Varnavas D. Mouchlis

Identification of putative estrogen receptor-mediated endocrine disrupting chemicals using QSAR- and structure-based virtual screening approaches.

Identification of endocrine disrupting chemicals is one of the important goals of environmental chemical hazard screening. We report on the development of validated in silico predictors of chemicals likely to cause estrogen receptor (ER)-mediated endocrine disruption to facilitate their prioritization for future screening. A database of relative binding affinity of a large number of ERα and/or ERβ ligands was assembled (546 for ERα and 137 for ERβ). Both single-task learning (STL) and multi-task learning (MTL) continuous quantitative structure-activity relationship (QSAR) models were developed for predicting ligand binding affinity to ERα or ERβ. High predictive accuracy was achieved for ERα binding affinity (MTL R(2)=0.71, STL R(2)=0.73). For ERβ binding affinity, MTL models were significantly more predictive (R(2)=0.53, p<0.05) than STL models. In addition, docking studies were performed on a set of ER agonists/antagonists (67 agonists and 39 antagonists for ERα, 48 agonists and 32 antagonists for ERβ, supplemented by putative decoys/non-binders) using the following ER structures (in complexes with respective ligands) retrieved from the Protein Data Bank: ERα agonist (PDB ID: 1L2I), ERα antagonist (PDB ID: 3DT3), ERβ agonist (PDB ID: 2NV7), and ERβ antagonist (PDB ID: 1L2J). We found that all four ER conformations discriminated their corresponding ligands from presumed non-binders. Finally, both QSAR models and ER structures were employed in parallel to virtually screen several large libraries of environmental chemicals to derive a ligand- and structure-based prioritized list of putative estrogenic compounds to be used for in vitro and in vivo experimental validation.

Membranes serve as allosteric activators of phospholipase A2, enabling it to extract, bind, and hydrolyze phospholipid substrates.

Significance The catalytic mechanisms of interfacial enzymes acting directly on the interfacial surface of the membrane are notoriously difficult to study experimentally with X-ray crystallography and other biophysical methods. This scientific study is, to our knowledge, the first to highlight similarities and differences in the extraction and binding of a phospholipid molecule into the substrate binding pocket of two human phospholipases A2 (PLA2s): the cytosolic Group IVA cPLA2 and the calcium-independent Group VIA iPLA2. Molecular dynamics simulations, guided by deuterium exchange experiments, are used to show that pathways to the active sites of these PLA2s are opened upon allosteric interaction with the membrane to facilitate entry of the substrate lipid. These enzymes are involved in various diseases, and understanding their mechanisms will aid in the discovery of therapeutics. Defining the molecular details and consequences of the association of water-soluble proteins with membranes is fundamental to understanding protein–lipid interactions and membrane functioning. Phospholipase A2 (PLA2) enzymes, which catalyze the hydrolysis of phospholipid substrates that compose the membrane bilayers, provide the ideal system for studying protein–lipid interactions. Our study focuses on understanding the catalytic cycle of two different human PLA2s: the cytosolic Group IVA cPLA2 and calcium-independent Group VIA iPLA2. Computer-aided techniques guided by deuterium exchange mass spectrometry data, were used to create structural complexes of each enzyme with a single phospholipid substrate molecule, whereas the substrate extraction process was studied using steered molecular dynamics simulations. Molecular dynamic simulations of the enzyme–substrate–membrane systems revealed important information about the mechanisms by which these enzymes associate with the membrane and then extract and bind their phospholipid substrate. Our data support the hypothesis that the membrane acts as an allosteric ligand that binds at the allosteric site of the enzyme’s interfacial surface, shifting its conformation from a closed (inactive) state in water to an open (active) state at the membrane interface.

Binding of novel fullerene inhibitors to HIV-1 protease: insight through molecular dynamics and molecular mechanics Poisson-Boltzmann surface area calculations.

The objectives of this study include the design of a series of novel fullerene-based inhibitors for HIV-1 protease (HIV-1 PR), by employing two strategies that can also be applied to the design of inhibitors for any other target. Additionally, the interactions which contribute to the observed exceptionally high binding free energies were analyzed. In particular, we investigated: (1) hydrogen bonding (H-bond) interactions between specific fullerene derivatives and the protease, (2) the regions of HIV-1 PR that play a significant role in binding, (3) protease changes upon binding and (4) various contributions to the binding free energy, in order to identify the most significant of them. This study has been performed by employing a docking technique, two 3D-QSAR models, molecular dynamics (MD) simulations and the molecular mechanics Poisson–Boltzmann surface area (MM–PBSA) method. Our computed binding free energies are in satisfactory agreement with the experimental results. The suitability of specific fullerene derivatives as drug candidates was further enhanced, after ADMET (absorption, distribution, metabolism, excretion and toxicity) properties have been estimated to be promising. The outcomes of this study revealed important protein–ligand interaction patterns that may lead towards the development of novel, potent HIV-1 PR inhibitors.

Insertion of the Ca2+-Independent Phospholipase A2 into a Phospholipid Bilayer via Coarse-Grained and Atomistic Molecular Dynamics Simulations

Group VI Ca2+-independent phospholipase A2 (iPLA2) is a water-soluble enzyme that is active when associated with phospholipid membranes. Despite its clear pharmaceutical relevance, no X-ray or NMR structural information is currently available for the iPLA2 or its membrane complex. In this paper, we combine homology modeling with coarse-grained (CG) and all-atom (AA) molecular dynamics (MD) simulations to build structural models of iPLA2 in association with a phospholipid bilayer. CG-MD simulations of the membrane insertion process were employed to provide a starting point for an atomistic description. Six AA-MD simulations were then conducted for 60 ns, starting from different initial CG structures, to refine the membrane complex. The resulting structures are shown to be consistent with each other and with deuterium exchange mass spectrometry (DXMS) experiments, suggesting that our approach is suitable for the modeling of iPLA2 at the membrane surface. The models show that an anchoring region (residues 710–724) forms an amphipathic helix that is stabilized by the membrane. In future studies, the proposed iPLA2 models should provide a structural basis for understanding the mechanisms of lipid extraction and drug-inhibition. In addition, the dual-resolution approach discussed here should provide the means for the future exploration of the impact of lipid diversity and sequence mutations on the activity of iPLA2 and related enzymes.

The Application of Rational Design on Phospholipase A2 Inhibitors

The phospholipase A(2) (PLA(2)) superfamily consists of different groups of enzymes which are characterized by their ability to catalyze the hydrolysis of the sn-2 ester bond in a variety of phospholipid molecules. The products of PLA(2s) activity play divergent roles in a variety of physiological processes. There are four main types of PLA(2s): the secreted PLA(2s) (sPLA(2s)), the cytosolic PLA(2s) (cPLA(2s)), the calcium-independent PLA(2s) (iPLA(2)) and the lipoprotein-associated PLA(2s) (LpPLA(2s)). Various potent and selective PLA2 inhibitors have been reported up to date and have provided outstanding support in understanding the mechanism of action and elucidating the function of these enzymes. The current review focuses on the implementation of rational design through computer-aided drug design (CADD) on the discovery and development of new PLA(2) inhibitors.

Inhibition of secreted phospholipases A2 by 2-oxoamides based on α-amino acids: Synthesis, in vitro evaluation and molecular docking calculations

Group IIA secreted phospholipase A₂ (GIIA sPLA₂) is a member of the mammalian sPLA₂ enzyme family and is associated with various inflammatory conditions. In this study, the synthesis of 2-oxoamides based on α-amino acids and the in vitro evaluation against three secreted sPLA₂s (GIIA, GV and GX) are described. The long chain 2-oxoamide GK126 based on the amino acid (S)-leucine displayed inhibition of human and mouse GIIA sPLA₂s (IC₅₀ 300nM and 180nM, respectively). It also inhibited human GV sPLA₂ with similar potency, while it did not inhibit human GX sPLA₂. The elucidation of the stereoelectronic characteristics that affect the in vitro activity of these compounds was achieved by using a combination of simulated annealing to sample low-energy conformations before the docking procedure, and molecular docking calculations.

Membrane and inhibitor interactions of intracellular phospholipases A2

Studying phospholipases A2 (PLA2s) is a challenging task since they act on membrane-like aggregated substrates and not on monomeric phospholipids. Multidisciplinary approaches that include hydrogen/deuterium exchange mass spectrometry (DXMS) and computational techniques have been employed with great success in order to address important questions about the mode of interactions of PLA2 enzymes with membranes, phospholipid substrates and inhibitors. Understanding the interactions of PLA2s is crucial since these enzymes are the upstream regulators of the eicosanoid pathway liberating free arachidonic acid (AA) and other polyunsaturated fatty acids (PUFA). The liberation of AA by PLA2 enzymes sets off a cascade of molecular events that involves downstream regulators such as cyclooxygenase (COX) and lipoxygenase (LOX) metabolites leading to inflammation. Aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) work by inhibiting COX, while Zileuton inhibits LOX and both rely on PLA2 enzymes to provide them with AA. That means PLA2 enzymes can potentially also be targeted to diminish inflammation at an earlier point in the process. In this review we describe extensive efforts reported in the past to define the interactions of PLA2 enzymes with membranes, substrate phospholipids and inhibitors using DXMS, molecular docking, and molecular dynamics (MD) simulations.

Binding conformation of 2-oxoamide inhibitors to group IVA cytosolic phospholipase A2 determined by molecular docking combined with molecular dynamics.

The group IVA cytosolic phospholipase A(2) (GIVA cPLA(2)) plays a central role in inflammation. Long chain 2-oxoamides constitute a class of potent GIVA cPLA(2) inhibitors that exhibit potent in vivo anti-inflammatory and analgesic activity. We have now gained insight into the binding of 2-oxoamide inhibitors in the GIVA cPLA(2) active site through a combination of molecular docking calculations and molecular dynamics simulations. Recently, the location of the 2-oxoamide inhibitor AX007 within the active site of the GIVA cPLA(2) was determined using a combination of deuterium exchange mass spectrometry followed by molecular dynamics simulations. After the optimization of the AX007-GIVA cPLA(2) complex using the docking algorithm Surflex-Dock, a series of additional 2-oxoamide inhibitors have been docked in the enzyme active site. The calculated binding affinity presents a good statistical correlation with the experimental inhibitory activity (r(2) = 0.76, N = 11). A molecular dynamics simulation of the docking complex of the most active compound has revealed persistent interactions of the inhibitor with the enzyme active site and proves the stability of the docking complex and the validity of the binding suggested by the docking calculations. The combination of molecular docking calculations and molecular dynamics simulations is useful in defining the binding of small-molecule inhibitors and provides a valuable tool for the design of new compounds with improved inhibitory activity against GIVA cPLA(2).

Review of four major distinct types of human phospholipase A2

The phospholipase A2 superfamily of enzymes plays a significant role in the development and progression of numerous inflammatory diseases. Through their catalytic action on membrane phospholipids, phospholipases are the upstream regulators of the eicosanoid pathway releasing free fatty acids for cyclooxygenases, lipoxygenases, and cytochrome P450 enzymes which produce various well-known inflammatory mediators including leukotrienes, thromboxanes and prostaglandins. Elucidating the association of phospholipases A2 with the membrane, the extraction and binding of phospholipid substrates, and their interactions with small-molecule inhibitors is crucial for the development of new anti-inflammatory therapeutics. Studying phospholipases has been challenging because they act on the surface of cellular membranes and micelles. Multidisciplinary approaches including hydrogen/deuterium exchange mass spectrometry, molecular dynamics simulations, and other computer-aided drug design techniques have been successfully employed by our laboratory to study interactions of phospholipases with membranes, phospholipid substrates and inhibitors. This review summarizes the application of these techniques to study four human recombinant phospholipases A2.

Development of Potent and Selective Inhibitors for Group VIA Calcium-Independent Phospholipase A2 Guided by Molecular Dynamics and Structure–Activity Relationships

The development of inhibitors for phospholipase A2 (PLA2) is important in elucidating the enzymes implication in various biological pathways. PLA2 enzymes are an important pharmacological target implicated in various inflammatory diseases. Computational chemistry, organic synthesis, and in vitro assays were employed to develop potent and selective inhibitors for group VIA calcium-independent PLA2. A set of fluoroketone inhibitors was studied for their binding mode with two human cytosolic PLA2 enzymes: group IVA cPLA2 and group VIA iPLA2. New compounds were synthesized and assayed toward three major PLA2s. This study led to the development of four potent and selective thioether fluoroketone inhibitors as well as a thioether keto-1,2,4-oxadiazole inhibitor for GVIA iPLA2, which will serve as lead compounds for future development and studies. The keto-1,2,4-oxadiazole functionality with a thioether is a novel structure, and it will be used as a lead to develop inhibitors with higher potency and selectivity toward GVIA iPLA2.