Synthesis, NMR kinetics and dynamic structure of a 17-mer heptaloop RNA hairpin carrying a 3-N-methyluridine nucleotide residue in the loop region.

A 17-mer RNA hairpin (5'GGGAGUXAGCGGCUCCC3') carrying 3-N-methyluridine (m3U) at position X (m3U7-RNA), designed to represent the anticodon stem-loop (ACSL) region of tRNAs to study an open loop state (O-state), was synthesized, purified by HPLC, and characterized by MALDI-ToF_MS and NMR methods. 1H-NMR data revealed primary (P-state in 56.1%), secondary (S-state in 43.9%) and tertiary (∼5-6%) ACSL conformations. Exchange rate constant (kex) for interconversion between P and S states is 112 sec-1 (<Δω ∼454 rad/sec), confirming a slow exchange regime between the two states. Forward (kPS) and backward (kSP) rate constants are 49.166 sec-1 and 62.792 sec-1, respectively, leading to a longer life-time (20.339 msec) for the P-state and a shorter life-time (15.926 msec) for the S-state. In accordance with conformational populations determined by 1H-NMR, dynamics of the P/S/tertiary states of m3U7-RNA and its wild-type counterpart (wt-RNA) were studied by three independent MD production simulations. Cluster analysis revealed that wt-RNA reflects the structural characteristics of the ACSL region of tRNAs. The P-state of m3U7-RNA was found to be structurally similar to wt-RNA but lacks an intraloop H-bond between m3U7 and C10 (U33 and nt36 in tRNAs). In the S-state of m3U7-RNA, m3U7 flips out of the loop region. O-state loop conformations of m3U7-RNA were also clustered (4.8%), wherein the loop nucleotides m3U7.A8.G9.C10.G11 stack one after another. We propose that the O-state of m3U7-RNA is the most suitable conformation that makes the loop accessible for complementary nucleotides and for non-enzymatic primordial replication of small circular RNAs.Communicated by Ramaswamy H. Sarma.

Isothermal titration calorimetry binding properties of Cibacron Blue F3GA in complex with human serum albumin.

Binding interactions between Cibacron Blue-F3GA (CB-F3GA) and human serum albumin (HSA, at physiologically ten-fold lower concentration) was studied by isothermal titration calorimetry (ITC) and in-silico docking computations. ITC experiments revealed two separate binding sites on HSA with different binding affinities for CB-F3GA. The high-affinity binding site (PBS-II) on HSA binds CB-F3GA at nanomolar scale (KD1 = 118 ± 107 nM) with favorable binding enthalpy (ΔHo 1 = - 6.47 ± 0.44 kcal/mol) and entropy (-TΔSo 1 = -2.98 kcal/mol) energies. CB-F3GA binds to the low-affinity binding site (PBS-I) at µM scale (KD2 = 31.20 ± 18.40 µM) with favorable binding enthalpy (ΔHo 1 = - 5.03 ± 3.86 × 10-2 kcal/mol) and entropy (-TΔSo 1 = -1.12 kcal/mol) energies. ITC binding data strongly suggest that CB-F3GA binding to PBS-II site increases the formation of dimeric-HSA clusters (N1 = 2.43 ± 0.50), while binding to PBS-I leads to tetrameric-HSA clusters (N2 = 4.61 ± 0.90). These results suggest that a higher degree of HSA aggregation upon drug binding may be expected under physiological conditions, a notion that should be further investigated for the delivery and toxicity of drug-HSA interactions.

Cross Referencing 2D-LC Determination of Intact Gliptins in Urine.

Dipeptidyl peptidase-4 inhibitors, so-called gliptins, constitute a fairly novel class of oral hypoglycemic agents. The development and validation of an automated online SPE-LC-UV method to determine intact sitagliptin, saxagliptin, vildagliptin and metformin simultaneously in human urine samples were performed. For the two-dimensional chromatographic separation, a Gemini C18 (250.0 × 4.6 mm i.d., 110 A0, 5.0 µ) analytical column and a gradient elution with 10.0 mM o-phosphoric acid and methanol and for the online SPE analysis of urine samples, a LiChrospher® ADS SPE-column (20.0 mm × 2.0 mm i.d., 25.0 µm) were used through the study. The fractionation, transfer, elution and separation of the spiked urine samples were achieved in just 9.57 min runtime with 12.0 mL of solvent consumption which was green and economical compared to other sample preparation methods. The calibration curves were determined to be linear in a wide range of 0.10-100.00 µg/mL with satisfactory regression coefficients. Method developed for two-dimensional determination of gliptins would be useful as a reference in therapeutic drug monitoring and screening for forensic medical cases which involve the abuse, unintentional or misuse of multiple gliptins in terms of its practical use, easy detection and reliable results.

Quinoline-based promising anticancer and antibacterial agents, and some metabolic enzyme inhibitors.

A series of substituted quinolines was screened for their antiproliferative, cytotoxic, antibacterial activities, DNA/protein binding affinity, and anticholinergic properties by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide cell proliferation, lactate dehydrogenase cytotoxicity, and microdilution assays, the Wolfe-Shimmer equality method, the Ellman method, and the esterase assay, respectively. The results of the cytotoxic and anticancer activities of the compounds displayed that 6-bromotetrahydroquinoline (2), 6,8-dibromotetrahydroquinoline (3), 8-bromo-6-cyanoquinoline (10), 5-bromo-6,8-dimethoxyquinoline (12), the novel N-nitrated 6,8-dimethoxyquinoline (13), and 5,7-dibromo-8-hydroxyquinoline (17) showed a significant antiproliferative potency against the A549, HeLa, HT29, Hep3B, and MCF7 cancer cell lines (IC50 = 2-50 µg/ml) and low cytotoxicity (∼7-35%) as the controls, 5-fluorouracil and cisplatin. The compound-DNA linkages are hyperchromic or hypochromic, causing variations in their spectra. This situation shows that they can be bound to DNA with the groove-binding mode, with Kb value in the range of 2.0 × 103 -2.2 × 105 M-1 . Studies on human Gram(+) and Gram(-) pathogenic bacteria showed that the substituted quinolines exhibited selective antimicrobial activities with MIC values of 62.50-250 µg/ml. All tested quinoline derivatives were found to be effective inhibitors of acetylcholinesterase (AChE) and the human carbonic anhydrase I and II isoforms (hCA I and II), with Ki values of 46.04-956.82 nM for hCA I, 54.95-976.93 nM for hCA II, and 5.51-155.22 nM for AChE. As a result, the preliminary data showed that substituted quinolines displayed effective pharmacological features. Molecular docking studies were performed to investigate the binding modes and interaction energies for compounds 2-17 with AChE (PDB ID: 4EY6), hCA I (PDB ID: 1BMZ), and hCA II (PDB ID: 2ABE).

Seroprevalence of Crimean-Congo Hemorrhagic Fever in Turkey's Van Province.

Crimean-Congo hemorrhagic fever (CCHF) is an endemic tick-borne viral disease that affects both animals and humans. This study aims to determine the seroprevalence of CCHF in Turkey's Van province using analysis of blood samples obtained from people living in the region. Blood specimens were taken from healthy subjects living in Van province and some of the surrounding villages between January and July 2012. Blood samples were initially tested using a CCHF virus (CCHFV) IgM IgG kit for anti-CCHFV IgG, followed by anti-CCHFV IgM determination of any IgG positive blood samples. IgM-positive specimens were re-confirmed using real-time polymerase chain reaction (qPCR). One hundred and 7 men and 261 women were included in the study. Fifty-three blood specimens (14.4%) were anti-CCHFV IgG positive, and 2 of these were anti-CCHFV IgM positive. Two blood samples with anti-CCHFV IgM seropositivity tested negative using qPCR, indicating chronic infections. Locality, sex, and a history of tick bites did not significantly affect anti-CCHFV IgG seropositivity. Although the incidence of anti-CCHFV IgG in blood specimens was 14.4%, no deaths have yet been reported in Turkey's Van province. It is imperative that clinical CCHFV tests be implemented for people at high risk of developing CCHFV-related complications.

Characterisation of drug resistance of nosocomial ESBL-producing E. coli isolates obtained from a Turkish university hospital between 2009 and 2012 by pulsed field gel electrophoresis and antibiotic resistance tests.

In this study, drug resistance of 28 ESBL-producing Escherichia coli isolates obtained from 144 patients hospitalized at the Yüzüncüyil University Hospital at Van (YUH), Turkey, between 2009 and 2012 were characterized by pulsed field gel electrophoresis and antibiotic susceptibility tests. Antibiotic resistance profile was determined by Phoenix automated system (BD, USA). The ratio of ESBL-producing E. coli strains was determined to be 19.4% (28 out of 144 E. coli isolates). It was determined that the anaesthesiology, paediatrics and thoracic medicine intensive care units in YUH were cross-contaminated between 2009 and 2012 by ESBL-producing E. coli strains, which is a sign of nosocomial infection in YUH. Analysis of PFGE results gave rise to two main PFGE profiles, profile-A with four subprofiles and profile-B with three subprofiles, where profile-A predominates over profile-B (14%). Comparison of the antibiotic resistance profile with the PFGE profile yielded similarities while some differences also exist due to either identical restriction enzyme cutting sites with slightly different genetic sequences in between the cutting sites or newly formed restriction enzyme cutting sites that do not affect antibiotic resistance genes. Enterobacteriaceae, particularly E. coli, have developed resistance in YUH by producing ESBLs against oxyimino and non-oxyimino cephalosporins, and penicillin-type antibiotics. Therefore, more effective antibiotics such as cefoxitin or cefoperazone-sulbactam should be used for the treatment of future nosocomial infections in YUH while hospital staff should take care with hygiene, such as hand washing.

Compressed images for affinity prediction-2 (CIFAP-2): an improved machine learning methodology on protein-ligand interactions based on a study on caspase 3 inhibitors.

The aim of this study is to propose an improved computational methodology, which is called Compressed Images for Affinity Prediction-2 (CIFAP-2) to predict binding affinities of structurally related protein-ligand complexes. CIFAP-2 method is established based on a protein-ligand model from which computational affinity information is obtained by utilizing 2D electrostatic potential images determined for the binding site of protein-ligand complexes. The quality of the prediction of the CIFAP-2 algorithm was tested using partial least squares regression (PLSR) as well as support vector regression (SVR) and adaptive neuro-fuzzy inference system (ANFIS), which are highly promising prediction methods in drug design. CIFAP-2 was applied on a protein-ligand complex system involving Caspase 3 (CASP3) and its 35 inhibitors possessing a common isatin sulfonamide pharmacophore. As a result, PLSR affinity prediction for the CASP3-ligand complexes gave rise to the most consistent information with reported empirical binding affinities (pIC(50)) of the CASP3 inhibitors.

Synthesis, anticancer activities and molecular modeling studies of novel indole retinoid derivatives.

In this study, novel (E)-3-(5-substituted-1H-indol-3-yl)-1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydronaphthalen-2-yl)prop-2-en-1-one (5(a-e)) derivatives were synthesized and their anticancer effects were determined in vitro. Novel indole retinoid compounds except 5e have anti-proliferative capacity in liver, breast and colon cancer cell lines. This anti-proliferative effect was further analyzed in breast cancer cell line panel by using the most potent compound 5a. It was determined that 5a can inhibit proliferation at very low IC(50) concentrations in all of the breast cancer cell lines. Here, we present some evidence on apoptotic termination of cancer cell proliferation which may be primarily driven by the inhibition of RXRα and, to a lesser extent, RXRγ.

Use of the parmbsc0 force field and trajectory analysis to study the binding of netropsin to the DNA fragment (5'CCAATTGG)(2) in the presence of excess NaCl salt in aqueous solution.

The parmbsc0 force field was applied to study in detail the binding of netropsin, at a salt concentration of 0.28M Na(+), to the minor groove of an 8-mer (5'CCAATTGG)(2) DNA duplex forming a netropsin·DNA complex which previously has been characterized by X-ray crystallography, albeit with the use of closely related DNA duplexes. The X-ray structure revealed that the terminal guanidinium and amidinium groups of netropsin interact with the extreme ends of the palindromic AATT sequence of the receptor DNA. The parmbsc0 parameters of B-DNA and AMBER v9 parameters of netropsin generated a stable 6ns molecular dynamics (MD) trajectory for a 1:1 class I binding motif of this complex. Trajectory analysis for the salt and hydration effects on the binding of netropsin to the 8-mer DNA duplex revealed that 18 water molecules and 2 Na(+) are displaced from the DNA upon netropsin binding. A hydration density map of the complex parallels the X-ray data showing that two structured water molecules are localized near the netropsin guanidinium and amidinium groups forming H-bond bridges between the receptor and the ligand.

NMR and amber analysis of the neamine pharmacophore for the design of novel aminoglycoside antibiotics.

The dependence of the solution structure of neamine on pH was determined by NMR and AMBER molecular dynamics methods at pD 3.3, pD 6.5, and pD 7.4 in D(2)O at 25°C. Unlike neamine structures at pD 3.3 and 6.5, which essentially showed only one conformer, slowly exchanging primary, P-state, and secondary, S-state, neamine conformers populated on the NMR time scale at ~80% and ~20%, respectively, were detected at pD 7.4 with kinetic constants k(on(P→S))=1.9771s(-1) and k(off(S→P))=1.1319s(-1). A tertiary, T-state, neamine species populated at ~3% was also detected by NMR at pD 7.4. The pKa values determined by NMR titration experiments are pKa1 6.44±0.13 for N3 of ring-II, pKa2 7.23±0.09 for N2' of ring-I, pKa3 7.77±0.19 for N1 of ring-II, and pKa4 8.08±0.15 for N6' of ring-I. Ring-I and ring-II of the P-state neamine and ring-I of the S and T-states of neamine possess the (4)C(1) chair conformation between pD 3.3 and pD=7.4. In contrast, ring-II of the S and T-states of neamine most likely adopt the (6)rH(1) half-chair conformation. The P and S-states of neamine exhibit a negative syn-ψ glycosidic geometry. The exocyclic aminomethyl group of ring-I adopts the gt exocyclic rotamer conformation around physiological pHs while the gg exocyclic rotamer conformation predominates in acidic solutions near and below pH 4.5. Neamine exists in the P-state as a mixture of tetra-/tri-/di-protonated species between pD 4.5 and pD 7.4, while the S-state neamine exist only in a di-protonated species around physiological pDs. The existence of the S-state neamine may facilitate binding of neamine-like aminoglycosides by favorable entropy of binding to the A-site of 16S ribosomal RNA, suggesting that novel aminoglycoside compounds carrying a S-state neamine pharmacophore can be developed.

Molecular recognition-based detoxification of aluminum in human plasma.

Molecular recognition-based Al(3+)-imprinted poly(hydroxyethyl methacrylate-N-methacryloyl-L-glutamic acid) (PHEMAGA-Al(3+)) beads were prepared to be used in selective removal of Al(3+) out of human plasma overdosed with Al(3+) cations. The PHEMAGA-Al(3+) beads were synthesized by suspension polymerization in the presence of a template-monomer complex (MAGA-Al(3+)). The specific surface area of PHEMAGA-Al(3+) beads was found to be 55.6 m(2)/g on the average. The MAGA content in the PHEMAGA-Al(3+) beads were found to be 640 micgomol/g polymer. The template Al(3+) cations could be reversibly detached from the matrix to form PHEMAGA-Al(3+) using a 50 mM solution of EDTA. The Al(3+)-free PHEMAGA-Al(3+) beads were then exposed to a selective separation procedure of Al(3+) out of human plasma, which was implemented in a continuous system by packing the beads into a separation column (10 cm long with an inner diameter of 0.9 cm) equipped with a water jacket to control the temperature. The Al(3+) adsorption capacity of the PHEMAGA-Al(3+) beads decreased drastically from 0.76 mg/g polymer to 0.22 mg/g polymer as the flow rate was increased from 0.3 ml/min to 1.5 ml/min. The relative selectivity coefficients of the PHEMAGA-Al(3+) beads for Al(3+)/Fe(3+), Al(3+)/Cu(2+) and Al(3+)/Zn(2+) were found to be 4.49, 8.95 and 32.44 times greater than those of the non-imprinted PHEMAGA beads, respectively. FT-IR analyses on the synthesized PHEMAGA-Al(3+) beads reveals monodentate and bidentate binding modes of Al(3+) in complex with the carboxylate groups of the glutamate residues. Density functional theory computations at the B3LYP/6-31G(d,p) basis set suggests that structured water molecules play essential role in the stability of the monodentate binding mode in 1:1 PHEMAGA-Al(3+) complexes. The PHEMAGA-Al(3+) beads were recovered and reused many times, with no significant decrease in their adsorption capacities.

Predicting the binding properties of cibacron blue F3GA in affinity separation systems.

The binding properties of cibacron blue F3GA (CB-F3GA) bound to a model NAD(P)H/FAD(H2)-dependent protein system, namely cytosolic quinone reductase (QR), was characterized by AMBER in an attempt to address the binding properties of immobilized CB-F3GA used in the separation of serum albumin. A favorable binding free energy of -4.52kcal/mol (KD=5.09 x 10(-4)kcal/mol) was determined for CB-F3GA binding by MM-PBSA method, which was found to be a ballpark estimate of empirical values reported in literature (DeltaG approximately -6kcal/mol). We propose that CB-F3GA primarily follows a class III binding motif in presence of FAD in the binding site of QR in solution, while a class II binding motif is observed in the crystal form. It was found that favorable van der Waals/hydrophobic interactions take place in the binding site making a major contribution to a favorably dominating enthalpy of binding (DeltaHtot=-25.87kcal/mol) as compared to a disfavorable binding entropy term (TDeltaStot=-21.35kcal/mol). Additional MM-PBSA experiments in the absence of FAD gave rise to a disfavorable binding free energy for CB in complex with QR, suggesting that FAD is an essential determinant of CB-F3GA binding. This is in contrast to an earlier observation of Denizli et al. on separation of human serum albumin (HSA) by immobilized CB-F3GA in the absence of FAD. Therefore, a class I binding model for CB-F3GA is proposed here to account for the efficient separation of HSA in affinity chromatography systems.