Computer-aided identification of potential drug targets and vaccine candidates in bacterial proteomes

Comunicaciones a congreso

Assessing the structural conservation of protein pockets to study functional and allosteric sites: implications for drug discovery

Background: With the classical, active-site oriented drug-development approach reaching its limits, protein ligand-binding sites in general and allosteric sites in particular are increasingly attracting the interest of medicinal chemists in the search for new types of targets and strategies to drug development. Given that allostery represents one of the most common and powerful means to regulate protein function, the traditional drug discovery approach of targeting active sites can be extended by targeting allosteric or regulatory protein pockets that may allow the discovery of not only novel drug-like inhibitors, but activators as well. The wealth of available protein structural data can be exploited to further increase our understanding of allosterism, which in turn may have therapeutic applications. A first step in this direction is to identify and characterize putative effector sites that may be present in already available structural data. Results: We performed a large-scale study of protein cavities as potential allosteric and functional sites, by integrating publicly available information on protein sequences, structures and active sites for more than a thousand protein families. By identifying common pockets across different structures of the same protein family we developed a method to measure the pocket's structural conservation. The method was first parameterized using known active sites. We characterized the predicted pockets in terms of sequence and structural conservation, backbone flexibility and electrostatic potential. Although these different measures do not tend to correlate, their combination is useful in selecting functional and regulatory sites, as a detailed analysis of a handful of protein families shows. We finally estimated the numbers of potential allosteric or regulatory pockets that may be present in the data set, finding that pockets with putative functional and effector characteristics are widespread across protein families. Conclusions: Our results show that structurally conserved pockets are a common feature of protein families. The structural conservation of protein pockets, combined with other characteristics, can be exploited in drug discovery procedures, in particular for the selection of the most appropriate target protein and pocket for the design of drugs against entire protein families or subfamilies (e.g. for the development of broad-spectrum antimicrobials) or against a specific protein (e.g. in attempting to reduce side effects)

Calculation of NMR-relaxation parameters for flexible molecules from molecular dynamics simulations

Comparatively small molecules such as peptides can show a high internal mobility with transitions between several conformational minima and sometimes coupling between rotational and internal degrees of freedom. In those cases the interpretation of NMR relaxation data is difficult and the use of standard methods for structure determination is questionable. On the other hand, in the case of those system sizes, the timescale of both rotational and internal motions is accessible by molecular dynamics (MD) simulations using explicit solvent. Thus a comparison of distance averages (〈r −6〉−1/6 or 〈r −3〉1/3) over the MD trajectory with NOE (or ROE) derived distances is no longer necessary, the (back)calculation of the complete spectra becomes possible. In the present study we use two 200ns trajectories of a heptapeptide of β-amino acids in methanol at two different temperatures to obtain theoretical ROESY spectra by calculating the exact spectral densities for the interproton vectors and the full relaxation matrix. Those data are then compared with the experimental ones. This analysis permits to test some of the assumptions and approximations that generally have to be made to interpret NMR spectra, and to make a more reliable prediction of the conformational equilibrium that leads to the experimental spectru

antibacTR : dynamic antibacterial-drug-target ranking integrating comparative genomics, structural analysis and experimental annotation

Background : development of novel antibacterial drugs is both an urgent healthcare necessity and a partially neglected field. The last decades have seen a substantial decrease in the discovery of novel antibiotics, which combined with the recent thrive of multi-drug-resistant pathogens have generated a scenario of general concern. The procedures involved in the discovery and development of novel antibiotics are economically challenging, time consuming and lack any warranty of success. Furthermore, the return-on-investment for an antibacterial drug is usually marginal when compared to other therapeutics, which in part explains the decrease of private investment. - Results : in this work we present antibacTR, a computational pipeline designed to aid researchers in the selection of potential drug targets, one of the initial steps in antibacterial-drug discovery. The approach was designed and implemented as part of two publicly funded initiatives aimed at discovering novel antibacterial targets, mechanisms and drugs for a priority list of Gram-negative pathogens: Acinetobacter baumannii, Escherichia coli, Helicobacter pylori, Pseudomonas aeruginosa and Stenotrophomonas maltophilia. However, at present this list has been extended to cover a total of 74 fully sequenced Gram-negative pathogens. antibacTR is based on sequence comparisons and queries to multiple databases (e.g. gene essentiality, virulence factors) to rank proteins according to their potential as antibacterial targets. The dynamic ranking of potential drug targets can easily be executed, customized and accessed by the user through a web interface which also integrates computational analyses performed in-house and visualizable on-site. These include three-dimensional modeling of protein structures and prediction of active sites among other functionally relevant ligand-binding sites. - Conclusions : given its versatility and ease-of-use at integrating both experimental annotation and computational analyses, antibacTR may effectively assist microbiologists, medicinal-chemists and other researchers working in the field of antibacterial drug-discovery. The public web-interface for antibacTR is available at 'http://bioinf.uab.cat/antibactr'

Effect of Oxidative Damage on the Stability and Dimerization of Superoxide Dismutase 1

During their life cycle, proteins are subject to different modifications involving reactive oxygen species. Such oxidative damage to proteins may lead to the formation of insoluble aggregates and cytotoxicity and is associated with age-related disorders including neurodegenerative diseases, cancer, and diabetes. Superoxide dismutase 1 (SOD1), a key antioxidant enzyme in human cells, is particularly susceptible to such modifications. Moreover, this homodimeric metalloenzyme has been directly linked to both familial and sporadic amyotrophic lateral sclerosis (ALS), a devastating, late-onset motor neuronal disease, with more than 150 ALS-related mutations in the SOD1 gene. Importantly, oxidatively damaged SOD1 aggregates have been observed in both familial and sporadic forms of the disease. However, the molecular mechanisms as well as potential implications of oxidative stress in SOD1-induced cytotoxicity remain elusive. In this study, we examine the effects of oxidative modification on SOD1 monomer and homodimer stability, the key molecular properties related to SOD1 aggregation. We use molecular dynamics simulations in combination with thermodynamic integration to study microscopic-level site-specific effects of oxidative "mutations" at the dimer interface, including lysine, arginine, proline and threonine carbonylation, and cysteine oxidation. Our results show that oxidative damage of even single residues at the interface may drastically destabilize the SOD1 homodimer, with several modifications exhibiting a comparable effect to that of the most drastic ALS-causing mutations known. Additionally, we show that the SOD1 monomer stability decreases upon oxidative stress, which may lead to partial local unfolding and consequently to increased aggregation propensity. Importantly, these results suggest that oxidative stress may play a key role in development of ALS, with the mutations in the SOD1 gene being an additional factor

Irreproducibility in Research. What can we do about it?

We all would agree with Karl Popper's statement: 'Non-reproducible single occurrences are of no significance to science.' But what if a substantial percentage of published scientific facts are of the irreproducible category? Such an alarming scenario may be close to reality, according to a number of recent reports. Indeed, some shocking statistics suggest that irreproducibility has gone awry in the last years. For instance, pharma and biotech companies can only reproduce between 11 and 25% high-impact research papers in the field of cancer research

Characterization of lithic raw materials from the Paleolithic site of Dolina de l’Esquerda de les Alzines (Garraf, Barcelona)

El presente estudio se centra en la caracterización macroscópica y microscópica de las materias primas silíceas del yacimiento de la Dolina de l’Esquerda de les Alzines, un yacimiento del Pleistoceno superior ubicado en el macizo del Garraf. El objetivo ha sido establecer distintas variedades de sílex, mediante la descripción macroscópica y microscópica de los elementos del conjunto lítico, para disponer, por vez primera, de unas categorías definidas de los recursos abióticos silíceos disponibles y explotados durante la prehistoria en este macizo. Además, mediante el presente estudio se ha evaluado también la posible procedencia y el área captación de dichas materias.The present study focuses on the macroscopic and microscopic characterization of siliceous raw materials from the archeological site of Dolina de l’Esquerda de les Alzines, an Upper Pleistocene deposit located in the Garraf Massif. The aim of this study has been to establish different varieties of chert, by a precise description of the elements of the lithic assemblage, to provide for the first time a few categories defined from the siliceous abiotic resources available and exploited during Prehistory in the massif. Furthermore, through the present study we also assessed the possible origin and procurement area of such materials

Free energy calculations offer insights into the influence of receptor flexibility on ligand-receptor binding affinities

Docking algorithms for computer-aided drug discovery and design often ignore or restrain the flexibility of the receptor, which may lead to a loss of accuracy of the relative free enthalpies of binding. In order to evaluate the contribution of receptor flexibility to relative binding free enthalpies, two host-guest systems have been examined: inclusion complexes of α-cyclodextrin (αCD) with 1-chlorobenzene (ClBn), 1-bromobenzene (BrBn) and toluene (MeBn), and complexes of DNA with the minor-groove binding ligands netropsin (Net) and distamycin (Dist). Molecular dynamics simulations and free energy calculations reveal that restraining of the flexibility of the receptor can have a significant influence on the estimated relative ligand-receptor binding affinities as well as on the predicted structures of the biomolecular complexes. The influence is particularly pronounced in the case of flexible receptors such as DNA, where a 50% contribution of DNA flexibility towards the relative ligand-DNA binding affinities is observed. The differences in the free enthalpy of binding do not arise only from the changes in ligand-DNA interactions but also from changes in ligand-solvent interactions as well as from the loss of DNA configurational entropy upon restrainin

Caracterización de las amterias primas líticas del yacimiento paleolítico de la Dolina de l'Esquerda de ls Alzines (macizo del Garraf, Barcelona)

[spa] El presente estudio se centra en la caracterización macroscópica y microscópica de las materias primas silíceas del yacimiento de la Dolina de l'Esquerda de les Alzines, un yacimiento del Pleistoceno superior ubicado en el macizo del Garraf. El objetivo ha sido establecer distintas variedades de sílex, mediante la descripción macroscópica y microscópica de los elementos del conjunto lítico, para disponer, por vez primera, de unas categorías definidas de los recursos abióticos silíceos disponibles y explotados durante la prehistoria en este macizo. Además, mediante el presente estudio se ha evaluado también la posible procedencia y el área captación de dichas materias. [eng]The present study focuses on the macroscopic and microscopic characterization of siliceous raw materials from the archeological site of Dolina de l'Esquerda de les Alzines, an Upper Pleistocene deposit located in the Garraf Massif. The aim of this study has been to establish different varieties of chert, by a precise description of the elements of the lithic assemblage, to provide for the first time a few categories defined from the siliceous abiotic resources available and exploited during Prehistory in the massif. Furthermore, through the present study we also assessed the possible origin and procurement area of such materials

Circular dichroism spectra of β-peptides: sensitivity to molecular structure and effects of motional averaging

Circular dichroism spectra of two β-peptides, i.e. peptides composed of β-amino acids, calculated using ensembles of configurations obtained by molecular dynamics simulation are presented. The calculations were based on 200ns simulations of a β-heptapeptide in methanol at 298K and 340K and a 50ns simulation of a β-hexapeptide in methanol at 340K. In the simulations the peptides sampled both folded (helical) and unfolded structures. Trajectory structures with common backbone conformations were identified and grouped into clusters. The CD spectra were calculated for individual structures, based on peptide-group dipole transition moments obtained from semi-empirical molecular orbital theory and using the so-called matrix method. The single-structure spectra were then averaged over entire trajectories and over clusters of structures. Although certain features of the experimental CD spectra of the β-peptides are reproduced by the trajectory-average spectra, there exist clear differences between the two sets of spectra in both wavelength and peak intensities. The analysis of individual contributions to the average spectra shows that, in general, the interpretation of a CD signal in terms of a single structure is not possible. Moreover, there is a large variation in the CD spectra calculated for a set of individual structures that belong to the same cluster, even when a structurally tight clustering criterion is used. This indicates that the CD spectra of these peptides are very sensitive to small local structural difference