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1.
Nat Genet ; 46(3): 261-269, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24441737

ABSTRACT

The hookworm Necator americanus is the predominant soil-transmitted human parasite. Adult worms feed on blood in the small intestine, causing iron-deficiency anemia, malnutrition, growth and development stunting in children, and severe morbidity and mortality during pregnancy in women. We report sequencing and assembly of the N. americanus genome (244 Mb, 19,151 genes). Characterization of this first hookworm genome sequence identified genes orchestrating the hookworm's invasion of the human host, genes involved in blood feeding and development, and genes encoding proteins that represent new potential drug targets against hookworms. N. americanus has undergone a considerable and unique expansion of immunomodulator proteins, some of which we highlight as potential treatments against inflammatory diseases. We also used a protein microarray to demonstrate a postgenomic application of the hookworm genome sequence. This genome provides an invaluable resource to boost ongoing efforts toward fundamental and applied postgenomic research, including the development of new methods to control hookworm and human immunological diseases.


Subject(s)
Genome, Helminth , Necator americanus/genetics , Animals , Caenorhabditis elegans/genetics , Female , Gene Expression Regulation, Developmental , Host-Parasite Interactions/immunology , Humans , Male , Molecular Sequence Data , Necator americanus/growth & development , Necator americanus/immunology , Necatoriasis/immunology , Necatoriasis/parasitology , Necatoriasis/prevention & control , Pregnancy , Species Specificity
2.
Chem Biol Drug Des ; 79(6): 1007-17, 2012 Jun.
Article in English | MEDLINE | ID: mdl-22339993

ABSTRACT

Two-component signal transduction (TCST) is the predominant signaling scheme used in bacteria to sense and respond to environmental changes in order to survive and thrive. A typical TCST system consists of a sensor histidine kinase to detect external signals and an effector response regulator to respond to external changes. In the signaling scheme, the histidine kinase phosphorylates and activates the response regulator, which functions as a transcription factor to modulate gene expression. One promising strategy toward antibacterial development is to target TCST regulatory systems, specifically the response regulators to disrupt the expression of genes important for virulence. In Salmonella enterica, the PhoQ/PhoP signal transduction system is used to sense and respond to low magnesium levels and regulates the expression for over 40 genes necessary for growth under these conditions, and more interestingly, genes that are important for virulence. In this study, a hybrid approach coupling computational and experimental methods was applied to identify drug-like compounds to target the PhoP response regulator. A computational approach of structure-based virtual screening combined with a series of biochemical and biophysical assays was used to test the predictability of the computational strategy and to characterize the mode of action of the compounds. Eight compounds from virtual screening inhibit the formation of the PhoP-DNA complex necessary for virulence gene regulation. This investigation served as an initial case study for targeting TCST response regulators to modulate the gene expression of a signal transduction pathway important for bacterial virulence. With the increasing resistance of pathogenic bacteria to current antibiotics, targeting TCST response regulators that control virulence is a viable strategy for the development of antimicrobial therapeutics with novel modes of action.


Subject(s)
Bacterial Proteins/antagonists & inhibitors , Salmonella enterica/metabolism , Bacterial Proteins/metabolism , Binding Sites , Computer Simulation , DNA/metabolism , Dimerization , Electrophoretic Mobility Shift Assay , Protein Interaction Mapping , Protein Structure, Tertiary , Salmonella enterica/drug effects , Signal Transduction/drug effects
3.
Methods Mol Biol ; 716: 1-22, 2011.
Article in English | MEDLINE | ID: mdl-21318897

ABSTRACT

The identification of small drug-like compounds that selectively inhibit the function of biological targets has historically been a major focus in the pharmaceutical industry, and in recent years, has generated much interest in academia as well. Drug-like compounds are valuable as chemical genetics tools to probe biological pathways in a reversible, dose- and time-dependent manner for drug target identification. In addition, small molecule compounds can be used to characterize the shape and charge preferences of macromolecular binding sites, for both structure-based and ligand-based drug design. High-throughput screening is the most common experimental method used to identify lead compounds. Because of the cost, time, and resources required for performing high-throughput screening for compound libraries, the use of alternative strategies is necessary for facilitating lead discovery. Virtual screening has been successful in prioritizing large chemical libraries to identify experimentally active compounds, serving as a practical and effective alternative to high-throughput screening. Methodologies used in virtual screening such as molecular docking and scoring have advanced to the point where they can rapidly and accurately identify lead compounds in addition to predicting native binding conformations. This chapter provides instructions on how to perform a virtual screen using freely available tools for structure-based lead discovery.


Subject(s)
Drug Design , Software , Ligands , Models, Molecular , Protein Binding
4.
J Chem Inf Model ; 51(2): 214-28, 2011 Feb 28.
Article in English | MEDLINE | ID: mdl-21214225

ABSTRACT

Binding affinity prediction is one of the most critical components to computer-aided structure-based drug design. Despite advances in first-principle methods for predicting binding affinity, empirical scoring functions that are fast and only relatively accurate are still widely used in structure-based drug design. With the increasing availability of X-ray crystallographic structures in the Protein Data Bank and continuing application of biophysical methods such as isothermal titration calorimetry to measure thermodynamic parameters contributing to binding free energy, sufficient experimental data exists that scoring functions can now be derived by separating enthalpic (ΔH) and entropic (TΔS) contributions to binding free energy (ΔG). PHOENIX, a scoring function to predict binding affinities of protein-ligand complexes, utilizes the increasing availability of experimental data to improve binding affinity predictions by the following: model training and testing using high-resolution crystallographic data to minimize structural noise, independent models of enthalpic and entropic contributions fitted to thermodynamic parameters assumed to be thermodynamically biased to calculate binding free energy, use of shape and volume descriptors to better capture entropic contributions. A set of 42 descriptors and 112 protein-ligand complexes were used to derive functions using partial least-squares for change of enthalpy (ΔH) and change of entropy (TΔS) to calculate change of binding free energy (ΔG), resulting in a predictive r2 (r(pred)2) of 0.55 and a standard error (SE) of 1.34 kcal/mol. External validation using the 2009 version of the PDBbind "refined set" (n = 1612) resulted in a Pearson correlation coefficient (R(p)) of 0.575 and a mean error (ME) of 1.41 pK(d). Enthalpy and entropy predictions were of limited accuracy individually. However, their difference resulted in a relatively accurate binding free energy. While the development of an accurate and applicable scoring function was an objective of this study, the main focus was evaluation of the use of high-resolution X-ray crystal structures with high-quality thermodynamic parameters from isothermal titration calorimetry for scoring function development. With the increasing application of structure-based methods in molecular design, this study suggests that using high-resolution crystal structures, separating enthalpy and entropy contributions to binding free energy, and including descriptors to better capture entropic contributions may prove to be effective strategies toward rapid and accurate calculation of binding affinity.


Subject(s)
Calorimetry , Computational Biology/methods , Drug Design , Crystallography, X-Ray , Databases, Protein , Least-Squares Analysis , Proteins/metabolism , Reproducibility of Results , Structure-Activity Relationship , Thermodynamics
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