Sviatlana Yahorava

A Look Inside HIV Resistance through Retroviral Protease Interaction Maps

Retroviruses affect a large number of species, from fish and birds to mammals and humans, with global socioeconomic negative impacts. Here the authors report and experimentally validate a novel approach for the analysis of the molecular networks that are involved in the recognition of substrates by retroviral proteases. Using multivariate analysis of the sequence-based physiochemical descriptions of 61 retroviral proteases comprising wild-type proteases, natural mutants, and drug-resistant forms of proteases from nine different viral species in relation to their ability to cleave 299 substrates, the authors mapped the physicochemical properties and cross-dependencies of the amino acids of the proteases and their substrates, which revealed a complex molecular interaction network of substrate recognition and cleavage. The approach allowed a detailed analysis of the molecular–chemical mechanisms involved in substrate cleavage by retroviral proteases.

Kinetic evidence for tandemly arranged ligand binding sites in melanocortin 4 receptor complexes.

The melanocortin 4 receptor (MC(4)R) binding of the peptide analogue of melanocyte stimulating hormone, [(125)I]NDP-MSH, and the low molecular weight radionucleid 1-(D-1,2,3,4-tetrahydroisoquinoline-3-carboxy-D-4-(125)iodophenylalanyl)-4-cyclohexyl-4-[(1,2,4-triazol-1-yl)methyl]piperidine trifluoroacetate ([(125)I]THIQ) were compared. Kinetic analysis indicated heterogeneity in the binding of both radioligands, the binding apparently proceeding to two tandemly arranged interconnected mutually dependent binding sites. Steric considerations and BRET analysis of Rluc and GFP tagged receptors proposed that these sites are located on different subunits of receptor dimers, which form receptor complexes. According to the minimal model proposed, ligand binding proceeds consecutively to the two binding sites of the dimer. After binding of the first ligand conformational transformations of the complex occur, which is followed by binding of the second ligand. When both receptor units have bound [(125)I]NDP-MSH, the radioligand can be released only from one unit. The [(125)I]NDP-MSH bound to the remaining unit stays practically irreversibly bound due to a very slow retransformation rate of the transformed complex. The considerably faster binding of [(125)I]THIQ did not allow accurate kinetic differentiation of the two binding sites. However, addition of NDP-MSH as well as a fragment of the human agouti protein, hAGRP(83-132) to the preformed [(125)I]THIQ-MC(4)R complex drastically retarded the release of [(125)I]THIQ from the complex, blocking conformational transformations in the complex by binding into the second binding site. The consecutive binding of ligands to the MC(4)R dimers has substantial impact on the apparent ligand potencies, when determined in competition with the two different radioligands applied herein; the apparent potencies of the same ligand differing up to three orders of magnitude when assayed in competition with [(125)I]NDP-MSH or [(125)I]THIQ.

Design and evaluation of substrate-based octapeptide and non substrate-based tetrapeptide inhibitors of dengue virus NS2B-NS3 proteases.

A series of 45 peptide inhibitors was designed, synthesized, and evaluated against the NS2B-NS3 proteases of the four subtypes of dengue virus, DEN-1-4. The design was based on proteochemometric models for Michaelis (Km) and cleavage rate constants (kcat) of protease substrates. This led first to octapeptides showing submicromolar or low micromolar inhibitory activities on the four proteases. Stepwise removal of cationic substrate non-prime side residues and variations in the prime side sequence resulted finally in an uncharged tetrapeptide, WYCW-NH2, with inhibitory Ki values of 4.2, 4.8, 24.4, and 11.2 μM for the DEN-1-4 proteases, respectively. Analysis of the inhibition data by proteochemometric modeling suggested the possibility for different binding poses of the shortened peptides compared to the octapeptides, which was supported by results of docking of WYCW-NH2 into the X-ray structure of DEN-3 protease.

2-(p-Hydroxybenzyl)indoles - side products formed upon cleavage of indole derivatives from carboxylated Wang polymer- an NMR study.

Treatment of carboxylated Wang polymer attached to a 2-unsubstituted indole derivative with a trifluoroacetic acid based mixture resulted in a side reaction: p-hydroxybenzylation at the 2-position of the indole ring. The structure of the resulting N-3-aminopropyl)-N-benzyl-4-[2-(4-hydroxybenzyl)-1H-indol-3-yl]-butyramide trifluoroacetate was ascertained by a full assignment of its 1H- and 13C-NMR spectra. The side reaction could be suppressed by the use of 1,2-ethanedithiol in high concentrations (16 %).

Improving the affinity of antigens for mutated antibodies by use of statistical molecular design

We demonstrate the use of statistical molecular design (SMD) in the selection of peptide libraries aimed to systematically investigate antigen‐antibody binding spaces. Earlier, we derived two novel antibodies by mutating the complementarity‐determining region of the anti‐p24 (HIV‐1) single chain Fv antibody, CB4‐1 that had lost their affinity for a p24 epitope‐homologous peptide by 8‐ and 60‐fold. The present study was devoted to explore how peptide libraries can be designed under experimental design criteria for effective screening of peptide antigens. Several small peptide–antigen libraries were selected using SMD principles and their activities were evaluated by their binding to SPOT‐synthesized peptide membranes and by fluorescence polarization (FP). The approach was able to reveal the most critical residues required for antigen binding, and finally to increase the binding activity by proper modifications of amino acids in the peptide antigen. A model of the active peptide binding pocket formed by the mutated scFv and the antigen was compatible with the information gained from the experimental data. Our results suggest that SMD approaches can be used to explore peptide antigen features essential for their interactions with antibodies. Copyright

Libiguins A and B : Novel Phragmalin Limonoids Isolated from Neobeguea mahafalensis Causing Profound Enhancement of Sexual Activity

In a screening programme directed towards the discovery of drugs that could enhance sexual activity, we found that a decoction of the root bark of Neobeguea mahafalensis displayed an extraordinarily high potency and remarkably long duration in augmenting sexual activity in male rodents. Bioassay-guided fractionation led to the isolation of two pharmacoactive constituents, which turned out to be novel 1,8,9-orthoacetate phragmalin limonoids that we named libiguins A and B, each with a C-16/30 δ-lactone ring. Chemical structures were established by the interpretation of their 1D and 2D NMR data. In vivo pharmacological tests showed that starting with a treatment from 0.004-0.4 mg/kg/day for three consecutive days, over a 3-h sampling period, these limonoids induced a long-lasting augmentation of frequency and sustainment of mounting behaviour in male rodents, with an effect lasting for up to 11 days post-treatment. Libiguin A proved to be markedly more potent than libiguin B. This report is the first of limonoids having such an effect, and the findings could lead to novel therapies for the treatment of sexual dysfunction. Moreover, the results can serve as an opening to elucidate the central physiological control of mating behaviour, which is still not well mapped out.

Semisynthesis of libiguin A and its analogues by trans-lactonization of phragmalin.

Libiguins are limonoids with highly potent sexual activity enhancing effects, originally isolated from the Madagascarian Meliaceae species Neobeguea mahafalensis, where they exist in only minute quantities. Their low natural abundance has hampered mapping of their biological effects. Here we describe an approach to the semisynthesis of libiguin A and its close analogues 1-3 starting from phragmalin, which is a limonoid present in high amounts in a commercially cultivated Meliaceae species, Chukrasia tabularis, allowing the preparation of libiguins in appreciable quantities.

A new protolimonoid from Capuronianthus mahafalensis

From stem barks of Capuronianthus mahafalensis (Meliaceae) endemic to Madagascar, a new protolimonoid named capulin containing a four membered ring in its side chain was isolated by repeated silica gel column chromatography. Its structure was determined by 1D and 2D NMR spectroscopy and high-resolution MS. To the best of our knowledge, this is the first time that a four-membered ring occurs in the side chain of protolimonoids.