Abstract: Quantitative structure-activity relationship (QSAR) methods aim to build quantitatively predictive models for the discovery of new molecules. It has been widely used in medicinal chemistry for drug discovery. Many QSAR techniques have been developed since Hansch's seminal work, and more are still being developed. Motivated by Hopfinger's receptor-dependent QSAR (RD-QSAR) formalism and the Lukacova-Balaz scheme to treat multimode issues, we have initiated studies that focus on a structure-based multimode QSAR (SBMM QSAR) method, where the structure of the target protein is used in characterizing the ligand, and the multimode issue of ligand binding is systematically treated with a modified Lukacova-Balaz scheme. All ligand molecules are first docked to the target binding pocket to obtain a set of aligned ligand poses. A structure-based pharmacophore concept is adopted to characterize the binding pocket. Specifically, we represent the binding pocket as a geometric grid labeled by pharmacophoric features. Each pose of the ligand is also represented as a labeled grid, where each grid point is labeled according to the atom types of nearby ligand atoms. These labeled grids or three-dimensional (3D) maps (both the receptor map (R-map) and the ligand map (L-map)) are compared to each other to derive descriptors for each pose of the ligand, resulting in a multimode structure-activity relationship (SAR) table. Iterative partial least-squares (PLS) is employed to build the QSAR models. When we applied this method to analyze PDE-4 inhibitors, predictive models have been developed, obtaining models with excellent training correlation (r(2) = 0.65-0.66), as well as test correlation (R(2) = 0.64-0.65). A comparative analysis with 4 other QSAR techniques demonstrates that this new method affords better models, in terms of the prediction power for the test set.
Abstract: The anthrax lethal factor is a zinc metalloprotease toxin secreted by Bacillus anthracis which cleaves at the N-terminal region of six mitogen activated protein kinase kinases (MEKs) in the cell. Additionally, it is known to cleave a nine residue peptide "LF10," 50-fold more efficiently than nine residues of MEK1. There is very little sequence similarity between the MEK N-termini, thus, it is unclear how the lethal factor can accommodate and cleave the diverse N-termini of the MEKs and whether there is a hierarchy in this interaction, as there is between LF10 and MEK1. To investigate this problem, we carried out multiple molecular dynamics simulations of the lethal factor with nine residues of each of the substrates. Our simulations reveal that like LF10, certain MEK substrates have residue compositions that favor beta-sheet formation with the lethal factor over others. The formation of this secondary structure maintains a catalytic conformation. Binding energetics using the MM-PBSA method was used to rank-order the substrates for their affinity to LF (K(M)). On the basis of the results, we conclude that the LF does not equally accommodate the MEK substrates and further predict that there will be differences between rates of cleavage among the nine residue MEK N-termini.
Abstract: Current anesthetics, especially the inhaled ones, have troublesome side effects and may be associated with durable changes in cognition. It is therefore highly desirable to develop novel chemical entities that reduce these effects while preserving or enhancing anesthetic potency. In spite of progress toward identifying protein targets involved in anesthesia, we still do not have the necessary atomic level structural information to delineate their interactions with anesthetic molecules. Recently, we have described a protein target, apoferritin, to which several anesthetics bind specifically and in a pharmacodynamically relevant manner. Further, we have reported the high resolution X-ray structure of two anesthetic/apoferritin complexes (Liu, R.; Loll, P. J.; Eckenhoff, R. G. FASEB J. 2005, 19, 567). Thus, we describe in this paper a structure-based approach to establish validated shape pharmacophore models for future application to virtual and high throughput screening of anesthetic compounds. We use the 3D structure of apoferritin as the basis for the development of several shape pharmacophore models. To validate these models, we demonstrate that (1) they can be used to effectively recover known anesthetic agents from a diverse database of compounds; (2) the shape pharmacophore scores afford a significant linear correlation with the measured binding energetics of several known anesthetic compounds to the apoferritin site; and (3) the computed scores based on the shape pharmacophore models also predict the trend of the EC(50) values of a set of anesthetics. Therefore, we have now obtained a set of structure-based shape pharmacophore models, using ferritin as the surrogate target, which may afford a new way to rationally discover novel anesthetic agents in the future.
Abstract: 3D molecular shape similarity search has recently become an attractive method for virtual screening and scaffold hopping in drug discovery and chemical genomics research. Among these 3D similarity methods is ROCS (Rapid Overlay of Chemical Structures), a popular tool because of its efficiency and effectiveness. However, searching a large multiconformer molecular database remains a very challenging task because of the nature of such calculations. To simplify shape similarity calculations and potentially increase the efficiency for large scale virtual screening, we have explored an alternative shape similarity approach that does not depend on multiconformers of molecules. The hypothesis underlying this approach is that similar chemical structures tend to have similar 2D chemical depictions and that shape comparison techniques can be utilized to effectively compare the shapes between chemical depictions. We use a 2D depiction program to generate 2-D chemical drawings for both the query molecule and database molecules. We have built a 2D shape comparison program based on the OESHAPE Toolkit (OE Scientific, NM) that compares the molecular depictions and quantifies the shape similarity between the molecules. We demonstrate that this unconventional 2D shape similarity method performs fairly well in virtual screening experiments compared to the 3D Shape method ROCS, with an added advantage of its computational efficiency.
Abstract: Phosphodiesterase-4 (PDE-4) is an important drug target for several diseases, including COPD (chronic obstructive pulmonary disorder) and neurodegenerative diseases. In this paper, we describe the development of improved QSAR (quantitative structure-activity relationship) models using a novel multi-conformational structure-based pharmacophore key (MC-SBPPK) method. Similar to our previous work, this method calculates molecular descriptors based on the matching of a molecule's pharmacophore features with those of the target binding pocket. Therefore, these descriptors are PDE4-specific, and most relevant to the problem under study. Furthermore, this work expands our previous SBPPK QSAR method by explicitly including multiple conformations of the PDE-4 inhibitors in the regression analysis, and thus addresses the issue of molecular flexibility. The nonlinear regression problem resulted from including multiple conformations has been transformed into a linear equation and solved by an iterative partial least square (iPLS) procedure, according to the Lukacova-Balaz scheme. 35 PDE-4 inhibitors have been analyzed with this new method, and predictive models have been developed. Based on the prediction statistics for both the training set and the test set, these new models are more robust and predictive than those obtained by traditional ligand-based QSAR techniques as well as that obtained with the SBPPK method reported in our previous work. As a result, multiple predictive models have been added to the collection of QSAR models for PDE4 inhibitors. Collectively, these models will be useful for the discovery of new drug candidates targeting the PDE-4 enzyme.
Abstract: Our earlier studies have shown that the Vibrio harveyi flavin reductase FRP undergoes a monomer-dimer equilibrium, and luciferase forms a functional complex with the FRP monomer but not significantly with the dimer. This work is aimed at further investigating the nature and regulation of FRP subunit interactions by computation and site-directed mutagenesis approaches. In silico mutations of a number of residues were performed, and energetic analyses led us to target residue E99, which interacts directly with R113 and R225 from the second subunit of the FRP homodimer, for detailed investigation. E99 was found non-essential to the binding of either the FMN cofactor or the substrates. However, in comparison with the native enzyme, the E99K variant was shown to have an enhanced subunit dissociation as evident from a 44-fold higher K(d) for the monomer-dimer equilibrium. The critical role of E99 in the formation of the FRP dimer has thus been demonstrated.
Abstract: Computationally efficient structure-based virtual screening methods have recently been reported that seek to find effective means to utilize experimental structure information without employing detailed molecular docking calculations. These tools can be coupled with efficient experimental screening technologies to improve the probability of identifying hits and leads for drug discovery research. Commercial software ROCS (rapid overlay of chemical structures) from Open Eye Scientific is such an example, which is a shape-based virtual screening method using the 3D structure of a ligand, typically from a bound X-ray costructure, as the query. We report here the development of a new structure-based pharmacophore search method (called Shape4) for virtual screening. This method adopts a variant of the ROCS shape technology and expands its use to work with an empty crystal structure. It employs a rigorous computational geometry method and a deterministic geometric casting algorithm to derive the negative image (i.e., pseudoligand) of a target binding site. Once the negative image (or pseudoligand) is generated, an efficient shape comparison algorithm in the commercial OE SHAPE Toolkit is adopted to compare and match small organic molecules with the shape of the pseudoligand. We report the detailed computational protocol and its computational validation using known biologically active compounds extracted from the WOMBAT database. Models derived for five selected targets were used to perform the virtual screening experiments to obtain the enrichment data for various virtual screening methods. It was found that our approach afforded similar or better enrichment ratios than other related methods, often with better diversity among the top ranking computational hits.
Abstract: Human immunodeficiency virus (HIV)-1 integrase (IN) is an attractive target for development of acquired immunodeficiency syndrome chemotherapy. In this study, conventional and coupled quantum mechanical and molecular mechanical (QM/MM) molecular dynamics (MD) simulations of HIV-1 IN complexed with 5CITEP (IN-5CITEP) were carried out. In addition to differences in the bound position of 5CITEP, significant differences at the two levels of theory were observed in the metal coordination geometry and the areas involving residues 116-119 and 140-166. In the conventional MD simulation, the coordination of Mg(2+) was found to be a near-perfect octahedral geometry whereas a distorted octahedral complex was observed in QM/MM. All of the above reasons lead to a different pattern of protein-ligand salt link formation that was not observed in the classical MD simulation. Furthermore to provide a theoretical understanding of inhibition mechanisms of 5CITEP and its derivative (DKA), hybrid QM/MM MD simulations of the two complexes (IN-5CITEP and IN-DKA) have been performed. The results reveal that areas involving residues 60-68, 116-119, and 140-149 were substantially different among the two systems. The two systems show similar pattern of metal coordination geometry, i.e., a distorted octahedron. In IN-DKA, both OD1 and OD2 of Asp-64 coordinate the Mg(2+) in a monodentate fashion whereas only OD1 is chelated to the metal as observed in IN-5CITEP. The high potency of DKA as compared to 5CITEP is supported by a strong salt link formed between its carboxylate moiety and the ammonium group of Lys-159. Detailed comparisons between HIV-1 IN complexed with DKA and with 5CITEP provide information about ligand structure effects on protein-ligand interactions in particular with the Lys-159. This is useful for the design of new selective HIV-1 IN inhibitors.
Abstract: The electrostatic environments near the acetylcholine binding sites on the nicotinic acetylcholine receptor (nAChR) and acetylcholinesterase were measured by diffusion-enhanced fluorescence energy transfer (DEFET) to determine the influence of long-range electrostatic interactions on ligand binding kinetics and net binding energy. Changes in DEFET from variously charged Tb3+ -chelates revealed net potentials of -20 mV at the nAChR agonist sites and -14 mV at the entrance to the AChE active site, in physiological ionic strength conditions. The potential at the alphadelta-binding site of the nAChR was determined independently in the presence of d-tubocurarine to be -14 mV; the calculated potential at the alphagamma-site was approximately threefold stronger than at the alphadelta-site. By determining the local potential in increasing ionic strength, Debye-Hückel theory predicted that the potentials near the nAChR agonist binding sites are constituted by one to three charges in close proximity to the binding site. Examination of the binding kinetics of the fluorescent acetylcholine analog dansyl-C6-choline at ionic strengths from 12.5 to 400 mM revealed a twofold decrease in association rate. Debye-Hückel analysis of the kinetics revealed a similar charge distribution as seen by changes in the potentials. To determine whether the experimentally determined potentials are reflected by continuum electrostatics calculations, solutions to the nonlinear Poisson-Boltzmann equation were used to compute the potentials expected from DEFET measurements from high-resolution models of the nAChR and AChE. These calculations are in good agreement with the DEFET measurements for AChE and for the alphagamma-site of the nAChR. We conclude that long-range electrostatic interactions contribute -0.3 and -1 kcal/mol to the binding energy at the nAChR alphadelta- and alphagamma-sites due to an increase in association rates.
Abstract: Electrostatic surface potentials in the vestibule of the nicotinic acetylcholine receptor (nAChR) were computed from structural models using the University of Houston Brownian Dynamics program to determine their effect on ion conduction and ionic selectivity. To further determine whether computed potentials accurately reflect the electrostatic environment of the channel, the potentials were used to predict the rate constants for diffusion-enhanced fluorescence energy transfer; the calculated energy transfer rates are directly comparable with those determined experimentally (see companion article by Meltzer et al. in this issue). To include any effects on the local potentials by the bound acceptor fluorophore crystal violet, its binding site was first localized within the pore by fluorescence energy transfer measurements from dansyl-C6-choline bound to the agonist sites and also by simulations of binding using Autodock. To compare the computed potentials with those determined experimentally, we used the predicted energy transfer rates from Tb3+ chelates of varying charge to calculate an expected potential using the Boltzmann relationship. This expected potential (from -20 to -40 mV) overestimates the values determined experimentally (from -10 to -25 mV) by two- to fourfold at similar conditions of ionic strength. Although the results indicate a basic discrepancy between experimental and computed surface potentials, both methods demonstrate that the vestibular potential has a relatively small effect on conduction and selectivity.
Abstract: We report the crystal structure of alanine racemase from Mycobacterium tuberculosis (Alr(Mtb)) at 1.9 A resolution. In our structure, Alr(Mtb) is found to be a dimer formed by two crystallographically different monomers, each comprising 384 residues. The domain makeup of each monomer is similar to that of Bacillus and Pseudomonas alanine racemases and includes both an alpha/beta-barrel at the N-terminus and a C-terminus primarily made of beta-strands. The hinge angle between these two domains is unique for Alr(Mtb), but the active site geometry is conserved. In Alr(Mtb), the PLP cofactor is covalently bound to the protein via an internal aldimine bond with Lys42. No guest substrate is noted in its active site, although some residual electron density is observed in the enzyme's active site pocket. Analysis of the active site pocket, in the context of other known alanine racemases, allows us to propose the inclusion of conserved residues found at the entrance to the binding pocket as additional targets in ongoing structure-aided drug design efforts. Also, as observed in other alanine racemase structures, PLP adopts a conformation that significantly distorts the planarity of the extended conjugated system between the PLP ring and the internal aldimine bond.
