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Zsila F.,Institute of Biomolecular Chemistry | Kaman J.,BioBlocks Magyarorszag Kft. | Boganyi B.,BioBlocks Magyarorszag Kft. | Jozsvai D.,Institute of Biomolecular Chemistry
Organic and Biomolecular Chemistry | Year: 2011

Non-covalent binding of planar aromatic molecules into the S1 specificity pocket of the serine protease α-chymotrypsin (αCHT) can be detected by measuring induced circular dichroism (CD) spectroscopic signals. Utilizing this phenomenon, αCHT association of proflavine (PRF), the well known serine protease inhibitor has been investigated together with plant-derived compounds including isoquinoline, pyridocarbazole and indoloquinoline alkaloids, of which αCHT binding has never been reported. Non-degenerate exciton coupling between π-π* transitions of the ligand molecules and two tryptophan residues (Trp172 and Trp215) near to the binding site is proposed to be responsible for the induced CD activity. The association constants calculated from CD titration data indicated strong αCHT association of sanguninarine, ellipticine, desmethyl-isocryptolepine and isoneocryptolepine (Ka ≈ 105 M-1) while berberine, coptisine and chelerythrine bind to the enzyme with lower, PRF-like affinity (K a ≈ 104 M-1). PRF-trypsin and ellipticine-trypsin binding interactions have also been demonstrated. The binding of the alkaloids into the S1 pocket of αCHT has been confirmed by CD competition experiments. Molecular docking calculations showed the inclusion of PRF as well as the alkaloid molecules in the S1 cavity where they are stabilized by hydrophobic and H-bonding interactions. These novel nonpeptidic scaffolds can be used for developing selective inhibitors of serine proteases having chymotrypsin-like folds. Furthemore, the results provide a novel, CD spectroscopic based approach for probing the ligand binding of αCHT and related proteases. © 2011 The Royal Society of Chemistry.

Jemnitz K.,Institute of Biomolecular Chemistry | Heredi-Szabo K.,SOLVO Biotechnology | Janossy J.,SOLVO Biotechnology | Ioja E.,SOLVO Biotechnology | And 2 more authors.
Drug Metabolism Reviews | Year: 2010

ABCC2/Abcc2 (MRP2/Mrp2) is expressed at major physiological barriers, such as the canalicular membrane of liver cells, kidney proximal tubule epithelial cells, enterocytes of the small and large intestine, and syncytiotrophoblast of the placenta. ABCC2/Abcc2 always localizes in the apical membranes. Although ABCC2/Abcc2 transports a variety of amphiphilic anions that belong to different classes of molecules, such as endogenous compounds (e.g., bilirubin- glucuronides), drugs, toxic chemicals, nutraceuticals, and their conjugates, it displays a preference for phase II conjugates. Phenotypically, the most obvious consequence of mutations in ABCC2 that lead to Dubin-Johnson syndrome is conjugate hyperbilirubinemia. ABCC2/Abcc2 harbors multiple binding sites and displays complex transport kinetics. © 2010 Informa UK Ltd.

Zsila F.,Institute of Biomolecular Chemistry | Bikadi Z.,Virtua Drug Ltd. | Malik D.,Delta Services Ltd. | Hari P.,Delta Services Ltd. | And 3 more authors.
Bioinformatics | Year: 2011

Motivation: Human serum albumin (HSA), the most abundant plasma protein is well known for its extraordinary binding capacity for both endogenous and exogenous substances, including a wide range of drugs. Interaction with the two principal binding sites of HSA in subdomain IIA (site 1) and in subdomain IIIA (site 2) controls the free, active concentration of a drug, provides a reservoir for a long duration of action and ultimately affects the ADME (absorption, distribution, metabolism, and excretion) profile. Due to the continuous demand to investigate HSA binding properties of novel drugs, drug candidates and drug-like compounds, a support vector machine (SVM) model was developed that efficiently predicts albumin binding. Our SVM model was integrated to a free, web-based prediction platform (http://albumin.althotas.com). Automated molecular docking calculations for prediction of complex geometry are also integrated into the web service. The platform enables the users (i) to predict if albumin binds the query ligand, (ii) to determine the probable ligand binding site (site 1 or site 2), (iii) to select the albumin X-ray structure which is complexed with the most similar ligand and (iv) to calculate complex geometry using molecular docking calculations. Our SVM model and the potential offered by the combined use of in silico calculation methods and experimental binding data is illustrated. © The Author 2011. Published by Oxford University Press. All rights reserved.

Kardos J.,Institute of Biomolecular Chemistry | Pallo A.,Institute of Biomolecular Chemistry | Bencsura A.,Hungarian Academy of Sciences | Simon A.,Institute of Biomolecular Chemistry
Current Medicinal Chemistry | Year: 2010

Ambient level of γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter of the brain is mediated by neuronal and glial GABA transporters (GATs), members of the sodium and chloride ion-dependent solute carrier family. The neuronal GABA transporter subtype (GAT-1) has already been proven to be the target for the antiepileptic drug Tiagabine. However, druggability of glial GAT-2 and GAT-3 is yet to be established. Recent advances in structure elucidation of a bacterial orthologue leucine transporter in complex with different substrates substantiate homology modeling of human GATs (hGATs). These modeling studies can provide mechanistic clues for structure-based prediction of the potential of medicinal chemistry campaigns. A recently identified characteristic structural feature of the occluded conformation of hGATs is that similar extra- and intracellular gates are formed by middle-broken transmembrane helices TM1 and TM6. Binding crevice formed by unwound segments of broken helices facilitates symport of GABA with Na+ ion via fitting of GABA to TM1-bound Na+(1) closely inside. Favored accommodation of substrate inhibitors with high docking score predicts efficient inhibition of the neuronal hGAT-1 if the TM1-TM8 binding prerequisite for GABA was used. Docking, molecular dynamics and transport data indicate, that amino acids participating in substrate binding of the neuronal hGAT-1 and the glial hGAT-2 and hGAT-3 subtypes are different. By contrast, substrate binding crevices of hGAT-2 and hGAT-3 cannot be distinguished, avoiding sensible prediction of efficient selective substrate inhibitors. Glial subtypes might be specifically distinguished by interfering Zn2+ binding in the second extracellular loop of hGAT-3. Formation of the unique ring-like Na+-GABA complex in the occluded binding crevices anticipates family member symporters exploring chemiosmotic energy via reversible chemical coupling of Na+ ion. © 2010 Bentham Science Publishers Ltd.

Promiscuous ligand binding by hen egg-white avidin has been demonstrated and studied by using circular dichroism (CD) spectroscopy complemented by molecular docking calculations. It has been shown that the biotin-binding pocket of avidin is able to accommodate a wide variety of chemical compounds including therapeutic drugs (e.g., thalidomide, NSAIDs, antihistamines), natural compounds (bilirubin, myristic acid), and synthetic agents (xanthenone dyes). The cluster of aromatic residues located at the biotin-binding pocket renders the intrinsic CD spectrum of avidin sensitive to ligand binding that results in the increase of the vibronic components of the 1L b transition of the Trp residues. Extrinsic (induced) CD bands measured with chemically diverse avidin ligands are generated by intramolecular coupled oscillator (e.g., bilirubin) or by intermolecular ligand-Trp exciton coupling mechanism [e.g., 2-(4′-hydroxyazobenzene)-benzoic acid (HABA)]. Among the compounds of which avidin-binding affinity constants have been calculated, two novel high-affinity ligands, flufenamic acid and an enzyme inhibitor thiazole derivative have been identified (K d ≈ 1 μM). Avidin binding mode of the ligand molecules has been discussed in the light of docking results. The induced CD profile of the thiazole derivative has been correlated with the stereochemistry of its docked conformation. The important role in the ligand binding of a polar side-chain cluster at the bottom of the biotin-binding cavity as well as the analogous avidin-binding mode of HABA and fenamic acid type NSAIDs have been proposed. Copyright © 2011 John Wiley & Sons, Ltd. Copyright © 2011 John Wiley & Sons, Ltd.

Nyitrai G.,Institute of Biomolecular Chemistry | Lasztoczi B.,Institute of Biomolecular Chemistry | Kardos J.,Institute of Biomolecular Chemistry
Brain Research | Year: 2010

A wide range of data support a role for ambient glutamate (Glu) in epilepsy, although temporal patterns associated with the cellular uptake of Glu have not been addressed in detail. We report on the effects of Glu uptake inhibitors on recurrent seizure-like events (SLEs) evoked by low-[Mg2+] condition in juvenile rat hippocampal slices. Effects were compared for inhibitors such as l-trans-pyrrolidine-2,4-dicarboxylate (tPDC), dl-threo-β-benzyloxyaspartate (dl-TBOA) and dihydrokainic acid (DHK), representing different transporter specificity and transportability profiles. Latency to the first SLE after drug application was shortened by the inhibitors (in % of control: 500 μM tPDC: 54 ± 7, 15 μM dl-TBOA: 74 ± 5, 50 μM dl-TBOA: 70 ± 6, 100 μM DHK: 69 ± 4, 300 μM DHK: 71 ± 7). Further SLEs were frequently aborted by higher inhibitor concentrations applied (500 μM tPDC: 2/6, 50 μM TBOA: 5/5, 100 μM DHK: 6/8, 300 μM DHK: 3/3). Simultaneous field potential and whole-cell voltage recordings showed depolarization-induced inactivation of CA3 pyramidal neurons during inhibitor application. In the presence of inhibitors, the amplitude of forthcoming SLE was also decreased (in % of control: 500 μM tPDC: 66 ± 9, 15 μM dl-TBOA: 88 ± 5, 50 μM dl-TBOA: 59 ± 6, 100 μM DHK: 67 ± 4, 300 μM DHK: 68 ± 1). Dependent on type and concentration of the inhibitor, the duration of the first SLE of drug application either increased (100 μM DHK: 375 ± 90 %; 100 μM tPDC: 137 ± 13 %) or decreased (50 μM TBOA: 62 ± 13 %; 300 μM DHK: 60 ± 15 %) reflecting differences in subtype-specificity or mechanism of action of the inhibitors. Our findings suggest a role for ambient Glu in the genesis and maintenance of recurrent epileptiform discharges. © 2009 Elsevier B.V. All rights reserved.

In vitro chaperone-like activity of the serpin family member and plasma acute-phase component human α1-antitrypsin (AAT) has been shown for the first time. Results of light-scattering experiments demonstrated that AAT efficiently inhibits both heat- and chemical-induced aggregation of various test proteins including alcohol dehydrogenase, aldolase, carbonic anhydrase, catalase, citrate synthase, enolase, glutathione S-transferase, l-lactate dehydrogenase, and βL-crystallin. The results suggest that the unique metastable serpin architecture enables dual function, protease inhibiton as well as chaperone activity and highlight the serpin superfamily as a possible source of additional intra- and extracellular chaperones (e.g. α1-antichymotrypsin). The present finding is surprising in the light of the well-known role of mutated forms of AAT and other serpins in the pathogenesis of diseases called serpinopathies that featured with aberrant conformational transitions and consequent self-aggregation of serpin proteins. © 2010 Elsevier Inc. All rights reserved.

Zsila F.,Institute of Biomolecular Chemistry
Biomacromolecules | Year: 2011

Noncovalent complex formation of unconjugated bilirubin with various enzymes has been demonstrated by measuring induced circular dichroism (ICD) peaks associated with the pigment VIS absorption band. Preferential binding of the P-or M-helical conformer of bilirubin to dehydrogenases, catalase, alkaline phosphatase, and α-chymotrypsin is responsible for the characteristic exciton CD couplet that undergoes remarkable changes upon the addition of enzymatic cofactors (NADH, AMP) and an inhibitor (acridine). Alterations of the ICD spectra refer to a direct binding competition between bilirubin and NADH for a common binding site on alcohol dehydrogenase and catalase, suggesting a potential mechanism for the inhibitory effect of BR reported on NAD(P)H dependent enzymes. NADH and bilirubin form a ternary complex with glutamate dehydrogenase indicated by peculiar CD spectral changes that are proposed to be generated by allosteric mechanism. α-chymotrypsin binds bilirubin in its catalytic site, as indicated by CD displacement experiments performed with the competitive inhibitor acridine. Surprisingly, the closely related trypsin does not induce any CD signal with bilirubin. Taking into consideration the clinically relevant but controversial and poorly understood areas of bilirubin biochemistry, the fast and simple CD spectroscopic approach presented here may help to unfold diverse physiological and pathophysiological roles of BR on a molecular level. © 2010 American Chemical Society.

In vitro chaperone-like activity of the acute-phase component and plasma drug transporter human α1-acid glycoprotein (AAG) has been shown for the first time. AAG suppressed thermal aggregation of a variety of unrelated enzymatic (e.g., aldolase, catalase, enolase, carbonic anhydrase) and non-enzymatic proteins (β-lactoglobulin, ovotransferrin) and it also prevented dithiothreitol induced aggregation of insulin. The anti-aggregation ability of AAG was abolished/reduced upon drug binding suggesting that protein-protein interactions established between the lipocalin β-barrel fold of AAG and hydrophobic surfaces of the stressed proteins are involved in the chaperone-like activity. The results shed some light on the possible biological function of this enigmatic protein and suggest that besides haptoglobin, clusterin, fibrinogen and α2-macroglobulin AAG can be considered as a novel member of the extracellular molecular chaperones found in human body fluids. © 2009 Elsevier Ltd. All rights reserved.

High-affinity human serum albumin (HSA) binding of the C3-substituted antimalarial 2-hydroxy-1,4-naphthoquinone derivative atovaquone (ATQ) has been demonstrated and studied by circular dichroism (CD), UV/VIS absorption, fluorescence spectroscopy and affinity chromatography methods. The analysis of induced CD data generated upon HSA binding of ATQ revealed two high-affinity binding sites (Ka ≈ 2 × 106 M-1). CD interaction studies and displacement of specific fluorescent and radioactive marker ligands indicated the contribution of both principal drug binding sites of HSA to complexation of ATQ, and also suggested the possibility of simultaneous binding of ATQ and some other drugs (e.g. warfarin, phenylbutazone, diazepam). Comparison of UV/VIS spectra of ATQ measured in aqueous solutions indicated the prevalence of the anionic species formed by dissociation of the 2-hydroxyl group. HSA binding of related natural hydroxynaphthoquinones, lapachol and lawsone also induces similar CD spectra. The much weaker binding affinity of lawsone (Ka ≈ 104 M-1) bearing no C3 substituent highlights the importance of hydrophobic interactions in the strong HSA binding of ATQ and lapachol. Since neither drug exhibited significant binding to serum α1-acid glycoprotein, HSA must be the principal plasma protein for the binding and transportation of 2-hydroxy-1,4-naphthoquinone-type compounds which are ionized at physiological pH values. © 2010 The Royal Society of Chemistry.

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