Trias Thireou

Crystal and Solution Studies of the “Plus-C” Odorant-binding Protein 48 from Anopheles gambiae CONTROL OF BINDING SPECIFICITY THROUGH THREE-DIMENSIONAL DOMAIN SWAPPING

Background: Mosquito odorant-binding proteins constitute molecular targets for structure-based discovery of novel host-seeking disruptors. Results: “Plus-C” AgamOBP48 exists as a three-dimensional domain-swapped dimer containing a combined binding site. Conclusion: Domain swapping has important implications for AgamOBP48 binding specificity. Significance: OBP dimerization should be considered in a successful OBP-based discovery strategy. Much physiological and behavioral evidence has been provided suggesting that insect odorant-binding proteins (OBPs) are indispensable for odorant recognition and thus are appealing targets for structure-based discovery and design of novel host-seeking disruptors. Despite the fact that more than 60 putative OBP-encoding genes have been identified in the malaria vector Anopheles gambiae, the crystal structures of only six of them are known. It is therefore clear that OBP structure determination constitutes the bottleneck for structure-based approaches to mosquito repellent/attractant discovery. Here, we describe the three-dimensional structure of an A. gambiae “Plus-C” group OBP (AgamOBP48), which exhibits the second highest expression levels in female antennae. This structure represents the first example of a three-dimensional domain-swapped dimer in dipteran species. A combined binding site is formed at the dimer interface by equal contribution of each monomer. Structural comparisons with the monomeric AgamOBP47 revealed that the major structural difference between the two Plus-C proteins localizes in their N- and C-terminal regions, and their concerted conformational change may account for monomer-swapped dimer conversion and furthermore the formation of novel binding pockets. Using a combination of gel filtration chromatography, differential scanning calorimetry, and analytical ultracentrifugation, we demonstrate the AgamOBP48 dimerization in solution. Eventually, molecular modeling calculations were used to predict the binding mode of the most potent synthetic ligand of AgamOBP48 known so far, discovered by ligand- and structure-based virtual screening. The structure-aided identification of multiple OBP binders represents a powerful tool to be employed in the effort to control transmission of the vector-borne diseases.

Synthesis and study of 2-(pyrrolesulfonylmethyl)-N-arylimines: a new class of inhibitors for human glutathione transferase A1-1.

Overexpression of human GSTA1-1 in tumor cells is part of MDR mechanisms. We report on the synthesis of 11 pyrrole derivatives as hGSTA1-1 inhibitors starting from 1-methyl-2-[(2-nitrobenzylsulfanyl]-1H-pyrrole. Molecular modeling revealed two locations in the enzyme H binding site: the catalytic primary one accommodating shorter and longer derivatives and the secondary one, where shorter derivatives can occupy. Derivative 9, displaying the highest inhibition and bearing a p-nitroarylimino moiety, and derivative 4, lacking this moiety, were studied kinetically. Derivative 9 binds (K(i(9)) = 71 ± 4 μM) at the primary site competitively vs CDNB. Derivative 4 binds (K(i(4)) = 135 ± 27 μM) at the primary and secondary sites, allowing the binding of a second molecule (4 or CDNB) leading to formation of unreactive and reactive complexes, respectively. The arylmethylsulfonylpyrrole core structure is a new pharmacophore for hGSTA1-1, whereas its derivative 9 may serve as a lead structure.

Mapping the Anopheles gambiae Odorant Binding Protein 1 (AgamOBP1) using modeling techniques, site directed mutagenesis, circular dichroism and ligand binding assays

The major malaria vector in Sub-Saharan Africa is the Anopheles gambiae mosquito. This species is a key target of malaria control measures. Mosquitoes find humans primarily through olfaction, yet the molecular mechanisms associated with host-seeking behavior remain largely unknown. To further understand the functionality of A. gambiae odorant binding protein 1 (AgamOBP1), we combined in silico protein structure modeling and site-directed mutagenesis to generate 16 AgamOBP1 protein analogues containing single point mutations of interest. Circular dichroism (CD) and ligand-binding assays provided data necessary to probe the effects of the point mutations on ligand binding and the overall structure of AgamOBP1. Far-UV CD spectra of mutated AgamOBP1 variants displayed both substantial decreases to ordered α-helix structure (up to22%) and increases to disordered α-helix structure(up to 15%) with only minimal changes in random coil (unordered) structure. In mutations Y54A, Y122A and W114Q, aromatic side chain removal from the binding site significantly reduced N-phenyl-1-naphthylamine binding. Several non-aromatic mutations (L15T, L19T, L58T, L58Y, M84Q, M84K, H111A, Y122A and L124T) elicited changes to protein conformation with subsequent effects on ligand binding. This study provides empirical evidence for the in silico predicted functions of specific amino acids in AgamOBP1 folding and ligand binding characteristics.

A survey of the availability of primary bioinformatics web resources.

The explosive growth of the bioinformatics field has led to a large amount of data and software applications publicly available as web resources. However, the lack of persistence of web references is a barrier to a comprehensive shared access. We conducted a study of the current availability and other features of primary bioinformatics web resources (such as software tools and databases). The majority (95%) of the examined bioinformatics web resources were found running on UNIX/Linux operating systems, and the most widely used web server was found to be Apache (or Apache-related products). Of the overall 1,130 Uniform Resource Locators (URLs) examined, 91% were highly available (more than 90% of the time), while only 4% showed low accessibility (less than 50% of the time) during the survey. Furthermore, the most common URL failure modes are presented and analyzed.

Isoenzyme- and allozyme-specific inhibitors: 2,2'-dihydroxybenzophenones and their carbonyl N-analogues that discriminate between human glutathione transferase A1-1 and P1-1 allozymes.

The selectivity of certain benzophenones and their carbonyl N‐analogues was investigated towards the human GSTP1‐1 allozymes A, B and C involved in MDR. The allozymes were purified from extracts derived from E. coli harbouring the plasmids pEXP5‐CT/TOPO‐TA‐hGSTP1*A, pOXO4‐hGSTP1*B or pOXO4‐hGSTP1*C. Compound screening with each allozyme activity indicated three compounds with appreciable inhibitory potencies, 12 and 13 with P1‐1A 62% and 67%, 11 and 12 with P1‐1C 51% and 70%, whereas that of 15 fell behind with P1‐1B (41%). These findings were confirmed by IC50 values (74–125 μm). Enzyme inhibition kinetics, aided by molecular modelling and docking, revealed that there is competition with the substrate CDNB for the same binding site on the allozyme (Ki(13/A) = 63.6 ± 3.0 μm, Ki(15/B) = 198.6 ± 14.3 μm, and Ki(11/C) = 16.5 ± 2.7 μm). These data were brought into context by an in silico structural comparative analysis of the targeted proteins. Although the screened compounds showed moderate inhibitory potency against hGSTP1‐1, remarkably, some of them demonstrated absolute isoenzyme and/or allozyme selectivity.

Designer Xanthone: An Inhibitor Scaffold for MDR-Involved Human Glutathione Transferase Isoenzyme A1-1

Glutathione transferases (GSTs) are cell detoxifiers involved in multiple drug resistance (MDR), hampering the effectiveness of certain anticancer drugs. To our knowledge, this is the first report on well-defined synthetic xanthones as GST inhibitors. Screening 18 xanthones revealed three derivatives bearing a bromomethyl and a methyl group (7) or two bromomethyl groups (8) or an aldehyde group (17), with high inhibition potency (>85%), manifested by low IC50 values (7: 1.59 ± 0.25 µM, 8: 5.30 ± 0.30 µM, and 17: 8.56 ± 0.14 µM) and a competitive modality of inhibition versus CDNB (Ki(7) = 0.76 ± 0.18 and Ki(17) = 1.69 ± 0.08 µM). Of them, derivative 17 readily inhibited hGSTA1-1 in colon cancer cell lysate (IC50 = 10.54 ± 2.41 µM). Furthermore, all three derivatives were cytotoxic to Caco-2 intact cells, with 17 being the least cytotoxic (LC50 = 151.3 ± 16.3 µM). The xanthone scaffold may be regarded as a pharmacophore for hGSTA1-1 and the three derivatives, especially 17, as potent precursors for the synthesis of new inhibitors and conjugate prodrugs for human GSTs.

Glutathione analogues as substrates or inhibitors that discriminate between allozymes of the MDR-involved human glutathione transferase P1-1.

Glutathione (GSH) structure‐guided tripeptide analogues were designed and synthesized by solid phase technology, purified (≥95%) by RP and/or GF column chromatography, to identify those that, compared with GSH, exhibited similar or higher binding and catalytic efficiency toward the MDR‐involved human GSTP1‐1 isoenzyme, and could discriminate between the allozymic expression products of the polymorphic human GSTP1 gene locus, designated as hGSTP1*A (Ile104/Ala113), hGSTP1*B (Val104/Ala113), and hGSTP1*C (Val104/Val113). The analogues bear single amino acid alterations as well as alterations in more than one position. Some analogues showed remarkable allozyme selectivity, binding catalytically to A (I, II, IV, XII), to C (V and XVI), to A and C (III, VII, XIV) or to all three allozymes (XV). A heterocyclic substituent at positions 1 or 2 of GSH favors inhibition of A, whereas a small hydrophobic/hydrophilic amide substituent at position 2 (Cys) favors inhibition of B and C. Heterocyclic substituents at position 1, only, produce catalytic analogues for A, whereas less bulky and more flexible hydrophobic/hydrophilic substituents, at positions 1 or 3, lead to effective substrates with C. When such substituents were introduced simultaneously at positions 1 and 3, the analogues produced have no catalytic potential but showed appreciable inhibitory effects, instead, with all allozymes. It is anticipated that when GSH analogues with selective inhibitory or catalytic binding, were conjugated to allozyme‐selective inhibitors of hGSTP1‐1, the derived leads would be useful for the designing of novel chimeric inhibitors against the MDR‐involved hGSTP1‐1 allozymes.

Concluding the trilogy: The interaction of 2,2′-dihydroxy-benzophenones and their carbonyl N-analogues with human glutathione transferase M1-1 face to face with the P1-1 and A1-1 isoenzymes involved in MDR

A series of 2,2′‐dihydroxybenzophenones and their carbonyl N‐analogues were studied as potential inhibitors against human glutathione transferase M1‐1 (hGSTM1‐1) purified from recombinant E. coli. Their screening revealed an inhibition against hGSTM1‐1 within a range of 0‐42% (25 μM). The IC50 values for the two stronger ones, 16 and 13, were 53.5 ± 5.6 μΜ and 28.5 ± 2.5 μΜ, respectively. The results were compared with earlier ones for isoenzymes hGSTP1‐1 and hGSTA1‐1 involved in MDR. All but one bind more strongly to A1‐1, than M1‐1 and P1‐1, the latter being a poor binder. An order of potency A1‐1 > > M1‐1 > P1‐1 meritted 13, 14 and 16 as the most potent inhibitors with hGSTM1‐1. Enzyme kinetics with hGSTM1‐1 (Km(CDNB) 213 ± 10 μΜ and Km(GSH) 303 ± 11 μΜ) revealed a competitive modality for 16 (Ki(16) = 22.3 ± 1.1 μΜ) and a mixed one for 13 versus CDNB (Ki(13) = 33.3 ± 1.6 μM for the free enzyme and Ki(13)′ = 17.7 ± 1.7 μM for the enzyme‐CDNB complex). 5‐ or 5′‐Bromo‐ or phenyl‐substituted (but not in combination) inhibitors, having a H‐bonded oxime weakly acidic group of a small volume, are optimal candidates for binding hGSTM1‐1. The outcome of the isoenzyme trilogy identified good binder leads for the investigated GSTs involved in MDR.

Quantitative Prediction of Critical Amino Acid Positions for Protein Folding

The MIR algorithm provides an ab initio prediction of a proteins core residues. An improved version, the MIR2, is presented and validated on 3203 proteins from PDB. Structures are decomposed in Closed Loops, their limits constituting the observed core residues. They are predicted by MIR2 with an accuracy approaching 80%.

CrystTwiV: a webserver for automated phase extension and refinement in X-ray crystallography.

An important stage in macromolecular crystallography is that of phase extension and refinement when initial phase estimates are available from isomorphous replacement or anomalous scattering or other methods. For this purpose, an alternative method called the twin variables (TwiV) method has been proposed. The algorithm is based on alternately transferring the phase information between the twin variable sets. The phase extension and refinement is evaluated with the crystallographic symmetry test by deliberately sacrificing the space-group symmetry in the starting set, then using its re-appearance as a criterion for correctness. Here we present a software program (CrysTwiV) that runs on the web (freely available at: http://btweb.aua.gr/crystwiv/) implementing the above-mentioned method.An important stage in macromolecular crystallography is that of phase extension and refinement when initial phase estimates are available from isomorphous replacement or anomalous scattering or other methods. For this purpose, an alternative method called the twin variables (TwiV) method has been proposed. The algorithm is based on alternately transferring the phase information between the twin variable sets. The phase extension and refinement is evaluated with the crystallographic symmetry test by deliberately sacrificing the space-group symmetry in the starting set, then using its re-appearance as a criterion for correctness. Here we present a software program (CrysTwiV) that runs on the web (freely available at: http://btweb.aua.gr/crystwiv/) implementing the above-mentioned method.