Synthesis of an allosteric modulator of ionotropic glutamate receptors

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Molecular modelling prediction of ligand binding site flexibility

We have investigated the efficacy of generating multiple sidechain conformations using a rotamer library in order to find the experimentally observed ligand binding site conformation of a protein in the presence of a bound ligand. We made use of a recently published algorithm that performs an exhaustive conformational search using a rotamer library to enumerate all possible sidechain conformations in a binding site. This approach was applied to a dataset of proteins whose structures were determined by X-ray and NMR methods. All chosen proteins had two or more structures, generally involving different bound ligands. By taking one of these structures as a reference, we were able in most cases to successfully reproduce the experimentally determined conformations of the other structures, as well as to suggest alternative low-energy conformations of the binding site. In those few cases where this procedure failed, we observed that the bound ligand had induced a high-energy conformation of the binding site. These results suggest that for most proteins that exhibit limited backbone motion, ligands tend to bind to low energy conformations of their binding sites. Our results also reveal that it is possible in most cases to use a rotamer search-based approach to predict alternative low-energy protein binding site conformations that can be used by different ligands. This opens the possibility of incorporating alternative binding site conformations to improve the efficacy of docking and structure-based drug design algorithms.     

Buried solvent determines both anion-binding selectivity and binding stoichiometry with hydrogen-bonding receptors

[reaction: see text] The crystal structure of a tetraurea picket porphyrin-chloride anion complex has previously shown the anion to be situated between two adjacent ureas and hydrogen bonded via four NH protons (J. Am. Chem. Soc. 1998, 120, 11684-11692). The porphyrin receptor also binds a DMSO molecule and utilizes it as a participant in its anion recognition unit, in a manner similar to enzymes that bind water for use as part of their substrate recognition unit. The bound solvent molecule determines the anion-binding affinity, selectivity, and stoichiometry of binding. With a bound DMSO molecule, the tetraurea picket porphyrin is a highly selective receptor for chloride anion and binds all anions with a 1:1 binding stoichiometry. Absent the buried DMSO molecule, the receptor is selective for phosphate anion and binds chloride and phosphate anions with both 1:1 and 1:2 receptor-anion stoichiometries. Additionally, a remarkable reversal in the selectivity of anion complexation between various picket porphyrin receptors is observed, wherein the binding constant ratios change over 3 orders of magnitude as the receptor's number of urea pickets change from four to two. The latter receptor has no urea pickets available to bind to solvent after complexation with an anion. The results demonstrate that anion complexation with hydrogen-bonding receptors in a competitive solvent is enhanced when a ubiquitous solvent molecule is incorporated into the binding motif. In this way, competitive solvent adds to the overall complexation energy and thereby strengthens binding rather than weakens it, as commonly believed. The results are pertinent to drug design, for they suggest that pharmaceuticals need not be completely desolvated to selectively bind to their biological target when water can be included in the binding motif.     

Functional characterisation of homomeric ionotropic glutamate receptors GluR1-GluR6 in a fluorescence-based high throughput screening assay

We have constructed stable HEK293 cell lines expressing the rat ionotropic glutamate receptor subtypes GluR1(i), GluR2Q(i), GluR3(i), GluR4(i), GluR5Q and GluR6Q and characterised the pharmacological profiles of the six homomeric receptors in a fluorescence-based high throughput screening assay using Fluo-4/AM as a fluorescent Ca2+ indicator. In this assay, the pharmacological properties of nine standard GluR ligands correlated nicely with those previously observed in electrophysiology studies of GluRs expressed in Xenopus oocytes or mammalian cells. The potencies and efficacies displayed by the agonists (S)-glutamate, (S)-quisqualate, kainate, (RS)-AMPA, (RS)-ATPA, (RS)-ACPA] and (S)-4-AHCP at the six GluRs were in concordance with electrophysiological studies. Furthermore, the Ki values exhibited by the competitive antagonists NBQX and (RS)-ATPO were also in agreement with findings of previous studies. Finally, the effects of various concentrations of Ca2+ in the assay buffer and of the allosteric modulators cyclothiazide and concanavalin A on GluR signalling were examined. This study represents the most elaborate functional characterisation of multiple AMPA and KA receptor subtypes in the same assay reported to date. We propose that high throughput screening of compound libraries at the six GluR-HEK293 cell lines could be helpful in the search for structurally and pharmacologically novel ligands acting at the receptors.     

Enantioselective synthetic thalidomide receptors based upon DNA binding motifs

A series of molecularly imprinted polymer (MIP) synthetic receptors selective for the sedative thalidomide (5) have been designed and synthesized based upon the functional monomer 9-(2'-methacryloyloxyethyl)adenine (2). (1)H-NMR studies were used to establish the existence of DNA-like binding interactions between 2 and the template (5). A series of ethylene glycol dimethacrylate cross-linked copolymers was synthesized using either 2 or methacrylic acid, or a combination of these functional monomers. Zonal HPLC studies demonstrated enantioselectivity (alpha = 2.11) and ligand selectivity which could be attributed to the interaction of 2 with the imide moiety of 5. Compound 2 provided a more significant contribution to the binding of 5 than methacrylic acid, though a combination of these two functional monomers resulted in improved enantioselectivity. Frontal chromatographic and batch binding studies confirmed the observed differences in affinity of the imprinted and reference polymers for the template.     

Molecular dynamics simulation of the ligand binding domain of mGluR1 in response to agonist and antagonist binding

The interdomain movements of the ligand binding domain (LBD) of mGluR1 in response to agonist or antagonist binding are studied by 2 ns molecular dynamics (MD) simulations. Our results indicate that MD is able to reproduce many of the experimentally determined features of the open and closed conformations of LBD. Analysis of the ligand behavior over time allows to delineate some of the molecular determinants responsible for the agonist-induced or antagonist-blocked LBD responses.     

NMR spectroscopy techniques for screening and identifying ligand binding to protein receptors

Binding events of ligands to receptors are the key for an understanding of biological processes. Gaining insight into protein-protein and protein-ligand interactions in solution has recently become possible on an atomic level by new NMR spectroscopic techniques. These experiments identify binding events either by looking at the resonance signals of the ligand or the protein. Ideally, both techniques together deliver a complete picture of ligand binding to a receptor. The approaches discussed in this review allow screening of compound libraries as well as a detailed identification of the groups involved in the binding events. Also, characterization of the binding strength and kinetics is possible, competitive binding as well as allosteric effects can be identified, and it has even been possible to identify ligand binding to intact viruses and membrane-bound proteins.     

Kinetics of ligand binding to membrane receptors from equilibrium fluctuation analysis of single binding events

Equilibrium fluctuation analysis of single binding events has been used to extract binding kinetics of ligand interactions with cell-membrane bound receptors. Time-dependent total internal reflection fluorescence (TIRF) imaging was used to extract residence-time statistics of fluorescently stained liposomes derived directly from cell membranes upon their binding to surface-immobilized antibody fragments. The dissociation rate constants for two pharmaceutical relevant antibodies directed against different B-cell expressed membrane proteins was clearly discriminated, and the affinity of the interaction could be determined by inhibiting the interaction with increasing concentrations of soluble antibodies. The single-molecule sensitivity made the analysis possible without overexpressed membrane proteins, which makes the assay attractive in early drug-screening applications.     

A single hydroxyl group governs ligand site selectivity in human ileal bile acid binding protein

The recognition between proteins and their native ligands is fundamental to biological function. In vivo, human ileal bile acid binding protein (I-BABP) encounters a range of bile salts that vary in the number and position of steroidal hydroxyl groups and the presence and type of side-chain conjugation. Therefore, it is necessary to understand how chemical variability in the ligand affects the energetic and structural aspects of its recognition. Here we report studies of the binding site selectivity of I-BABP for glycocholic (GCA) and glycochenodeoxycholic (GCDA) acids using isotope-enriched bile salts along with two-dimensional heteronuclear NMR methods. When I-BABP is presented with either GCA or GCDA alone, the ligands bind to both sites. However, when presented with an equimolar mixture of the two bile salts, GCDA binds exclusively to site 1 and GCA to site 2. This remarkable selectivity is governed by the presence or absence of a single hydroxyl group at the C-12 position of the steroid tetracycle. The basis for this site selectivity appears to be energetic rather then steric.     

Network dimensionality and ligand flexibility in lanthanide terephthalate hydrates

Various lanthanide open framework materials incorporating the terephthalate (TP) entity were prepared using hydrothermal synthesis methods at a moderate temperature of 170 °C. The compounds Nd₂(TP)₃(H₂O)₄(1), Er₂(TP)₃(H₂O)₄(2), Yb₂(TP)₃(H₂O)₂(3), Yb₂(TP)₃(H₂O)₆(4), and Yb₂(TP)₃(H₂O)₈·2H₂O (5), were characterized by single crystal structural analysis and FT-IR spectroscopy. While compounds 1 and 2 have been reported before on the basis of powder X-ray diffraction, the structural characterization of any ytterbium terephthalate species is unprecedented. Compounds 1-5 crystallize in triclinic settings with space group P-1. The compounds are compared with their previously reported Er and Tb-counterparts and the reduction of the dimensionality of the resulting networks from 3D over 2D to 1D with increasing level of hydration is discussed. Compounds 1, 2, and 3 with the lowest water content assemble in three-dimensional network lattices. Compounds 4 and 5, however, form 2D layered systems and 1D rod like chains, respectively, which are held together by hydrogen bonds originating from coordinating H₂O. The crystal lattices of the 3D networks experience higher levels of tension as can be seen by increasing out-of-plane torsion with regard to the terephthalate carboxylate groups. Moreover, there seems to be a correlation between the level of strain on the aromatic ligands and the reduction of the number of carboxylate oxygen atoms that are part of the coordination polyhedra.     

Subtype selectivity of dopamine receptor ligands: insights from structure and ligand-based methods

Subtype selective dopamine receptor ligands have long been sought after as therapeutic and/or imaging agents for the treatment and monitoring of neurologic disorders. We report herein on a combined structure- and ligand-based approach to explore the molecular mechanism of the subtype selectivity for a large class of D?-like dopamine receptor ligands (163 ligands in total). Homology models were built for both human D(?L) and D? receptors in complex with haloperidol. Other ligands, which included multiple examples of substituted phenylpiperazines, were aligned against the binding conformations of haloperidol, and three-dimensional quantitative structure activity relationship (3D-QSAR) analyses were carried out. The receptor models show that although D? and D? share highly similar folds and 3D conformations, the slight sequence differences at their extracellular loop regions result in the binding cavity in D? being comparably shallower than in D?, which may explain why some larger ligands bind with greater affinity at D? compared to D? receptors. The QSAR models show excellent correlation and high predictive power even when evaluated by the most stringent criteria. They confirm that the origins of subtype selectivity for the ligands arise primarily due to differences in the contours of the two binding sites. The predictive models suggest that while both steric and electrostatic interactions contribute to the compounds' binding affinity, the major contribution arises from hydrophobic interactions, with hydrogen bonding conferring binding specificity. The current work provides clues for the development of more subtype selective dopamine receptor ligands. Furthermore, it demonstrates the possibility of being able to apply similar modeling methods to other subtypes or classes of receptors to study GPCR receptor-ligand interactions at a molecular level.     

Three-dimensional models of glutamate receptors

Structural models of glutamate receptors have been produced as part of a multidisciplinary study of neuronal function--both ligand/receptor interactions and ion transport--at the atomic level. The models have concentrated on the agonist binding and transmembrane domains of ionotropic (i.e. ligand-gated) glutamate receptors (iGluRs), and have aided our understanding of the molecular determinants of (1) ligand binding and (2) channel activity. The model building process involved a combination of homology modelling, distance geometry, molecular mechanics, protein-ligand and protein-protein docking, electrostatic calculations and manual adjustment, in conjunction with restraints from site-directed mutagenesis, ligand binding and electrophysiological studies. The initial models were used to produce hypotheses which were tested experimentally; these models have been subsequently refined as part of an extremely effective multidisciplinary study using an iterative molecular modelling/experimental verification cycle in which restraints derived from experimental studies are used at all stages, and the findings from one round of modelling are used as restraints in the next. By studying a variety of agonists and antagonists, details have been built up of (1) those residues involved in ligand binding and (2) the role of agonist binding (i.e. agonist-induced conformational change) in channel gating. The models also aid our understanding of the conductance properties of the channels.     

Changes in calmodulin main-chain dynamics upon ligand binding revealed by cross-correlated NMR relaxation measurements

The fast dynamics of protein backbones are often investigated by nuclear magnetic relaxation experiments that report on the degree of spatial restriction of the amide bond vector. By comparing calmodulin in the peptide-bound and peptide-free states with these classical methods, we observe little difference in the dynamics of the polypeptide main chain (average order parameter decrease of 0.01 unit upon binding). However, when using NMR methods that monitor the mobility of the CO-Calpha bond vector, we reveal a significant reduction of dynamics of the protein main chain (average order parameter decrease of 0.048 units). Previous investigations have suggested that the side-chain dynamics is reduced by an average of 0.07 order parameter units upon ligand binding (Lee, A. L.; Kinnear, S. A.; Wand, A. J. Nat. Struct. Biol. 2000, 7, 72-77). The current findings suggest that the change of the CO-Calpha bond vector dynamics is intermediate between the changes in NH and side-chain dynamics and report a previously undetected loss of main-chain entropy. Weak site-to-site correlations between the different motional indicators are also observed.     

A molecular dynamics investigation of CDK8/CycC and ligand binding: conformational flexibility and implication in drug discovery

Abnormal activity of cyclin-dependent kinase 8 (CDK8) along with its partner protein cyclin C (CycC) is a common feature of many diseases including colorectal cancer. Using molecular dynamics (MD) simulations, this study determined the dynamics of the CDK8-CycC system and we obtained detailed breakdowns of binding energy contributions for four type-I and five type-II CDK8 inhibitors. We revealed system motions and conformational changes that will affect ligand binding, confirmed the essentialness of CycC for inclusion in future computational studies, and provide guidance in development of CDK8 binders. We employed unbiased all-atom MD simulations for 500 ns on twelve CDK8-CycC systems, including apoproteins and protein–ligand complexes, then performed principal component analysis (PCA) and measured the RMSF of key regions to identify protein dynamics. Binding pocket volume analysis identified conformational changes that accompany ligand binding. Next, H-bond analysis, residue-wise interaction calculations, and MM/PBSA were performed to characterize protein–ligand interactions and find the binding energy. We discovered that CycC is vital for maintaining a proper conformation of CDK8 to facilitate ligand binding and that the system exhibits motion that should be carefully considered in future computational work. Surprisingly, we found that motion of the activation loop did not affect ligand binding. Type-I and type-II ligand binding is driven by van der Waals interactions, but electrostatic energy and entropic penalties affect type-II binding as well. Binding of both ligand types affects protein flexibility. Based on this we provide suggestions for development of tighter-binding CDK8 inhibitors and offer insight that can aid future computational studies.     

Multiple-step ligand injection affinity capillary electrophoresis for determining binding constants of ligands to receptors

This work demonstrates the use of multiple-step ligand injection affinity capillary electrophoresis (ACE) using two model systems: vancomycin from Streptomyces orientalis and carbonic anhydrase B (CAB, EC 4.2.1.1). In this technique a sample plug of receptor and non-interacting standards is injected by pressure and electrophoresed in a buffer containing a given concentration of ligand. The sequence is repeated for all concentrations of ligand generating a single electropherogram containing a series of individual sample plugs superimposed on environments of buffer containing increasing concentrations of ligand. Analysis of the change in the relative migration time ratio, RMTR, relative to the non-interacting standards, as a function of the concentration of the ligand, yields a value for the binding constant. A competitive assay using the technique is also demonstrated using neutral ligands for CAB. These values agree well with those estimated using other binding and ACE techniques. Data demonstrating the quantitative potential of this method are presented.