SEPIa, a knowledge-driven algorithm for predicting conformational B-cell epitopes from the amino acid sequence.

BACKGROUND: The identification of immunogenic regions on the surface of antigens, which are able to be recognized by antibodies and to trigger an immune response, is a major challenge for the design of new and effective vaccines. The prediction of such regions through computational immunology techniques is a challenging goal, which will ultimately lead to a drastic limitation of the experimental tests required to validate their efficiency. However, current methods are far from being sufficiently reliable and/or applicable on a large scale. RESULTS: We developed SEPIa, a B-cell epitope predictor from the protein sequence, which is sufficiently fast to be applicable on a large scale. The originality of SEPIa lies in the combination of two classifiers, a naïve Bayesian and a random forest classifier, through a voting algorithm that exploits the advantages of both. It is based on 13 sequence-based features, whose values in a 9-residue sequence window are compiled to predict the epitope/non-epitope state of the central residue. The features are related to the type of amino acid, its conservation in homologous proteins, and its tendency of being exposed to the solvent, soluble, flexible, and disordered. The highest signal is obtained from statistical amino acid preferences, but all 13 features contribute non-negligibly in the predictor. SEPIa's average prediction accuracy is limited, with an AUC score (area under the receiver operating characteristic curve) that reaches 0.65 both in 10-fold cross-validation and on an independent test set. It is nevertheless slightly higher than that of other methods evaluated on the same test set. CONCLUSIONS: SEPIa was applied to a test protein whose epitopes are known, human ß2 adrenergic G-protein-coupled receptor, with promising results. Although the actual AUC score is rather low, many of the predicted epitopes cluster together and overlap the experimental epitope region. The reasons underlying the limitations of SEPIa and of all other B-cell epitope predictors are discussed.

State-of-the-art technology in modern computer-aided drug design.

The quest for small drug-like compounds that selectively inhibit the function of biological targets has always been a major focus in the pharmaceutical industry and in academia as well. High-throughput screening of compound libraries requires time, cost and resources. Therefore, the use of alternative methods is necessary for facilitating lead discovery. Computational techniques that dock small molecules into macromolecular targets and predict the affinity and activity of the small molecule are widely used in drug design and discovery, and have become an integral part of the industrial and academic research. In this review, we present an overview of some state-of-the-art technologies in modern drug design that have been developed for expediting the search for novel drug candidates.

Cation-π, amino-π, π-π, and H-bond interactions stabilize antigen-antibody interfaces.

The identification of immunogenic regions on the surface of antigens, which are able to stimulate an immune response, is a major challenge for the design of new vaccines. Computational immunology aims at predicting such regions--in particular B-cell epitopes--but is far from being reliably applicable on a large scale. To gain understanding into the factors that contribute to the antigen-antibody affinity and specificity, we perform a detailed analysis of the amino acid composition and secondary structure of antigen and antibody surfaces, and of the interactions that stabilize the complexes, in comparison with the composition and interactions observed in other heterodimeric protein interfaces. We make a distinction between linear and conformational B-cell epitopes, according to whether they consist of successive residues along the polypeptide chain or not. The antigen-antibody interfaces were shown to differ from other protein-protein interfaces by their smaller size, their secondary structure with less helices and more loops, and the interactions that stabilize them: more H-bond, cation-π, amino-π, and π-π interactions, and less hydrophobic packing; linear and conformational epitopes can clearly be distinguished. Often, chains of successive interactions, called cation/amino-π and π-π chains, are formed. The amino acid composition differs significantly between the interfaces: antigen-antibody interfaces are less aliphatic and more charged, polar and aromatic than other heterodimeric protein interfaces. Moreover, paratopes and epitopes-albeit to a lesser extent-have amino acid compositions that are distinct from general protein surfaces. This specificity holds promise for improving B-cell epitope prediction.

A computational investigation on the role of glycosylation in the binding of alpha1 nicotinic acetylcholine receptor with two alpha-neurotoxins.

Based on the crystal structure of the extracellular domain (ECD) of the mouse nicotinic acetylcholine receptor (nAChR) alpha1 subunit bound to α-bungarotoxin (α-Btx) we have generated in silico models of the human nAChR α1 bound to α-Btx and α-cobratoxin (α-Cbtx), both in the presence and in the absence of the N-linked carbohydrate chain. To gain further insight into the structural role of glycosylation molecular dynamics (MD) simulations were carried out in explicit solvent so as to compare the conformational dynamics of the binding interface between nAChR α1 and the two toxins. An interesting observation during the course of the MD simulations is the strengthening of the receptor-toxin interaction in the presence of the carbohydrate chain, mediated through a shift in the position of the sugars towards the bound toxin. Critical protein-sugar interactions implicate residues Ser187 and Trp184 of nAChR and Thr6, Ser9, and Thr15 of α-Btx, as well as Thr6 and Pro7 of α-Cbtx. Analysis of the predicted residue-specific intermolecular interactions is intended to inspire biophysical studies on the functional role of glycosylation in the gating mechanism.

Conformational dynamics of the anthrax lethal factor catalytic center.

Anthrax lethal factor (LF) is a zinc-metalloprotease that together with the protective antigen constitutes anthrax lethal toxin, which is the most prominent virulence factor of the anthrax disease. The solution nuclear magnetic resonance and in silico conformational dynamics of the 105 C-terminal residues of the LF catalytic core domain in its apo form are described here. The polypeptide adopts a compact structure even in the absence of the Zn(2+) cofactor, while the 40 N-terminal residues comprising the metal ligands and residues that participate in substrate and inhibitor recognition exhibit more flexibility than the C-terminal region.

Purification and biophysical characterization of the core protease domain of anthrax lethal factor.

Anthrax lethal toxin (LeTx) stands for the major virulence factor of the anthrax disease. It comprises a 90kDa highly specific metalloprotease, the anthrax lethal factor (LF). LF possesses a catalytic Zn(2+) binding site and is highly specific against MAPK kinases, thus representing the most potent native biomolecule to alter and inactivate MKK [MAPK (mitogen-activated protein kinase) kinases] signalling pathways. Given the importance of the interaction between LF and substrate for the development of anti-anthrax agents as well as the potential treatment of nascent tumours, the analysis of the structure and dynamic properties of the LF catalytic site are essential to elucidate its enzymatic properties. Here we report the recombinant expression and purification of a C-terminal part of LF (LF(672-776)) that harbours the enzyme's core protease domain. The biophysical characterization and backbone assignments ((1)H, (13)C, (15)N) of the polypeptide revealed a stable, well folded structure even in the absence of Zn(2+), suitable for high resolution structural analysis by NMR.

A computational approach to the study of the binding mode of dual ACE/NEP inhibitors.

Combined blockade of the renin-angiotensin-aldosterone system (RAAS) is an attractive therapeutic strategy for the treatment of cardiovascular diseases. Vasopeptidase inhibitors are a group of compounds capable of inhibiting more than one enzyme, which leads to potentiation of natriuretic peptide actions and suppression of the RAAS. In this study, molecular modeling has been used to elucidate key structural features that govern the binding and/or selectivity of a single compound toward the zinc catalytic sites of the N- and C-domains of the angiotensin-converting enzyme (ACE) and the neutral endopeptidase (NEP). Eleven dual inhibitors were categorized in three classes, according to their zinc binding groups. Analysis of their docked conformers revealed the molecular environment of the catalytic sites and the specific interactions between the inhibitors and amino acid residues that are important for selectivity and cooperativity. In addition, inhibitors were predicted to bind to the C-domain of the ACE with greater affinity than the N-domain, with an average difference in the free energy of binding approximately 2-3 kcal mol(-1). Residues that were identified to actively participate in the binding and stabilizing of the enzyme-inhibitor complexes were analyzed in a consensus way for both the ACE and the NEP. These atomic-level insights into enzyme-ligand binding can be used to drive new structure-based drug design processes in the quest for more selective and effective vasopeptidase inhibitors.

Study of a lipophilic captopril analogue binding to angiotensin I converting enzyme.

Human ACE is a central component of the renin-angiotensin system and a major therapeutic target for cardiovascular diseases. The somatic form of the enzyme (sACE) comprises two homologous metallopeptidase domains (N and C), each bearing a zinc active site with similar but distinct substrate and inhibitor specificities. In this study, we present the biological activity of silacaptopril, a silylated analogue of captopril, and its binding affinity towards ACE. Based on the recently determined crystal structures of both the ACE domains, a series of docking calculations were carried out in order to study the structural characteristics and the binding properties of silacaptopril and its analogues with ACE.

Low molecular weight inhibitors of the protease anthrax lethal factor.

Anthrax Lethal Factor (LF) is a zinc-dependent metalloprotease that together with the protective antigen constitute the anthrax lethal toxin, the most prominent virulence factor of the disease anthrax. This review summarizes the current knowledge on anthrax toxicity and defense in relation to LF. Particular emphasis is placed on the structural aspects of LF, the properties of its substrates and the achievements in the design of low molecular weight inhibitors of the catalytic activity of the metalloenzyme.

Putative bioactive conformations of amide linked cyclic myelin basic protein peptide analogues associated with experimental autoimmune encephalomyelitis.

The solution models of cyclo(87-99) MBP87-99, cyclo(87-99) [Ala91,96] MBP87-99, and cyclo(87-99) [Arg91, Ala96] MBP87-99 have been determined through 2D NMR spectroscopy in DMSO-d6. Chemical shift analysis has been performed in an attempt to elucidate structural changes occurring upon substitution of native residues. NMR-derived geometrical constraints have been used in order to calculate high-resolution conformers of the above peptides. Conformational analysis of the three synthetic analogues show that the bioactivity, or the lack of it, may possibly be due to the distinct local structure observed and the subsequent differences in the overall topology and exposed area after binding with Major Histocompatibility Complex II (MHC II). It is believed that an overall larger solvent accessible area blocks the approach and binding of the T-cell receptor (TCR) on the altered peptide ligand (APL)-MHC complex, whereas more compact structures do not occlude weak interactions with an approaching TCR and can cause Experimental Autoimmune Encephalomyelitis (EAE) antagonism. A pharmacophore model based on the structural data has been generated.

Zn(II)/pyridyloxime complexes as potential reactivators of OP-inhibited acetylcholinesterase: in vitro and docking simulation studies.

In order to investigate the ability of metal complexes to act as reactivators of organophosphorus compounds (OP)-inhibited acetylcholinesterase (AChE), we have synthesized and crystallographically characterized three novel mononuclear Zn(II) complexes formulated as [ZnCl2{(4-py)CHNOH}2] (1), [ZnBr2{(4-py)CHNOH}2] (2) and [Zn(O2CMe)2{(4-py)CHNOH}2]∙2MeCN (3∙2MeCN), where (4-py)CHNOH is 4-pyridinealdoxime. Their reactivation potency was tested in vitro with a slight modification of the Ellman's method using Electric eel acetylcholinesterase and the insecticide paraoxon (diethyl 4-nitrophenyl phosphate) as inhibitor. The activity of the already reported complex [Zn2(O2CPh)2{(4-py)CHNOH}2]·2MeCN (4·2MeCN) and of the clinically used drug obidoxime 1,1'-[oxybis(methylene)]bis{4-[(E)- (hydroxyimino)methyl]pyridinium} was also examined. The results of the in vitro experiments demonstrate moderate reactivation of the metal complexes compared to the drug obidoxime. On the other hand, it is clearly shown that the metal complex is the responsible molecular entity for the observed activity, as the reactivation efficacy of the organic ligand (4-pyridinealdoxime) is found to be inconsequential. Docking simulation studies were performed in the light of predicted complex-enzyme interactions using the paraoxon-inhibited enzyme along with the four Zn(II) complexes and obidoxime as a reference reactivator. The results showed that the three mononuclear metal complexes possess the required characteristics to be accommodated into the active site of AChE, while the entrance of the dinuclear Zn(II) compound is unsuccessful. An interesting outcome of docking simulations is the fact that the mononuclear compounds accommodate into the active site of AChE in a similar mode as obidoxime.

Insights into the anthrax lethal factor-substrate interaction and selectivity using docking and molecular dynamics simulations.

The anthrax toxin of the bacterium Bacillus anthracis consists of three distinct proteins, one of which is the anthrax lethal factor (LF). LF is a gluzincin Zn-dependent, highly specific metalloprotease with a molecular mass of approximately 90 kDa that cleaves most isoforms of the family of mitogen-activated protein kinase kinases (MEKs/MKKs) close to their amino termini, resulting in the inhibition of one or more signaling pathways. Previous studies on the crystal structures of uncomplexed LF and LF complexed with the substrate MEK2 or a MKK-based synthetic peptide provided structure-activity correlations and the basis for the rational design of efficient inhibitors. However, in the crystallographic structures, the substrate peptide was not properly oriented in the active site because of the absence of the catalytic zinc atom. In the current study, docking and molecular dynamics calculations were employed to examine the LF-MEK/MKK interaction along the catalytic channel up to a distance of 20 A from the zinc atom. This residue-specific view of the enzyme-substrate interaction provides valuable information about: (i) the substrate selectivity of LF and its inactivation of MEKs/MKKs (an issue highly important not only to anthrax infection but also to the pathogenesis of cancer), and (ii) the discovery of new, previously unexploited, hot-spots of the LF catalytic channel that are important in the enzyme/substrate binding and interaction.