Terry R. Stouch

Progress in understanding the structure-activity relationships of P-glycoprotein.

Efflux out of cells by P-glycoprotein (P-gp) represents a serious liability for pharmaceuticals, particularly for anti-cancer drugs. Consequently, identification of compounds as potential substrates is important for understanding their bioavailability. Also, the development of agents which reverse this multi-drug resistance phenotype has received considerable attention. Assays for determining these activities are reviewed. Recent literature and studies into the structure-activity relationships (SAR) of the resulting data are discussed. Multiple binding sites and other complicating factors have prevented the development of a truly general, conclusive SAR either for substrate or inhibitory activities. Consequently, many models have tended to address only very general properties, such as lipophilicity and size. However, progress has been made in the last few years toward more specific SAR suggesting well-defined structural features responsible for both activities. The future of understanding the details of P-gp SAR lies in more specific assays that target specific binding sites and mechanisms of action.

The limiting behavior of water hydrating a phospholipid monolayer: A computer simulation study

We report molecular dynamics simulations of water hydrating a lipid (dimyristoylphosphatidylcholine) monolayer under conditions chosen to eliminate simulation artifacts. These simulations provide a description of the behavior of the membrane–water interface that agrees with recent experimental studies. In particular, we find that the hydrating water orients to contribute the positive end of its dipole to the substantially positive electrostatic potential of the membrane interior, consistent with interpretations of recent experiments. In addition, recent experiments show that this water reorients rapidly on the NMR time scale. Our results concur, however the relatively rapid water motion does not preclude the preferential ordering that we observe. The limiting behavior of the system shows three hydration shells about the lipid PC headgroups and significant hydrogen bonding of water to the phosphate groups. The choline group experiences different environments, and the structure of the first hydration shell ...

Lipid Membrane Structure and Dynamics Studied by All-Atom Molecular Dynamics Simulations of Hydrated Phospholipid Bilayers

Abstract The structure and dynamics of phosphatidylcholine bilayers are examined by reviewing the results of several nanoseconds of molecular dynamics simulations on a number of bilayer and monolayer models. The lengths of these simulations, the longest single one of which was 2 nanoseconds, were sufficiently long to effectively sample many of the longer-scale motions governing the behaviour of biomembranes. These simulations reproduce many experimental observables well and provide a degree of resolution currently unavailable experimentally.

Computer simulation of a phospholipid monolayer‐water system: The influence of long range forces on water structure and dynamics

Molecular dynamics simulations of water above a lipid (dimyristoylphosphatidylcholine) monolayer show that the structure and dynamics of the water are quite sensitive to the treatment of the long range forces. To obtain a quantitative estimate of the perturbations introduced into the water behavior by truncation of the potential, we calculate various structural and dynamical properties of the water for simulations employing different cutoffs. When potential cutoffs on the order of 10–14 A are employed, the properties of water near the monolayer exhibit perturbations of up to 20%–25% that extend 30–35 A into the water. Employing cutoffs on the order of 30 A leads to water behavior that is essentially uniform above the monolayer. We also present a quantitative description of the difference in the force experienced by individual atoms in simulations employing different cutoffs. Despite the significant difference in the water properties between simulations employing 14 A and effective 30 A cutoffs, the forces...

Conformational dependence of electrostatic potential-derived charges: studies of the fitting procedure

Atomic monopole “point charges” are routinely determined through a least squares fit to molecular electrostatic potentials [potential‐derived (PD) charges]. Previously, it has been shown that these charges vary with variation in molecular conformation. Also, it has been observed that these swings in charges are highly correlated between neighboring atoms. Here, we examine the least squares variance–covariance data matrices for a set of data in the literature and find further indications of high colinearity within the data. These colinearities effectively reduce the dimensionality of the data to a value well below the number of atoms in the molecules. This suggests that the data is not of sufficient dimensionality to support calculation of the charges for all of the atoms in a statistically significant way. We experiment with fixing the charges of atoms whose PD charges reflect large errors in the fit. The resulting estimates of fit of the remaining charges are little degraded from the estimates of fit when the charges of all of the atoms are fit. In addition, the charges that are fit take what would be considered more reasonable and “chemically intuitive” values, often of smaller magnitude. Although most of the free charges continue to vary with molecular conformation, their range is no larger than when all charges were fit and, in some cases, the ranges of the charges for the fit atoms is actually reduced over those that are found when all of the atoms take part in the fitting procedure. The errors of fit are lower and the unconstrained charges appear more reasonable when more chemically “reasonable” charges are used for the fixed values. This suggests that in many cases charges are transferable between molecules. Further, it shows a way to justifiably reduce the large fluctuations in PD charges that occur with variations in conformation.

Analysis and optimization of structure-based virtual screening protocols. 2. Examination of docked ligand orientation sampling methodology: mapping a pharmacophore for success.

An important element of any structure-based virtual screening (SVS) technique is the method used to orient the ligands in the target active site. This has been a somewhat overlooked issue in recent SVS validation studies, with the assumption being made that the performance of an algorithm for a given set of orientation sampling settings will be representative for the general behavior of said technique. Here, we analyze five different SVS targets using a variety of sampling paradigms within the DOCK, GOLD and PROMETHEUS programs over a data set of approximately 10,000 noise compounds, combined with data sets containing multiple active compounds. These sets have been broken down by chemotype, with chemotype hit rate used to provide a measure of enrichment with a potentially improved relevance to real world SVS experiments. The variability in enrichment results produced by different sampling paradigms is illustrated, as is the utility of using pharmacophores to constrain sampling to regions that reflect known structural biology. The difference in results when comparing chemotype with compound hit rates is also highlighted.

DRUG DISCOVERY INTERFACE: Functional Group Dependence of Solute Partitioning to Various Locations within a DOPC Bilayer: A Comparison of Molecular Dynamics Simulations with Experiment

Atomic-level molecular dynamics simulations of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers containing small, amphiphilic, drug-like molecules were carried out to examine the influence of polar functionality on membrane partitioning and transport. Three related molecules (tyramine, phenethylamine, and 4-ethylphenol) were chosen to allow a detailed study of the isolated effects of the amine and hydroxyl functionalities on the preferred solute location, free energies of transfer, and the effect of combining both functional groups in a same molecule. Transfer free energy profiles (from water) generated from molecular dynamics (MD) simulations as a function of bilayer depth compared favorably to comparable experimental results. The simulations allowed the determination of the location of the barrier domain for permeability where the transfer free energy is highest and the preferred binding region at which the free energy is a minimum for each of the three solutes. Comparisons of the free energy profiles reveal that the hydrocarbon chain interior is the region most selective to chemical structure of different solutes because the free energies of transfer in that region vary to a significantly greater extent than in other regions of the bilayer. The contributions of the hydroxyl and amino groups to the free energies of solute transfer from water to the interfacial region were close to zero in both the MD simulations and experimental measurements. This suggests that the free energy decrease observed for solute transfer into the head group region occurs with minimal loss in solvation by hydrogen bonding to polar functional groups on the solute and is largely driven by hydrophobicity. Overall, the joint experimental and simulation studies suggest that the assumption of additivity of free energy contributions from multiple polar functional groups on the same molecule may hold for predictions of passive bilayer permeability coefficients providing that the groups are well isolated. However, this assumption does not hold for predictions of relative liposome-binding affinities.

Chemical Genetics Reveals an RGS/G-Protein Role in the Action of a Compound

We report here on a chemical genetic screen designed to address the mechanism of action of a small molecule. Small molecules that were active in models of urinary incontinence were tested on the nematode Caenorhabditis elegans, and the resulting phenotypes were used as readouts in a genetic screen to identify possible molecular targets. The mutations giving resistance to compound were found to affect members of the RGS protein/G-protein complex. Studies in mammalian systems confirmed that the small molecules inhibit muscarinic G-protein coupled receptor (GPCR) signaling involving G-αq (G-protein alpha subunit). Our studies suggest that the small molecules act at the level of the RGS/G-αq signaling complex, and define new mutations in both RGS and G-αq, including a unique hypo-adapation allele of G-αq. These findings suggest that therapeutics targeted to downstream components of GPCR signaling may be effective for treatment of diseases involving inappropriate receptor activation.

An atomic and molecular view of the depth dependence of the free energies of solute transfer from water into lipid bilayers.

Molecular interactions and orientations responsible for differences in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayer partitioning of three structurally related drug-like molecules (4-ethylphenol, phenethylamine, and tyramine) were investigated. This work is based on previously reported molecular dynamics (MD) simulations that determined their transverse free energy profiles across the bilayer. Previously, the location where the transfer free energy of the three solutes is highest, which defines the barrier domain for permeability, was found to be the bilayer center, while the interfacial region was found to be the preferred binding region. Contributions of the amino (NH2) and hydroxyl (OH) functional groups to the transfer free energies from water to the interfacial region were found to be very small both experimentally and by MD simulation, suggesting that the interfacial binding of these solutes is hydrophobically driven and occurs with minimal loss of hydrogen-bonding interactions of the polar functional groups which can occur with either water or phospholipid head groups. Therefore, interfacial binding is relatively insensitive to the number or type of polar functional groups on the solute. In contrast, the relative solute free energy in the barrier domain is highly sensitive to the number of polar functional groups on the molecule. The number and types of hydrogen bonds formed between the three solutes and polar phospholipid atoms or with water molecules were determined as a function of solute position in the bilayer. Minima were observed in the number of hydrogen bonds formed by each solute at the center of the bilayer, coinciding with a decrease in the number of water molecules in DOPC as a function of distance away from the interfacial region. In all regions, hydrogen bonds with water molecules account for the majority of hydrogen-bonding interactions observed for each solute. Significant orientational preferences for the solutes are evident in certain regions of the bilayer (e.g., within the ordered chain region and near the interfacial region 20-25 Å from the bilayer center). The preferred orientations are those that preserve favorable molecular interactions for each solute, which vary with the solute structure.

Force field studies of cholesterol and cholesteryl acetate crystals and cholesterol–cholesterol intermolecular interactions

To model the physical properties of sterols and related species, an all‐atom Class II force field has been derived based on the recently reported CFF93 force field for hydrocarbons. It has been tested using both energy minimization and molecular dynamics (MD) simulations of the low‐temperature neutron‐diffraction structure of cholesteryl acetate crystals and the X‐ray diffraction crystal structure of cholesterol. Thus these studies test the techniques and limitations of high‐accuracy crystal simulations as well. Employing energy minimization, all cell vectors and volumes were reproduced to within 2.4% of experimental values. For cholesteryl acetate, the root mean square (rms) deviations between the calculated and experimental bond lengths, angles, and torsions of nonhydrogen atoms are 0.013 Å, 1.2°, and 2.4°, respectively. The corresponding maximum deviations are also very small: 0.027 Å for bond length, 3.2° for angle, and 7.6° for torsion. For cholesterol, good agreement between the calculated and experimental structures was found only when the comparison was limited to atoms with relatively small thermal factors (Beq < 15 Å2). It was found that for both systems, the MD averaged structures were in better agreement with the experimental ones than the energy minimized structures, since the rms deviations in atom positions are smaller for the MD‐averaged structures (0.064 Å for cholesteryl acetate and 0.152 Å for cholesterol) than those for the minimized structures (0.178 Å for cholesteryl acetate and 0.189 Å for cholesterol). The force field was then applied to isolated molecules focusing on the rigidity of the cholesteryl ring and cholesterol–cholesterol interaction energies. It is concluded that the cholesteryl ring is fairly rigid since no major conformational change was observed during an MD simulation of a single cholesterol molecule in vacuo at 500 K, in agreement with condensed phase experiments. Calculations of cholesterol–cholesterol pairs suggest that there are only four low‐energy configurations and that it is more useful to describe each molecule as having a plane (flat face) and two grooves rather than as having two (one flat and one rough) faces. This provides some insight into the equilibrium crystal structures. Limited results from a modified Class I (CVFF) force field are presented for comparison.