Anthony Ivetac

A combinatorial TIR1/AFB–Aux/IAA co-receptor system for differential sensing of auxin

The plant hormone auxin regulates virtually every aspect of plant growth and development. Auxin acts by binding to the F-box protein TIR1 and promotes the degradation of the Aux/IAA transcriptional repressors. Here, we show that efficient auxin binding requires assembly of an auxin co-receptor complex consisting of TIR1 and an Aux/IAA protein. Heterologous experiments in yeast and quantitative IAA binding assays using purified proteins showed that different combinations of TIR1 and Aux/IAA proteins form co-receptor complexes with a wide range of auxin-binding affinities. Auxin affinity appears to be largely determined by the Aux/IAA. As there are 6 TIR1/AFBs and 29 Aux/IAA proteins in Arabidopsis thaliana, combinatorial interactions may result in many co-receptors with distinct auxin sensing properties. We also demonstrate that the AFB5-Aux/IAA co-receptor selectively binds the auxinic herbicide picloram. This co-receptor system broadens the effective concentration range of the hormone and may contribute to the complexity of auxin response.

Coarse-grained MD simulations of membrane protein-bilayer self-assembly

Complete determination of a membrane protein structure requires knowledge of the protein position within the lipid bilayer. As the number of determined structures of membrane proteins increases so does the need for computational methods which predict their position in the lipid bilayer. Here we present a coarse-grained molecular dynamics approach to lipid bilayer self-assembly around membrane proteins. We demonstrate that this method can be used to predict accurately the protein position in the bilayer for membrane proteins with a range of different sizes and architectures.

Mapping the Druggable Allosteric Space of G-Protein Coupled Receptors: a Fragment-Based Molecular Dynamics Approach

To address the problem of specificity in G‐protein coupled receptor (GPCR) drug discovery, there has been tremendous recent interest in allosteric drugs that bind at sites topographically distinct from the orthosteric site. Unfortunately, structure‐based drug design of allosteric GPCR ligands has been frustrated by the paucity of structural data for allosteric binding sites, making a strong case for predictive computational methods. In this work, we map the surfaces of the β1 (β1AR) and β2 (β2AR) adrenergic receptor structures to detect a series of five potentially druggable allosteric sites. We employ the FTMAP algorithm to identify ‘hot spots’ with affinity for a variety of organic probe molecules corresponding to drug fragments. Our work is distinguished by an ensemble‐based approach, whereby we map diverse receptor conformations taken from molecular dynamics (MD) simulations totaling approximately 0.5 μs. Our results reveal distinct pockets formed at both solvent‐exposed and lipid‐exposed cavities, which we interpret in light of experimental data and which may constitute novel targets for GPCR drug discovery. This mapping data can now serve to drive a combination of fragment‐based and virtual screening approaches for the discovery of small molecules that bind at these sites and which may offer highly selective therapies.

Cystic Fibrosis Transmembrane Conductance Regulator: Using Differential Reactivity toward Channel-Permeant and Channel-Impermeant Thiol-Reactive Probes To Test a Molecular Model for the Pore

The sixth transmembrane segment (TM6) of the CFTR chloride channel has been intensively investigated. The effects of amino acid substitutions and chemical modification of engineered cysteines (cysteine scanning) on channel properties strongly suggest that TM6 is a key component of the anion-conducting pore, but previous cysteine-scanning studies of TM6 have produced conflicting results. Our aim was to resolve these conflicts by combining a screening strategy based on multiple, thiol-directed probes with molecular modeling of the pore. CFTR constructs were screened for reactivity toward both channel-permeant and channel-impermeant thiol-directed reagents, and patterns of reactivity in TM6 were mapped onto two new, molecular models of the CFTR pore: one based on homology modeling using Sav1866 as the template and a second derived from the first by molecular dynamics simulation. Comparison of the pattern of cysteine reactivity with model predictions suggests that nonreactive sites are those where the TM6 side chains are occluded by other TMs. Reactive sites, in contrast, are generally situated such that the respective amino acid side chains either project into the predicted pore or lie within a predicted extracellular loop. Sites where engineered cysteines react with both channel-permeant and channel-impermeant probes occupy the outermost extent of TM6 or the predicted TM5−6 loop. Sites where cysteine reactivity is limited to channel-permeant probes occupy more cytoplasmic locations. The results provide an initial validation of two, new molecular models for CFTR and suggest that molecular dynamics simulation will be a useful tool for unraveling the structural basis of anion conduction by CFTR.

Predictive Power of Molecular Dynamics Receptor Structures in Virtual Screening

Molecular dynamics (MD) simulation is a well-established method for understanding protein dynamics. Conformations from unrestrained MD simulations have yet to be assessed for blind virtual screening (VS) by docking. This study presents a critical analysis of the predictive power of MD snapshots to this regard, evaluating two well-characterized systems of varying flexibility in ligand-bound and unbound configurations. Results from such VS predictions are discussed with respect to experimentally determined structures. In all cases, MD simulations provide snapshots that improve VS predictive power over known crystal structures, possibly due to sampling more relevant receptor conformations. Additionally, MD can move conformations previously not amenable to docking into the predictive range.

Molecular recognition in the case of flexible targets.

A proteins flexibility is well recognized to underlie its capacity to engage in critical functions, such as signal transduction, biomolecular transport and biochemical reactivity. Molecular recognition is also tightly linked to the dynamics of the binding partners, yet protein flexibility has largely been ignored by the growing field of structure-based drug design (SBDD). In combination with experimentally determined structures, a number of computational methods have been proposed to model protein movements, which may be important for small molecule binding. Such techniques have the ability to expose new binding site conformations, which may in turn recognize and lead to the discovery of more potent and selective drugs through molecular docking. In this article, we discuss various methods and focus on the Relaxed Complex Scheme (RCS), which uses Molecular Dynamics (MD) simulations to model full protein flexibility and enhance virtual screening programmes. We review practical applications of the RCS and use a recent study of the HIV-1 reverse transcriptase to illustrate the various phases of the scheme. We also discuss some encouraging developments, aimed at addressing current weaknesses of the RCS.

Locating a Plausible Binding Site for an Open-Channel Blocker, GlyH-101, in the Pore of the Cystic Fibrosis Transmembrane Conductance Regulator

High-throughput screening has led to the identification of small-molecule blockers of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, but the structural basis of blocker binding remains to be defined. We developed molecular models of the CFTR channel on the basis of homology to the bacterial transporter Sav1866, which could permit blocker binding to be analyzed in silico. The models accurately predicted the existence of a narrow region in the pore that is a likely candidate for the binding site of an open-channel pore blocker such as N-(2-naphthalenyl)-[(3,5-dibromo-2,4-dihydroxyphenyl)methylene]glycine hydrazide (GlyH-101), which is thought to act by entering the channel from the extracellular side. As a more-stringent test of predictions of the CFTR pore model, we applied induced-fit, virtual, ligand-docking techniques to identify potential binding sites for GlyH-101 within the CFTR pore. The highest-scoring docked position was near two pore-lining residues, Phe337 and Thr338, and the rates of reactions of anionic, thiol-directed reagents with cysteines substituted at these positions were slowed in the presence of the blocker, consistent with the predicted repulsive effect of the net negative charge on GlyH-101. When a bulky phenylalanine that forms part of the predicted binding pocket (Phe342) was replaced with alanine, the apparent affinity of the blocker was increased ∼200-fold. A molecular mechanics-generalized Born/surface area analysis of GlyH-101 binding predicted that substitution of Phe342 with alanine would substantially increase blocker affinity, primarily because of decreased intramolecular strain within the blocker-protein complex. This study suggests that GlyH-101 blocks the CFTR channel by binding within the pore bottleneck.

Simulations of Biased Agonists in the β2 Adrenergic Receptor with Accelerated Molecular Dynamics

The biased agonism of the G protein-coupled receptors (GPCRs), where in addition to a traditional G protein-signaling pathway a GPCR promotes intracellular signals though β-arrestin, is a novel paradigm in pharmacology. Biochemical and biophysical studies have suggested that a GPCR forms a distinct ensemble of conformations signaling through the G protein and β-arrestin. Here we report on the dynamics of the β2 adrenergic receptor bound to the β-arrestin and G protein-biased agonists and the empty receptor to further characterize the receptor conformational changes caused by biased agonists. We use conventional and accelerated molecular dynamics (aMD) simulations to explore the conformational transitions of the GPCR from the active state to the inactive state. We found that aMD simulations enable monitoring of the transition within the nanosecond time scale while capturing the known microscopic characteristics of the inactive states, such as the ionic lock, the inward position of F6.44, and water clusters. Distinct conformational states are shown to be stabilized by each biased agonist. In particular, in simulations of the receptor with the β-arrestin-biased agonist N-cyclopentylbutanepherine, we observe a different pattern of motions in helix 7 when compared to simulations with the G protein-biased agonist salbutamol that involves perturbations of the network of interactions within the NPxxY motif. Understanding the network of interactions induced by biased ligands and the subsequent receptor conformational shifts will lead to development of more efficient drugs.

Discovery of Novel Inhibitors of HIV‐1 Reverse Transcriptase Through Virtual Screening of Experimental and Theoretical Ensembles

Non‐nucleoside reverse transcriptase inhibitors (NNRTIs) are potent anti‐HIV chemotherapeutics. Although there are FDA‐approved NNRTIs, challenges such as the development of resistance have limited their utility. Here, we describe the identification of novel NNRTIs through a combination of computational and experimental approaches. Based on the known plasticity of the NNRTI binding pocket (NNIBP), we adopted an ensemble‐based virtual screening strategy: coupling receptor conformations from 10 X‐ray crystal structures with 120 snapshots from a total of 480 ns of molecular dynamics (MD) trajectories. A screening library of 2864 National Cancer Institute (NCI) compounds was built and docked against the ensembles in a hierarchical fashion. Sixteen diverse compounds were tested for their ability to block HIV infection in human tissue cultures using a luciferase‐based reporter assay. Three promising compounds were further characterized, using a HIV‐1 RT‐based polymerase assay, to determine the specific mechanism of inhibition. We found that 2 of the three compounds inhibited the polymerase activity of RT (with potency similar to the positive control, the FDA‐approved drug nevirapine). Through a computational approach, we were able to discover two compounds which inhibit HIV replication and block the activity of RT, thus offering the potential for optimization into mature inhibitors.

Binding of GlyH-101 in the Pore of the CFTR Chloride Channel

GlyH-101 is a small molecule (MW: 493) that carries a single negative charge (pKa: 5.5) under physiological conditions (∼ pH 7.4) and blocks the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel by entering from the extracellular side and binding to a site or sites within the pore (Muanprasat et al., J. Gen. Physiol. 124: 125-137). However, the precise binding sites for this molecule have yet to be identified. We used virtual ligand docking software, “Glide” (Schrodinger Inc.) to identify potential GlyH-101 binding sites within molecular models of CFTR derived by means of molecular dynamics simulation from a homology model based on Sav 1866 (Alexander et al., Biochemistry 48: 10078-10088). These sites were evaluated by determining the extent of occlusion of reactive cysteines by the blocker. The results suggest that the binding of GlyH-101 near a narrow portion of the pore could reduce the reactivity of T338C with [Au(CN)2]- and MTSES- by a repulsive charge-charge interaction. We tested the efficacy and potency of GlyH-101 for CFTR mutant channels and discovered a single amino acid substitution that significantly increases the potency of GlyH-101. Supported by NIH, Cystic Fibrosis Foundation, American Lung Association, the Wellcome Trust, and the BBSRC.