Ensemble-based docking: From hit discovery to metabolism and toxicity predictions

This paper describes and illustrates the use of ensemble-based docking, i.e., using a collection of protein structures in docking calculations for hit discovery, the exploration of biochemical pathways and toxicity prediction of drug candidates. We describe the computational engineering work necessary to enable large ensemble docking campaigns on supercomputers. We show examples where ensemble-based docking has significantly increased the number and the diversity of validated drug candidates. Finally, we illustrate how ensemble-based docking can be extended beyond hit discovery and toward providing a structural basis for the prediction of metabolism and off-target binding relevant to pre-clinical and clinical trials

ESTUDIO PRELIMINAR DE COLECCIÓN DE SEMEN EN OSO DE ANTEOJOS (Tremarctos ornatus)

Semen was collected in a Spectacled Bear (Tremarctos ornatus) reared in captivity using the electroejaculation technique. Four series of 6 volt discharges by 15 seconds each plus manual stimulation were carried out. An effective penis erection and small volume of ejaculate was obtained in the last series of electrical stimulus. Seminal motility was 50%. Further studies are required to optimize the use of the electroejaculator in order to obtain higher volumes and better semen quality.Semen was collected in a Spectacled Bear (Tremarctos ornatus) reared in captivity using the electroejaculation technique. Four series of 6 volt discharges by 15 seconds each plus manual stimulation were carried out. An effective penis erection and small volume of ejaculate was obtained in the last series of electrical stimulus. Seminal motility was 50%. Further studies are required to optimize the use of the electroejaculator in order to obtain higher volumes and better semen quality

EQUINE CHORIONIC GONADOTROPHIN (ECG) STIMULATION DURING THE LUTEAL AND NON-LUTEAL PHASES ON OVARIAN RESPONSE AND EMBRYO QUALITY IN LLAMAS

Se evaluó el efecto del tratamiento superovulatorio en las dos fases del ciclo ovárico sobre la respuesta folicular y la calidad embrionaria en 45 llamas hembras adultas. Se incluyeron en el estudio aquellos animales que a la ecografía presentaron un folículo preovulatorio >7 mm. Los animales se distribuyeron en tres grupos: T0 (no estimulado), T1 (tratamiento superovulatorio en fase no luteal) y T2 (tratamiento superovulatorio en fase luteal). Los animales de T1 y T2 recibieron 1 ml de LH (día 0) para sincronizar la onda folicular y 1000 UI de eCG (día 3) como tratamiento superovulatorio. Se utilizaron esponjas vaginales impregnadas con progesterona entre el día 3 y 7 para simular la fase luteal en el T2. La inducción de la ovulación se hizo mediante monta natural y aplicación de 1 ml de GnRH (día 8). La colección y evaluación de embriones se realizó 7 días post cópula (día 15) en T1 y T2. En el grupo T0 se realizó monta natural y aplicación de GnRH y 7 días después se realizó la colección de embriones. El número de folículos preovulatorios fue mayor en T1 (11.07 ± 7.53) y T2 (6.13 ± 7.11) con respecto a T0 (1.07 ± 0.26) (p<0.05). El número de cuerpos lúteos fue mayor en T1 (9.27 ± 3.37) con respecto a T0 (1.07 ± 0.26) y T2 (6.47 ± 4.29) (p<0.05). Asimismo, el número de embriones recuperados fue mayor en T1 (3.47 ± 4.26) con respecto a T0 (0.33 ± 0.48) y T2 (1.33 ± 2.53). Los resultados permiten concluir que la aplicación del tratamiento superovulatorio durante una fase no luteal permiten obtener una mejor respuesta ovárica y embrionaria en comparación con tratamientos superovulatorios aplicados en fase luteal.The effect of superovulatory treatment during the two phases of the ovarian cycleon follicular growth and embryo quality was evaluated in 45 sexually adult llamas. Animals bearing a >7 mm follicle, observed by ultrasonography, were selected and allocated into 3 groups: T0 (non-stimulated), T1 (superovulatory treatment during the non luteal phase), and T2 (superovulatory treatment during the luteal phase). Animals in groups T1 and T2 received 1 ml of LH (day 0) for synchronization of the follicular wave and 1000 IU of eCG (day 3) as superovulatory treatment. Vaginal sponges impregnated with progesterone were used on days 3 to 7 in T2 to simulate the luteal phase. The induction of the ovulation (day 8) was done through natural mating and the application of GnRH (1 ml). Embryo recovery was done 7 days after natural mating (day 15) on T1 and T2. Similarly, embryo recovery was done 7 days after natural mating and application of GnRH in T0. The number of preovulatory follicles was larger in T1 (11.07 ± 7.53) and T2 (6.13 ± 7.11) than in T0 (1.07 ± 0.26) (p<0.05). The number of corpora lutea was larger in T1 (9.27 ± 3.37) than in T0 (1.07 ± 0.26) and T2 (6.47 ± 4.29) (p<0.05). The number of recovered embryos waslarger in T1 (3.47 ± 4.26) than in T0 (0.33 ± 0.48) and T2 (1.33 ± 2.53). The results showed that superovulatory treatment during the non luteal phase had a better response than superovulatory treatment during the luteal phase

Handheld computers for self-administered sensitive data collection: A comparative study in Peru

<p>Abstract</p> <p>Background</p> <p>Low-cost handheld computers (PDA) potentially represent an efficient tool for collecting sensitive data in surveys. The goal of this study is to evaluate the quality of sexual behavior data collected with handheld computers in comparison with paper-based questionnaires.</p> <p>Methods</p> <p>A PDA-based program for data collection was developed using Open-Source tools. In two cross-sectional studies, we compared data concerning sexual behavior collected with paper forms to data collected with PDA-based forms in Ancon (Lima).</p> <p>Results</p> <p>The first study enrolled 200 participants (18–29 years). General agreement between data collected with paper format and handheld computers was 86%. Categorical variables agreement was between 70.5% and 98.5% (Kappa: 0.43–0.86) while numeric variables agreement was between 57.1% and 79.8% (Spearman: 0.76–0.95). Agreement and correlation were higher in those who had completed at least high school than those with less education. The second study enrolled 198 participants. Rates of responses to sensitive questions were similar between both kinds of questionnaires. However, the number of inconsistencies (p = 0.0001) and missing values (p = 0.001) were significantly higher in paper questionnaires.</p> <p>Conclusion</p> <p>This study showed the value of the use of handheld computers for collecting sensitive data, since a high level of agreement between paper and PDA responses was reached. In addition, a lower number of inconsistencies and missing values were found with the PDA-based system. This study has demonstrated that it is feasible to develop a low-cost application for handheld computers, and that PDAs are feasible alternatives for collecting field data in a developing country.</p

The Role of Protein Structural Ensembles in Thermostability and Ligand Binding

The role of protein structural ensembles has been shown to be very important for different physical and chemical properties of proteins. The work presented in this dissertation explores two of these properties:i) Thermostability, by characterizing, at three different temperatures, the dynamics of aminoglycoside nucleotidyltransferase 4’ (ANT). This homodimeric enzyme detoxifies antibiotics. It possess two known variants, D80Y and T130K, with higher melting temperatures than the wild type. These mutations, however, would cause changes in the distributions of conformations in the ensemble and, consequently, on the dynamics of the protein. To test this hypothesis, the wild type and variants were examined by using molecular dynamics simulations and the results were compared with previous experimental information in order to characterize the similarities and differences between the, so-called, thermophilic and thermostable variants of this enzyme.ii) Ligand binding: Since proteins are in general dynamic structures, it would be expected that the effectiveness of ligand binding varies as the protein’s conformation changes. One of the most targeted protein family in the field of drug discovery/design is the G-Protein Coupled Receptor (GPCR) family. Over 30% of approved drugs target this family of proteins. This project examines, via in silico experiments, the differences in ligand binding between different conformations of GPCRs. To this end, GPCR ligand structures, actual binding (actives) and non-binding (decoys) ligands, were obtained from public databases, and eight GPCRs structures were selected to generate 5,000 conformational states for each protein. Ensemble-based docking was performed on representative structures of these 5,000 conformers and on a subset of 3,000 conformers from each of the eight proteins. Decoys and statistical analysis were incorporated in the docking simulations to test whether the sampled protein conformations can bind active ligands in greater numbers than the random selection from the pool of active and decoys. The results show that some conformations bind more ligands than other conformations, random selection, or the crystal structure. Characterizing the entire ensemble of protein conformations can improve the number of bound active ligands identified computationally, compared to random selection of compounds or docking using only a single crystal structure

Revisiting the Sweet Taste Receptor T1R2-T1R3 through Molecular Dynamics Simulations Coupled with a Noncovalent Interactions Analysis

It is nowadays widely accepted that sweet taste perception is elicited by the activation of the heterodimeric complex T1R2-T1R3, customarily known as sweet taste receptor (STR). However, the interplay between STR and sweeteners has not yet been fully clarified. Here through a methodology coupling molecular dynamics and the independent gradient model (igm) approach we determine the main interacting signatures of the closed (active) conformation of the T1R2 Venus flytrap domain (VFD) toward aspartame. The igm methodology provides a rapid and reliable quantification of noncovalent interactions through a score (Δginter) based on the attenuation of the electronic density gradient when two molecular fragments approach each other. Herein, this approach is coupled to a 100 ns molecular dynamics simulation (MD-igm) to explore the ligand-cavity contacts on a per-residue basis as well as a series of key inter-residue interactions that stabilize the closed form of VFD. We also apply an atomic decomposition scheme of noncovalent interactions to quantify the contribution of the ligand segments to the noncovalent interplay. Finally, a series of structural modification on aspartame are conducted in order to obtain guidelines for the rational design of novel sweeteners. Given that innovative methodologies to reliably quantify the extent of ligand-protein coupling are strongly demanded, this approach combining a noncovalent analysis and MD simulations represents a valuable contribution, that can be easily applied to other relevant biomolecular systems.Revisión por pare

Signal Transmission in Escherichia coli Cyclic AMP Receptor Protein for Survival in Extreme Acidic Conditions

El texto completo de este trabajo no está disponible en el Repositorio Académico UPC por restricciones de la casa editorial donde ha sido publicado.During the life cycle of enteric bacterium Escherichia coli, it encounters a wide spectrum of pH changes. The asymmetric dimer of the cAMP receptor protein, CRP, plays a key role in regulating the expressions of genes and the survival of E. coli. To elucidate the pH effects on the mechanism of signal transmission, we present a combination of results derived from ITC, crystallography, and computation. CRP responds to a pH change by inducing a differential effect on the affinity for the binding events to the two cAMP molecules, ensuing in a reversible conversion between positive and negative cooperativity at high and low pH, respectively. The structures of four crystals at pH ranging from 7.8 to 6.5 show that CRP responds by inducing a differential effect on the structures of the two subunits, particularly in the DNA binding domain. Employing the COREX/BEST algorithm, computational analysis shows the change in the stability of residues at each pH. The change in residue stability alters the connectivity between residues including those in cAMP and DNA binding sites. Consequently, the differential impact on the topology of the connectivity surface among residues in adjacent subunits is the main reason for differential change in affinity; that is, the pH-induced differential change in residue stability is the biothermodynamic basis for the change in allosteric behavior. Furthermore, the structural asymmetry of this homodimer amplifies the differential impact of any perturbations. Hence, these results demonstrate that the combination of these approaches can provide insights into the underlying mechanism of an apparent complex allostery signal and transmission in CRP.National Institutes of HealthRevisión por pare

The dynamic cycle of bacterial translation initiation factor IF3

Initiation factor IF3 is an essential protein that enhances the fidelity and speed of bacterial mRNA translation initiation. Here, we describe the dynamic interplay between IF3 domains and their alternative binding sites using pre-steady state kinetics combined with molecular modelling of available structures of initiation complexes. Our results show that IF3 accommodates its domains at velocities ranging over two orders of magnitude, responding to the binding of each 30S ligand. IF1 and IF2 promote IF3 compaction and the movement of the C-terminal domain (IF3C) towards the P site. Concomitantly, the N-terminal domain (IF3N) creates a pocket ready to accept the initiator tRNA. Selection of the initiator tRNA is accompanied by a transient accommodation of IF3N towards the 30S platform. Decoding of the mRNA start codon displaces IF3C away from the P site and rate limits translation initiation. 70S initiation complex formation brings IF3 domains in close proximity to each other prior to dissociation and recycling of the factor for a new round of translation initiation. Altogether, our results describe the kinetic spectrum of IF3 movements and highlight functional transitions of the factor that ensure accurate mRNA translation initiation