengthy enrichment in liquid medium. When applied to ground beef samples, this newly developed ELISA was capable of distinguishing beef samples spiked with a single bacterial cell.

Conclusions

A highly sensitive and universal assay for all subtypes of Stx1 and Stx2 was developed. It has significantly improved upon the current technologies by avoiding false negative results due to the narrow detection range of the assay. The assay developed in this study can be useful for prompt detection of new and emerging serotypes and screening ground beef samples for contamination of STEC at an early stage in the food supply chain, thus avoiding the need for possible recall.     

Shiga toxins induce autophagic cell death in intestinal epithelial cells via the endoplasmic reticulum stress pathway

Shiga toxins (Stxs) are a family of cytotoxic proteins that lead to the development of bloody diarrhea, hemolytic-uremic syndrome, and central nervous system complications caused by bacteria such as S. dysenteriae, E. coli O157:H7 and E. coli O104:H4. Increasing evidence indicates that macroautophagy (autophagy) is a key factor in the cell death induced by Stxs. However, the associated mechanisms are not yet clear. This study showed that Stx2 induces autophagic cell death in Caco-2 cells, a cultured line model of human enterocytes. Inhibition of autophagy using pharmacological inhibitors, such as 3-methyladenine and bafilomycin A₁, or silencing of the autophagy genes ATG12 or BECN1 decreased the Stx2-induced death in Caco-2 cells. Furthermore, there were numerous instances of dilated endoplasmic reticulum (ER) in the Stx2-treated Caco-2 cells, and repression of ER stress due to the depletion of viable candidates of DDIT3 and NUPR1. These processes led to Stx2-induced autophagy and cell death. Finally, the data showed that the pseudokinase TRIB3-mediated DDIT3 expression and AKT1 dephosphorylation upon ER stress were triggered by Stx2. Thus, the data indicate that Stx2 causes autophagic cell death via the ER stress pathway in intestinal epithelial cells.     

Exosomes released from Shiga toxin 2a–treated human macrophages modulate inflammatory responses and induce cell death in toxin receptor expressing human cells

Shiga toxins (Stxs) produced by Stx‐producing Escherichia coli are the primarily virulence factors of hemolytic uremic syndrome and central nervous system (CNS) impairment. Although the precise mechanisms of toxin dissemination remain unclear, Stxs bind to extracellular vesicles (EVs). Exosomes, a subset of EVs, may play a key role in Stx‐mediated renal injury. To test this hypothesis, we isolated exosomes from monocyte‐derived macrophages in the presence of Stx2a or Stx2 toxoids. Macrophage‐like differentiated THP‐1 cells treated with Stxs secreted Stx‐associated exosomes (Stx‐Exo) of 90–130 nm in diameter, which induced cytotoxicity in recipient cells in a toxin receptor globotriaosylceramide (Gb₃)‐dependent manner. Stx2‐Exo engulfed by Gb₃‐positive cells were translocated to the endoplasmic reticulum in the human proximal tubule epithelial cell line HK‐2. Stx2‐Exo contained pro‐inflammatory cytokine mRNAs and proteins and induced more severe inflammation than purified Stx2a accompanied by greater death of target cells such as human renal or retinal pigment epithelial cells. Blockade of exosome biogenesis using the pharmacological inhibitor GW4869 reduced Stx2‐Exo‐mediated human renal cell death. Stx2‐Exo isolated from human primary monocyte–derived macrophages activated caspase 3/7 and resulted in significant cell death in primary human renal cortical epithelial cells. Based on these results, we speculate that Stx‐containing exosomes derived from macrophages may exacerbate cytotoxicity and inflammation and trigger cell death in toxin‐sensitive cells. Therapeutic interventions targeting Stx‐containing exosomes may prevent or ameliorate Stx‐mediated acute vascular dysfunction.     

Selective inhibition of monoamine oxidase A by chelerythrine,an isoquinoline alkaloid

Chelerythrine, an isoquinoline alkaloid isolated from the herbaceous perennial Chelidonium majus, was found to potently and selectively inhibit an isoform of recombinant human monoamine oxidase-A (MAO-A) with an IC₅₀ value of 0.55?µM. Chelerythrine was a reversible competitive MAO-A inhibitor (K_i?=?0.22?µM) with a potency much greater than toloxatone (IC₅₀?=?1.10?µM), a marketed drug. Other isoquinoline alkaloids tested did not effectively inhibit MAO-A or MAO-B. A structural comparison with corynoline suggested the 1- and/or 2-methoxy groups of chelerythrine increase its inhibitory activity against MAO-A. Molecular docking simulations revealed that the binding affinity of chelerythrine for MAO-A (?9.7?kcal/mol) was greater than that for MAO-B (?4.6?kcal/mol). Docking simulation implied that Cys323 and Tyr444 of MAO-A are key residues for hydrogen-bond interaction with chelerythrine. Our findings suggest chelerythrine is one of the most reversible selective and potent natural inhibitor of MAO-A, and that it be regarded a potential lead compound for the design of novel reversible MAO-A inhibitors.     

Carbosilane dendrimers bearing globotriaoses: construction of a series of carbosilane dendrimers bearing globotriaoses

To enhance biological activities on the basis of the sugar cluster effect, a series of carbosilane dendrimers as core scaffolds for the construction of glycodendrimers was systematically synthesized from appropriate chlorosilanes by a combination of alkenylation and hydrosylation reactions. Those carbosilane dendrimers having terminal C=C double bonds underwent general hydroboration reactions to give corresponding primary polyols. Further transformations of the alcohols were then performed by mesylation followed by a displacement with NaBr to provide corresponding dendrimers with 4 to 36 bromine atoms at each terminal end. Assembly of trisaccharide moieties of globotriaosyl ceramide using alkyl halide-type carbosilane dendrimers as the core frame was conducted in liquid ammonia by a one-pot reaction involving selective removal of a benzyl group under the Birch reduction condition and subsequent S(N)2 reaction to yield a series of carbosilane dendrimers having appropriate numbers of trisaccharide moieties. These dendrimers have unique shapes and adequate numbers of terminal trisaccharide moieties. Some of the dendrimers showed unique biological activity against Stxs, which were produced by pathogenic Escherichia coli O157:H7.     

Selective inhibition of monoamine oxidase A by hispidol

Hispidol, an aurone, isolated from Glycine max Merrill, was found to potently and selectively inhibit an isoform of recombinant human monoamine oxidase-A (MAO-A), with an IC₅₀ value of 0.26?µM, and to inhibit MAO-B, but with lower potency (IC₅₀?=?2.45?µM). Hispidol reversibly and competitively inhibited MAO-A with a K_i value of 0.10?µM with a potency much greater than toloxatone (IC₅₀?=?1.10?µM), a marketed drug. It also reversibly and competitively inhibited MAO-B (K_i?= 0.51?µM). Sulfuretin, an analog of hispidol, effectively inhibited MAO-A (IC₅₀?=?4.16?µM) but not MAO-B (IC₅₀?>?80?µM). A comparison of their chemical structures showed that the 3′-hydroxyl group of sulfuretin might reduce its inhibitory activities against MAO-A and MAO-B. Flexible docking simulation revealed that the binding affinity of hispidol for MAO-A (?9.1?kcal/mol) was greater than its affinity for MAO-B (?8.7?kcal/mol). The docking simulation showed hispidol binds to the major pocket of MAO-A or MAO-B. The findings suggest hispidol is a potent, selective, reversible inhibitor of MAO-A, and that it be considered a novel lead compound for development of novel reversible inhibitors of MAO-A.     

A globotriaosylceramide (Gb3Cer) mimic peptide isolated from phage display library expressed strong neutralization to Shiga toxins

A Gb₃-trisaccharide mimic peptide was selected with biopanning from a phage display library against anti-Gb₃ antibody to neutralize Shiga toxins (Stxs). Biopanning was carried out on a microplate immobilized with a Fab fragment of anti-Gb₃ antibody and a subtraction procedure screening was applied to enhance specificity. The selected phage clones showed strong affinity to anti-Gb₃ antibody and to Stxs. Among these clones, a 9-mer sequence WHWTWLSEY was determined as the strongest Gb₃ mimic peptide and chemically synthesized. The peptide bound strongly to Stx-1 and Stx-2, though the binding was inhibited with Gb₃Cer. Surface plasmon resonance (SPR) and fluorescent spectroscopy determined that the affinity of the peptide to both Stxs was strong. Neutralization activity was confirmed by in vitro assay with HeLa cells. The Gb₃ mimic peptide potentially has great promise for use against Stxs.     

Molecular basis of differential B-pentamer stability of Shiga toxins 1 and 2

Escherichia coli strain O157:H7 is a major cause of food poisoning that can result in severe diarrhea and, in some cases, renal failure. The pathogenesis of E. coli O157:H7 is in large part due to the production of Shiga toxin (Stx), an AB₅ toxin that consists of a ribosomal RNA-cleaving A-subunit surrounded by a pentamer of receptor-binding B subunits. There are two major isoforms, Stx1 and Stx2, which differ dramatically in potency despite having 57% sequence identity. Animal studies and epidemiological studies show Stx2 is associated with more severe disease. Although the molecular basis of this difference is unknown, data suggest it is associated with the B-subunit. Mass spectrometry studies have suggested differential B-pentamer stability between Stx1 and Stx2. We have examined the relative stability of the B-pentamers in solution. Analytical ultracentrifugation using purified B-subunits demonstrates that Stx2B, the more deadly isoform, shows decreased pentamer stability compared to Stx1B (EC₅₀ = 2.3 µM vs. EC₅₀ = 0.043 µM for Stx1B). X-ray crystal structures of Stx1B and Stx2B identified a glutamine in Stx2 (versus leucine in Stx1) within the otherwise strongly hydrophobic interface between B-subunits. Interchanging these residues switches the stability phenotype of the B-pentamers of Stx1 and Stx2, as demonstrated by analytical ultracentrifugation and circular dichroism. These studies demonstrate a profound difference in stability of the B-pentamers in Stx1 and Stx2, illustrate the mechanistic basis for this differential stability, and provide novel reagents to test the basis for differential pathogenicity of these toxins.     

Synthesis and biological evaluation of isoxazolyl-sulfonamides: A non-cytotoxic scaffold active against Trypanosoma cruzi, Leishmania amazonensis and Herpes Simplex Virus

In this study we report the synthesis, characterization, biological evaluation, and druglikeness assessment of a series of 20 novel isoxazolyl-sulfonamides, obtained by a four-step synthetic route. The compounds had their activity against Trypanosoma cruzi, Leishmania amazonensis, Herpes Simplex Virus type 1 and cytotoxicity evaluated in phenotypic assays. All compounds have drug-like properties, showed low cytotoxicity and were promising regarding all other biological activities reported herein, especially the inhibitory activity against T. cruzi. The compounds 8 and 16 showed significant potency and selectivity against T. cruzi (GI₅₀?=?14.3?µM, SI?>?34.8 and GI₅₀?=?11.6?µM, SI?=?29.1, respectively). These values, close to the values of the reference drug benznidazole (GI₅₀?=?10.2?µM), suggest that compounds 8 and 16 represent promising candidates for further pre-clinical development targeting Chagas disease.     

Inhibition of protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase by xanthones from Cratoxylum cochinchinense, and their kinetic characterization

Cratoxylum cochinchinense displayed significant inhibition against protein tyrosine phosphatase 1B (PTP1B) and α-glucosidase, both of which are key target enzymes to attenuate diabetes and obesity. The compounds responsible for both enzymes inhibition were identified as twelve xanthones (1–12) among which compounds 1 and 2 were found to be new ones. All of them simultaneously inhibited PTP1B with IC₅₀s of (2.4–52.5?µM), and α-glucosidase with IC₅₀ values of (1.7–72.7?µM), respectively. Cratoxanthone A (3) and γ-mangostin (7) were estimated to be most active inhibitors against both PTP1B (IC₅₀?=?2.4?µM for 3, 2.8?µM for 7) and α-glucosidase (IC₅₀?=?4.8?µM for 3, 1.7?µM for 7). In kinetic studies, all isolated xanthones emerged to be mixed inhibitors of α-glucosidase, whereas they behaved as competitive inhibitors of PTP1B. In time dependent experiments, compound 3 showed isomerization inhibitory behavior with following kinetic parameters: K_i^app?=?2.4?µM; k₅?=?0.05001?µM^?1?S^?1 and k₆?=?0.02076?µM^?1?S^?1.     

Interaction of 70-kDa heat shock protein with glycosaminoglycans and acidic glycopolymers

Interaction of Hsp70 with natural and artificial acidic glycans is demonstrated based on the native PAGE analysis. Hsp70 interacts with acidic glycopolymers that contain clustered sulfated and di-sialylated glycan moieties on a polyacrylamide backbone, but not with neutral or mono-sialylated glycopolymers. Hsp70 also interacts and forms a large complex with heparin, heparan sulfate, and dermatan sulfate that commonly contain 2-O-sulfated iduronic acid residues, but not with other types of glycosaminoglycans (GAGs). Hsp70 consists of the N-terminal ATPase domain and the C-terminal peptide-binding domain. The interaction analyses using the recombinant N- and C-terminal half domains show that the ATPase domain mediates the direct interaction with acidic glycans, while the peptide-binding domain stabilizes the large complexes with particular GAGs. To our knowledge, this is the first demonstration of direct binding of Hsp70 to the particular GAGs. This property may be involved in the physiological functions of Hsp70 at the plasma membrane and extracellular environments.     

Uncommon membrane distribution of Shiga toxin glycosphingolipid receptors in toxin-sensitive human glomerular microvascular endothelial cells

Membrane microdomain association of the glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer) and globotetraosylceramide (Gb4Cer), the highly and less effective receptors, respectively, for Shiga toxins (Stxs), is assumed as a functional requirement for Stx-mediated cytotoxicity. In a previous study, we demonstrated predominant localization of Stx receptors in cholesterol-enriched membrane microdomains of moderately Stx-sensitive human brain microvascular endothelial cells (HBMECs) by means of detergent-resistant membranes (DRMs). Here we report a different preferential distribution of Stx receptors in non-DRM fractions of human glomerular microvascular endothelial cells (GMVECs), the major targets of Stxs in the human kidney. Full structural characterization of Stx receptors using electrospray ionization (ESI) mass spectrometry revealed Gb3Cer and Gb4Cer lipoforms with ceramide moieties mainly composed of C24:0/C24:1 or C16:0 fatty acid and sphingosine (d18:1) in GMVECs comparable to those previously found in HBMECs. Thin-layer chromatography immunostaining demonstrated an approximately 2-fold higher content of Gb3Cer and a 1.4-fold higher content of Gb4Cer in GMVECs than in HBMECs. However, this does not explain the remarkable higher cytotoxic action of Stx1 and Stx2 toward GMVECs as compared with HBMECs. Our finding opens new questions on the microdomain association of Stx receptors and the functional role of GSLs in the membrane assembly of GMVECs.     

Semisynthesis,cytotoxicity, antimalarial evaluation and structure-activity relationship of two series of triterpene derivatives

In this report, we describe the semisynthesis of two series of ursolic and betulinic acid derivatives through designed by modifications at the C-3 and C-28 positions and demonstrate their antimalarial activity against chloroquine-resistant P. falciparum (W2 strain). Structural modifications at C-3 were more advantageous to antimalarial activity than simultaneous modifications at C-3 and C-28 positions. The ester derivative, 3β-butanoyl betulinic acid (7b), was the most active compound (IC₅₀?=?3.4?µM) and it did not exhibit cytotoxicity against VERO nor HepG2 cells (CC₅₀?>?400?µM), showing selectivity towards parasites (selectivity index?>?117.47). In combination with artemisinin, compound 7b showed an additive effect (CI?=?1.14). While docking analysis showed a possible interaction of 7b with the Plasmodium protease PfSUB1, with an optimum binding affinity of ?7.02?kcal/mol, the rather low inhibition displayed on a Bacillus licheniformis subtilisin A protease activity assay (IC₅₀?=?93?µM) and the observed accumulation of ring forms together with a delay of appearance of trophozoites in vitro suggests that the main target of 3β-butanoyl betulinic acid on Plasmodium may be related to other molecules and processes pertaining to the ring stage. Therefore, compound 7b is the most promising compound for further studies on antimalarial chemotherapy. The results obtained in this study provide suitable information about scaffolds to develop novel antimalarials from natural sources.     

Production of Hybrid-IgG/IgA Plantibodies with Neutralizing Activity against Shiga Toxin 1

Shiga toxin 1 (Stx1) is a virulence factor of enterohemorrhagic Escherichia coli, such as the O157:H7 strain. In the intestines, secretory IgA (SIgA) is a major component of the immune defense against pathogens and toxins. To form SIgA, the production of dimeric IgA that retains biological activity is an important step. We previously established hybrid-IgG/IgA having variable regions of the IgG specific for the binding subunit of Stx1 (Stx1B) and the heavy chain constant region of IgA. If hybrid-IgG/IgA cDNAs can be expressed in plants, therapeutic or preventive effects may be expected in people eating those plants containing a “plantibody”. Here, we established transgenic Arabidopsis thaliana expressing dimeric hybrid-IgG/IgA. The heavy and light chain genes were placed under the control of a bidirectional promoter and terminator of the chlorophyll a/b-binding protein of Arabidopsis thaliana (expression cassette). This expression cassette and the J chain gene were subcloned into a single binary vector, which was then introduced into A. thaliana by means of the Agrobacterium method. Expression and assembly of the dimeric hybrid-IgG/IgA in plants were revealed by ELISA and immunoblotting. The hybrid-IgG/IgA bound to Stx1B and inhibited Stx1B binding to Gb₃, as demonstrated by ELISA. When Stx1 holotoxin was pre-treated with the resulting plantibody, the cytotoxicity of Stx1 was inhibited. The toxin neutralization was also demonstrated by means of several assays including Stx1-induced phosphatidylserine translocation on the plasma membrane, caspase-3 activation and 180 base-pair DNA ladder formation due to inter-nucleosomal cleavage. These results indicate that edible plants containing hybrid-IgG/IgA against Stx1B have the potential to be used for immunotherapy against Stx1-caused food poisoning.     

Design and synthesis of new fused carbazole-imidazole derivatives as anti-diabetic agents: In vitro α-glucosidase inhibition,kinetic, and in silico studies

Twenty three fused carbazole–imidazoles 6a–w were designed, synthesized, and screened as new α-glucosidase inhibitors. All the synthesized fused carbazole-imidazoles 6a-w were found to be more active than acarbose (IC₅₀?=?750.0?±?1.5?µM) against yeast α-glucosidase with IC₅₀ values in the range of 74.0?±?0.7–298.3?±?0.9?µM. Kinetic study of the most potent compound 6v demonstrated that this compound is a competitive inhibitor for α-glucosidase (K_i value?=?75?µM). Furthermore, the in silico studies of the most potent compounds 6v and 6o confirmed that these compounds interacted with the key residues in the active site of α-glucosidase.     

Comparison of Shiga Toxin Production by Hemolytic-Uremic Syndrome-Associated and Bovine-Associated Shiga Toxin-Producing Escherichia coli Isolates

There is considerable diversity among Shiga toxin (Stx)-producing Escherichia coli (STEC) bacteria, and only a subset of these organisms are thought to be human pathogens. The characteristics that distinguish STEC bacteria that give rise to human disease are not well understood. Stxs, the principal virulence determinants of STEC, are thought to account for hemolytic-uremic syndrome (HUS), a severe clinical consequence of STEC infection. Stxs are typically bacteriophage encoded, and their production has been shown to be enhanced by prophage-inducing agents such as mitomycin C in a limited number of clinical STEC isolates. Low iron concentrations also enhance Stx production by some clinical isolates; however, little is known regarding whether and to what extent these stimuli regulate Stx production by STEC associated with cattle, the principal environmental reservoir of STEC. In this study, we investigated whether toxin production differed between HUS- and bovine-associated STEC strains. Basal production of Stx by HUS-associated STEC exceeded that of bovine-associated STEC. In addition, following mitomycin C treatment, Stx2 production by HUS-associated STEC was significantly greater than that by bovine-associated STEC. Unexpectedly, mitomycin C treatment had a minimal effect on Stx1 production by both HUS- and bovine-associated STEC. However, Stx1 production was induced by growth in low-iron medium, and induction was more marked for HUS-associated STEC than for bovine-associated STEC. These observations reveal that disease-associated and bovine-associated STEC bacteria differ in their basal and inducible Stx production characteristics.     

Synthesis,computational studies and antiproliferative activities of coumarin-tagged 1,3,4-oxadiazole conjugates against MDA-MB-231 and MCF-7 human breast cancer cells

A novel library of coumarin tagged 1,3,4 oxadiazole conjugates was synthesized and evaluated for their antiproliferative activities against MDA-MB-231 and MCF-7 breast cancer cell lines. The evaluation studies revealed that compound 9d was the most potent molecule with an IC₅₀ value of <5?µM against the MCF-7 cell line. Interestingly, compounds 10b and 11a showed a similar trend with lower inhibitory concentration (IC₅₀?=?7.07?µM), in Estrogen Negative (ER?) cells than Estrogen Positive (ER+) cells. Structure–activity relationship (SAR) studies revealed that conjugates bearing benzyl moieties (9b, 9c and 9d) had superior activities compared to their alkyl analogues. The most potent compound 9d showed ～1.4?times more potent activity than tamoxifen against MCF-7 cell line; while the introduction of sulfone unit in compounds 11a, 11b and 11c resulted in significant cytotoxicity against both MCF-7 and MDA-MB-231 cell lines. These results were further supported by docking studies, which revealed that the stronger binding affinity of the synthesized conjugates is due to the presence of sulfone unit attached to the substituted benzyl moiety in their pharmacophores.     

Development and Characterization of Recombinant Antibody Fragments That Recognize and Neutralize In Vitro Stx2 Toxin from Shiga Toxin-Producing Escherichia coli

Background

Stx toxin is a member of the AB₅ family of bacterial toxins: the active A subunit has N-glycosidase activity against 28S rRNA, resulting in inhibition of protein synthesis in eukaryotic cells, and the pentamer ligand B subunits (StxB) bind to globotria(tetra)osylceramide receptors (Gb3/Gb4) on the cell membrane. Shiga toxin-producing Escherichia coli strains (STEC) may produce Stx1 and/or Stx2 and variants. Strains carrying Stx2 are considered more virulent and related to the majority of outbreaks, besides being usually associated with hemolytic uremic syndrome in humans. The development of tools for the detection and/or neutralization of these toxins is a turning point for early diagnosis and therapeutics. Antibodies are an excellent paradigm for the design of high-affinity, protein-based binding reagents used for these purposes.

Methods and Findings

In this work, we developed two recombinant antibodies; scFv fragments from mouse hybridomas and Fab fragments by phage display technology using a human synthetic antibody library. Both fragments showed high binding affinity to Stx2, and they were able to bind specifically to the GKIEFSKYNEDDTF region of the Stx2 B subunit and to neutralize in vitro the cytotoxicity of the toxin up to 80%. Furthermore, the scFv fragments showed 79% sensitivity and 100% specificity in detecting STEC strains by ELISA.

Conclusion

In this work, we developed and characterized two recombinant antibodies against Stx2, as promising tools to be used in diagnosis or therapeutic approaches against STEC, and for the first time, we showed a human monovalent molecule, produced in bacteria, able to neutralize the cytotoxicity of Stx2 in vitro.     

Targeting microbial resistance: Synthesis,antibacterial evaluation,DNA binding and modeling study of new chalcone-based dithiocarbamate derivatives

New dithiocarbamate chalcone-based derivatives were synthesized, their structures were elucidated using different spectroscopic techniques. They were subjected to antimicrobial screening against selected Gram negative bacteria focusing on microbial resistance. Bacterial resistance was targeted via phosphoethanolamine transferase enzyme. Most of the synthesized compounds showed equal or higher activity to colistin standard. Compound 24 proved to be the most active candidate with MIC of 8 µg/ml against both Ps12 and K4 and MBC of 32 µg/ml against Ps12 and 16 µg/ml against K4 Molecular docking study showed that 20, 22, 24 and 25 had good binding affinity with active site residues via Thr280. DNA macromolecule was further targeted. Compounds 28 and 34 were recorded to have better DNA binding than doxurubucin with IC₅₀ of 27.48 and 30.97 µg/ml respectively, suggesting that it could have a role in their higher antibacterial effect. Their docking into DNA has shown a clear intercalation matching with antibacterial data. Pharmacokinetics parameters of active compounds showed that they have better absorption through GIT.     

Binding mode prediction of conformationally restricted anandamide analogs within the CB1 receptor

Background

CB₁ cannabinoid receptors are G-protein coupled receptors for endocannabinoids including anandamide and 2-arachidonoylglycerol. Because these arachidonic acid metabolites possess a 20-carbon polyene chain as the alkyl terminal moiety, they are highly flexible with the potential to adopt multiple biologically relevant conformations, particularly those in a bent form. To better understand the molecular interactions associated with binding and steric trigger mechanisms of receptor activation, a series of conformationally-restricted anandamide analogs having a wide range of affinity and efficacy were evaluated.

Results

A CB₁ receptor model was constructed to include the extracellular loops, particularly extracellular loop 2 which possesses an internal disulfide linkage. Using both Glide (Schrödinger) and Affinity (Accelrys) docking programs, binding conformations of six anandamide analogs were identified that conform to rules applicable to the potent, efficacious and stereoselective non-classical cannabinoid CP55244. Calculated binding energies of the optimum structures from both procedures correlated well with the reported binding affinity values. The most potent and efficacious of the ligands adopted conformations characterized by interactions with both the helix-3 lysine and hydrophobic residues that interact with CP55244. The other five compounds formed fewer or less energetically favorable interactions with these critical residues. The flexibility of the tested anandamide analogs, measured by torsion angles around the benzene as well as the stretch between side chain moieties, could contribute to the differences in ability to interact with the CB₁ receptor.

Conclusion

Analyses of multiple poses of conformationally-restricted anandamide analogs permitted identification of favored amino acid interactions within the CB₁ receptor binding pocket. A ligand possessing both high affinity and cannabinoid agonist efficacy was able to interact with both polar and hydrophobic interaction sites utilized by the potent and efficacious non-classical cannabinoid CP55940. In contrast, other analogs characterized by reduced affinity or efficacy exhibited less favorable interactions with those key residues.