New Polyfunctional Phragmalin Limonoids from Neobeguea mahafalensis.

Neobeguea mahafalensis is used as a medicinal plant in Madagascar. A decoction of the stem bark of this species is reported to treat back pain. Recently, it was reported that a decoction of the root bark, containing two novel phragmalin limonoids as identified active constituents, exhibited an extraordinarily high potency and remarkably long duration in augmenting sexual activity in male rodents.From the dichloromethane extract of the root barks of N. mahafalensis, nine phragmalin limonoids were isolated, of which eight were novel compounds. The structures were established mainly by extensive use of 2D NMR spectroscopic techniques and high-resolution mass spectrometry. One of the new compounds named dodoguin displayed sleep-inducing activity in Swiss albino mice. The amount of 3-10 mg/kg of this compound induced sleep 18-22 min after its administration with a duration of 16-18 min.

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.

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.

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.

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.

Proteochemometrics mapping of the interaction space for retroviral proteases and their substrates.

Understanding the complex interactions of retroviral proteases with their ligands is an important scientific challenge in efforts to achieve control of retroviral infections. Development of drug resistance because of high mutation rates and extensive polymorphisms causes major problems in treating the deadly diseases these viruses cause, and prompts efforts to identify new strategies. Here we report a comprehensive analysis of the interaction of 63 retroviral proteases from nine different viral species with their substrates and inhibitors based on publicly available data from the past 17years of retroviral research. By correlating physico-chemical descriptions of retroviral proteases and substrates to their biological activities we constructed a highly statistically valid 'proteochemometric' model for the interactome of retroviral proteases. Analysis of the model indicated amino acid positions in retroviral proteases with the highest influence on ligand activity and revealed general physicochemical properties essential for tight binding of substrates across multiple retroviral proteases. Hexapeptide inhibitors developed based on the discovered general properties effectively inhibited HIV-1 proteases in vitro, and some exhibited uniformly high inhibitory activity against all HIV-1 proteases mutants evaluated. A generalized proteochemometric model for retroviral proteases interactome has been created and analysed in this study. Our results demonstrate the feasibility of using the developed general strategy in the design of inhibitory peptides that can potentially serve as templates for drug resistance-improved HIV retardants.

Proteochemometrics analysis of substrate interactions with dengue virus NS3 proteases.

The prime side specificity of dengue protease substrates was investigated by use of proteochemometrics, a technology for drug target interaction analysis. A set of 48 internally quenched peptides were designed using statistical molecular design (SMD) and assayed with proteases of four subtypes of dengue virus (DEN-1-4) for Michaelis (K(m)) and cleavage rate constants (k(cat)). The data were subjected to proteochemometrics modeling, concomitantly modeling all peptides on all the four dengue proteases, which yielded highly predictive models for both activities. Detailed analysis of the models then showed that considerably differing physico-chemical properties of amino acids contribute independently to the K(m) and k(cat) activities. For k(cat), only P1' and P2' prime side residues were important, while for K(m) all four prime side residues, P1'-P4', were important. The models could be used to identify amino acids for each P' substrate position that are favorable for, respectively, high substrate affinity and cleavage rate.

Oligomerization of indole derivatives with incorporation of thiols.

Two molecules of indole derivative, e.g. indole-5-carboxylic acid, reacted with one molecule of thiol, e.g. 1,2-ethanedithiol, in the presence of trifluoroacetic acid to yield adducts such as 3-[2-(2-amino-5-carboxyphenyl)-1-(2-mercaptoethylthio)ethyl]-1Hindole-5-carboxylic acid. Parallel formation of dimers, such as 2,3-dihydro-1H,1'H-2,3'-biindole-5,5'-dicarboxylic acid and trimers, such as 3,3'-[2-(2-amino-5-carboxyphenyl) ethane-1,1-diyl]bis(1H-indole-5-carboxylic acid) of the indole derivatives was also observed. Reaction of a mixture of indole and indole-5-carboxylic acid with 2-phenylethanethiol proceeded in a regioselective way, affording 3-[2-(2-aminophenyl)-1-(phenethylthio)ethyl]-1H-indole-5-carboxylic acid. An additional product of this reaction was 3-[2-(2-aminophenyl)-1-(phenethylthio)ethyl]-2,3-dihydro-1H,1'H-2,3'-biindole-5'-carboxylic acid, which upon standing in DMSO-d6 solution gave 3-[2-(2-aminophenyl)-1-(phenethylthio)ethyl]-1H,1'H-2,3'-biindole-5'-carboxylic acid. Structures of all compounds were elucidated by NMR, and a mechanism for their formation was suggested.

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.

Design and synthesis of a library of tertiary amides: evaluation as mimetics of the melanocortins' active core.

Two hundred and ten tertiary amides were prepared on solid phase. Diamines were coupled to activated carboxylated Wang polymer, and the polymeric substituted benzyloxycarbonyl protected diamines obtained were reacted with aldehydes or ketones in trimethyl orthoformate giving resin attached Schiff bases. Coupled resins were then reduced to secondary amines by sodium cyanoborohydride in 4% acetic acid/trimethyl orthoformate, followed by acylation with the carboxylic acid in the presence of PyBroP and diisopropylethylamine. Cleavage of tertiary amides from the resin was made by trifluoroacetic acid in the presence of scavengers (mainly 1,2-ethanedithiol). When indole derivatives were prepared, parallel alkylation with the linker fragment occurred, giving derivatives of 2-(4-hydroxybenzyl)-indole as side products. Solution synthesis or mixed liquid/solid phase preparation of title substances proved to be advantageous in cases when the above method did not give acceptable results. According to this approach an efficient formation of Schiff bases was achieved in the presence of TiCl(4). Substances were isolated by reversed phase chromatography; in some cases isomers were additionally separated by chiral chromatography on Chirobiotic T. When tested on human recombinant melanocortin receptors all the tertiary amides showed some binding affinities; for the highest affinity compounds the K(i)s reached 400 nM on MC(1), 2 microM on MC(3) and 1 microM on MC(4) and MC(5) receptors. cAMP assays of some of the title compounds showed that the tertiary amides are melanocortin receptor antagonists on the four MC receptor subtypes.

Proteochemometric modelling of antibody-antigen interactions using SPOT synthesised peptide arrays.

Proteochemometrics is a technology for the study of molecular recognition based on chemometric techniques. Here we applied it to analyse the amino acids and amino acid physico-chemical properties that are involved in antibodies' recognition of peptide antigens. To this end, we used a study system comprised by a diverse single chain antibody library derived from the murine mAb anti-p24 (HIV-1) antibody CB4-1, evaluated on peptide arrays manufactured by SPOT synthesis. The binding pattern obtained was correlated to physico-chemical descriptors (z-scales) of antibodies and peptides amino acids using partial least-squares projections to latent structures. Cross terms derived from antibody and antigen descriptors were included, which substantially improved the proteochemometric model. The final model was statistically highly satisfactory with a correlation coefficient R(2) = 0.73 and predictive ability Q(2) = 0.68. The physico-chemical properties of each interacting amino acid residue of both the peptides and the antibodies being essential for the antigen-antibody recognition could be retrieved from the model. The study shows for the first time the feasibility of using proteochemometrics to analyse the molecular recognition of antigens by antibodies.

QSAR of multiple mutated antibodies.

The aim of this study was to develop predictive quantitative structure-activity relationship (QSAR) modeling for antibody-peptide interactions. A small single chain antibody library was designed and manufactured around the murine anti-p24 (HIV-1) monoclonal antibody CB4-1 by use of statistical molecular design (SMD) principles and site directed mutagenesis, and its affinity for a p24 derived antigen was determined by fluorescence polarization. A satisfactory QSAR model (Q(2) = 0.74, R(2) = 0.88) was derived by correlating the affinity data to physicochemical property scales of the amino acids varied in the library. The model explains most of the antibody-antigen interactions of the studied set, and provides insights into the molecular mechanism involved in antigen binding.

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.

Probing the substrate specificity of the dengue virus type 2 NS3 serine protease by using internally quenched fluorescent peptides.

The NS3 (dengue virus non-structural protein 3) serine protease of dengue virus is an essential component for virus maturation, thus representing an attractive target for the development of antiviral drugs directed at the inhibition of polyprotein processing. In the present study, we have investigated determinants of substrate specificity of the dengue virus NS3 protease by using internally quenched fluorogenic peptides containing Abz (o-aminobenzoic acid; synonymous to anthranilic acid) and 3-nitrotyrosine (nY) representing both native and chimaeric polyprotein cleavage site sequences. By using this combinatorial approach, we were able to describe the substrate preferences and determinants of specificity for the dengue virus NS2B(H)-NS3pro protease. Kinetic parameters (kcat/K(m)) for the hydrolysis of peptide substrates with systematic truncations at the prime and non-prime side revealed a length preference for peptides spanning the P4-P3' residues, and the peptide Abz-RRRRSAGnY-amide based on the dengue virus capsid protein processing site was discovered as a novel and efficient substrate of the NS3 protease (kcat/K(m)=11087 M(-1) x s(-1)). Thus, while having confirmed the exclusive preference of the NS3 protease for basic residues at the P1 and P2 positions, we have also shown that the presence of basic amino acids at the P3 and P4 positions is a major specificity-determining feature of the dengue virus NS3 protease. Investigation of the substrate peptide Abz-KKQRAGVLnY-amide based on the NS2B/NS3 polyprotein cleavage site demonstrated an unexpected high degree of cleavage efficiency. Chimaeric peptides with combinations of prime and non-prime sequences spanning the P4-P4' positions of all five native polyprotein cleavage sites revealed a preponderant effect of non-prime side residues on the K(m) values, whereas variations at the prime side sequences had higher impact on kcat.

Proteochemometric mapping of the interaction of organic compounds with melanocortin receptor subtypes.

Proteochemometrics was applied in the analysis of the binding of organic compounds to wild-type and chimeric melanocortin receptors. Thirteen chimeric melanocortin receptors were designed based on statistical molecular design; each chimera contained parts from three of the MC(1,3-5) receptors. The binding affinities of 18 compounds were determined for these chimeric melanocortin receptors and the four wild-type melanocortin receptors. The data for 14 of these compounds were correlated to the physicochemical and structural descriptors of compounds, binary descriptors of receptor sequences, and cross-terms derived from ligand and receptor descriptors to obtain a proteochemometric model (correlation was performed using partial least-squares projections to latent structures; PLS). A well fitted mathematical model (R(2) = 0.92) with high predictive ability (Q(2) = 0.79) was obtained. In a further validation of the model, the predictive ability for ligands (Q(2)lig = 0.68) and receptors (Q(2)rec = 0.76) was estimated. The model was moreover validated by external prediction by using the data for the four additional compounds that had not at all been included in the proteochemometric model; the analysis yielded a Q(2)ext = 0.73. An interpretation of the results using PLS coefficients revealed the influence of particular properties of organic compounds on their affinity to melanocortin receptors. Three-dimensional models of melanocortin receptors were also created, and physicochemical properties of the amino acids inside the receptors' transmembrane cavity were correlated to the PLS modeling results. The importance of particular amino acids for selective binding of organic compounds was estimated and used to outline the ligand recognition site in the melanocortin receptors.

New substituted piperazines as ligands for melanocortin receptors. Correlation to the X-ray structure of "THIQ".

A series of piperazine analogues of the melanocortin 4 receptor (MC4R) specific small-molecule agonist "THIQ" was synthesized and characterized structurally and pharmacologically. First, several THIQ imitations lacking the triazole moiety were prepared. Syntheses included acylation of 4-phenylpiperazine or 4-cyclohexylpiperazine. In two cases the tertiary amine function was replaced by the corresponding N-oxide. To obtain more complex structures, a 4-substituted piperazine ring was formed by alkylation of the primary amino group of cyclohexane-derived amino alcohols with N,N-bis(2-chloroethyl)benzylamine. The hydroxylic group of the intermediate was first activated with methanesulfonyl chloride, and the sulfonic ester formed in situ was introduced into the reaction with the sodium salt of 1,2,4-triazole. In one case (i.e., preparation of 23c) introduction of the 1,2,4-triazole moiety was performed at a carbon of the cyclohexane ring. In addition, this intermediate contained a piperazine moiety connected via its nitrogen atom to a cyclohexane ring carbon neighboring the reaction center. As established in NMR and X-ray investigations herein, this substitution proceeded with retention of the initial trans configuration of 1,2-disubstituted cyclohexane. To obtain pure enantiomers of 23c, its precursor 21c was subjected to chiral chromatography on a Chirobiotic V column. The derivatives (R,R)-21cand (S,S)-21c obtained were introduced into further syntheses steps, giving (R,R)-23c and (S,S)-23c, respectively. Melanocortin MC(1,3-5) receptor binding studies showed that all tested piperazine derivatives were active. Several compounds showed clear selectivity for MC4R, with submicromolar affinities being obtained. Among them, one substance, (R,R)-23c, displayed a biphasic curve in displacement of [125I]NDP-MSH on MC4R [K(i)high = 1 nM and K(i)low = 260 nM]. This biphasic competition curve was similarly biphasic to the competition curve obtained herein using THIQ. An X-ray study performed on crystals of the THIQ sulfate salt revealed two closely related conformations, which resemble the shape of the letter "Y", where piperidine and 4-chlorophenyl groups are situated close to each other, but the 1,2,3,4-tetrahydroisoquinoline residue is remote, the triazole function being highly exposed to the environment. The crystals of the dinitrate salt of (R,R)-23c showed a different conformation, where parts of the molecule are spread out almost symmetrically around the central section. Molecular modeling, based on the THIQ crystal structure and the functional similarity of THIQ and (R,R)-23c, allowed us to suggest a possible "bioactive" conformation of (R,R)-23c that is similar to the crystal conformation of THIQ.