A molecular mechanism of P-loop pliability of Rho-kinase investigated by molecular dynamic simulation

Rho-kinase is a leading player in the regulation of cytoskeletal events involving smooth muscle contraction and neurite growth-cone collapse and retraction, and is a promising drug target in the treatment of both vascular and neurological disorders. Recent crystal structure of Rho-kinase complexed with a small-molecule inhibitor fasudil has revealed structural details of the ATP-binding site, which represents the target site for the inhibitor, and showed that the conserved phenylalanine on the P-loop occupies the pocket, resulting in an increase of protein–ligand contacts. Thus, the P-loop pliability is considered to play an important role in inhibitor binding affinity and specificity. In this study, we carried out a molecular dynamic simulation for Rho-kinase–fasudil complexes with two different P-loop conformations, i.e., the extended and folded conformations, in order to understand the P-loop pliability and dynamics at atomic level. A PKA–fasudil complex was also used for comparison. In the MD simulation, the flip-flop movement of the P-loop conformation starting either from the extended or folded conformation was not able to be observed. However, a significant conformational change in a long loop region covering over the P-loop, and also alteration of ionic interaction-manner of fasudil with acidic residues in the ATP binding site were shown only in the Rho-kinase–fasudil complex with the extended P-loop conformation, while Rho-kinase with the folded P-loop conformation and PKA complexes did not show large fluctuations, suggesting that the Rho-kinase–fasudil complex with the extended P-loop conformation represents a meta-stable state. The information of the P-loop pliability at atomic level obtained in this study could provide valuable clues to designing potent and/or selective inhibitors for Rho-kinase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The Conformation of a Peptidyl Methyl Ketone Inhibitor Bound to the Human Cytomegalovirus Protease

A weak inhibitor means faster exchange! Since the methyl ketone MK2 is a weak noncovalent peptidyl inhibitor of the human cytomegalovirus protease, exchange between the free and enzyme-bound forms is rapid. This allows for the use of transferred NOE NMR methods and molecular modeling, which show that the bound conformation of MK2 is an extended peptide. This is confirmed by the results of an X-ray crystallographic analysis of a related enzyme–inhibitor complex.     

Synthesis and biological evaluation of novel isopropanolamine derivatives as non-peptide human immunodeficiency virus protease inhibitors

Novel potential human immunodeficiency virus (HIV) protease inhibitors were designed by a combination of nelfinavir and amprenavir motifs. The designed compounds were prepared by a facile synthetic route and their stereochemistry was further confirmed by a stereospecific synthesis from commercially available (S)-2-oxiranylmethyl m-nitrobenzenesulfonate. All compounds were tested for their ability in inhibiting HIV type 1 protease activity with the published method of reference 19. Derivatives 1a--u exhibited moderate to significant inhibitory activities in preliminary bioassay. The best compound 1a has IC50 value of 0.02 microM, comparable to that of amprenavir. A docking study on compounds 1a--u was performed using the published X-ray crystal structure of HIV type 1 protease, all compounds bound to the HIV type 1 protease in an extended conformation and the scaffoldings of the binding conformations could be aligned quite well. Comparative molecular field analysis (CoMFA) study was performed to explore the specific contributions of electrostatic and steric effects in the binding of these new compounds to HIV type 1 protease and a predictive CoMFA model was built with thirteen compounds as training set. Test analysis of other five compounds as test set demonstrated that the CoMFA model has strong predictive ability to this series of compounds. It will be very useful to further optimize the designed inhibitors.     

Torsion angle preference and energetics of small-molecule ligands bound to proteins

Small organic molecules can assume conformations in the protein-bound state that are significantly different from those in solution. We have analyzed the conformations of 21 common torsion motifs of small molecules extracted from crystal structures of protein-ligand complexes and compared them with their torsion potentials calculated by an ab initio DFT method. We find a good correlation between the potential energy of the torsion motifs and their conformational distribution in the protein-bound state: The most probable conformations of the torsion motifs agree well with the calculated global energy minima, and the lowest torsion-energy state becomes increasingly dominant as the torsion barrier height increases. The torsion motifs can be divided into 3 groups based on torsion barrier heights: high (>4 kcal/mol), medium (2-4 kcal/mol), and low (<2 kcal/mol). The calculated torsion energy profiles are predictive for the most preferred bound conformation for the high and medium barrier groups, the latter group common in druglike molecules. In the high-barrier group of druglike ligands, >95% of conformational torsions occur in the energy region <4 kcal/mol. The conformations of the torsion motifs in the protein-bound state can be modeled by a Boltzmann distribution with a temperature factor much higher than room temperature. This high-temperature factor, derived by fitting the theoretical model to the experimentally observed conformation occurrence of torsions, can be interpreted as the perturbation that proteins inflict on the conformation of the bound ligand. Using this model, it is calculated that the average strain energy of a torsion motif in ligands bound to proteins is approximately 0.6 kcal/mol, a result which can be related to the lower binding efficiency of larger ligands with more rotatable bonds. The above results indicate that torsion potentials play an important role in dictating ligand conformations in both the free and the bound states.     

Docking flexible molecules: A case study of three proteins

A genetic algorithm (GA) conformation search method is used to dock a series of flexible molecules into one of three proteins. The proteins examined are thermolysin (tmn), carboxypeptidase A (cpa), and dihydrofolate reductase (dfr). In the latter two proteins, the crystal ligand was redocked. For thermolysin, we docked eight ligands into a protein conformation derived from a single crystal structure. The bound conformations of the other ligands in tmn are known. In the cpa and dfr cases, and in seven of the eight tmn ligands, the GA docking method found conformations within 1.6 Å root mean square (rms) of the relaxed crystal conformation. © 1995 John Wiley & Sons, Inc.     

Identifying conformational changes of the β2 adrenoceptor that enable accurate prediction of ligand/receptor interactions and screening for GPCR modulators

The new β₂ Adrenoceptor (β₂AR) crystal structures provide a high-resolution snapshot of receptor interactions with two particular partial inverse agonists, (−)-carazolol and timolol. However, both experimental and computational studies of GPCR structure are significantly complicated by the existence of multiple conformational states coupled to ligand type and receptor activity. Agonists and antagonists induce or stabilize distinct changes in receptor structure that mediate a range of pharmacological activities. In this work, we (1) established that the existing β₂AR crystallographic conformers can be extended to describe ligand/receptor interactions for additional antagonist types, (2) generated agonist-bound receptor conformations, and (3) validated these models for agonist and antagonist virtual ligand screening (VLS). Using a ligand directed refinement protocol, we derived a single agonist-bound receptor conformation that selectively retrieved a diverse set of full and partial β₂AR agonists in VLS trials. Additionally, the impact of extracellular loop two conformation on VLS was assessed by docking studies with rhodopsin-based β₂AR homology models, and loop-deleted receptor models. A general strategy for constructing and selecting agonist-bound receptor pocket conformations is presented, which may prove broadly useful in creating agonist and antagonist bound models for other GPCRs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Hydrothermal Synthesis,Conversion Reaction,and Crystal Structure of 2-D 2-bromo-1,4-benzenedicarboxylate Copper(II) Complex with 2,2′-bipyridine

A fully deprotonated 2-bromo-1,4-benzenedicarboxylate copper(II) complex, [Cu(bbdc)(2,2′-bipy)](H₂O) (bbdc= 2-bromo-1,4-benzenedicarboxylate dianion, 2,2′-bipy=2,2′-bipyridine), was synthesized under hydrothermal conditions. This compound also can be prepared from the partly deprotonated [Cu(Hbbdc)₂(2,2′-bipy)] under basic and hydrothermal conditions. The crystal structure consists of infinite chains of [Cu₂(μ₄-bbdc)]²⁺ building blocks connected by bis-monodentate bbdc ligands and exhibits an extended 2-D architecture. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Incorporating protein flexibility in structure-based drug discovery: using HIV-1 protease as a test case

We have developed a receptor-based pharmacophore method which utilizes a collection of protein structures to account for inherent protein flexibility in structure-based drug design. Several procedures were systematically evaluated to derive the most general protocol for using multiple protein structures. Most notably, incorporating more protein flexibility improved the performance of the method. The pharmacophore models successfully discriminate known inhibitors from drug-like non-inhibitors. Furthermore, the models correctly identify the bound conformations of some ligands. We used unliganded HIV-1 protease to develop and validate this method. Drug design is always initiated with a protein-ligand structure, and such success with unbound protein structures is remarkable - particularly in the case of HIV-1 protease, which has a large conformational change upon binding. This technique holds the promise of successful computer-based drug design before bound crystal structures are even discovered, which can mean a jump-start of 1-3 years in tackling some medically relevant systems with computational methods.     

Determination of drug–serum protein interactions via fluorescence polarization measurements

New fast methods for the determination of pharmacokinetic behaviour of potential drug candidates are receiving increasing interest. We present a new homogeneous method for the determination of drug binding and drug competition for human serum albumin and α₁-acid glycoprotein that is amenable to high-throughput-screening. It is based on selective fluorescent probes and the measurement of fluorescence polarization. This leads to decreased interference with fluorescent drugs as compared with previously published methods based on similar probes and the measurement of fluorescence intensity. The binding of highly fluorescent drugs that still interfere with the probes can be measured by simply titrating the drugs in a two-component system with the serum protein. The assay may also be used to discover strongly binding protein ligands that are interesting for drug-targeting strategies. Additionally, binding data could be obtained from larger libraries of compounds for in silico predictive pharmacokinetics. Figure Fluorescence polarization displacement titration of dansylsarcosine (3D-structure as insert) bound to human serum albumin (HSA) by naproxene Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Binding of ligands by copolymer globules: Mesoscopic simulation

A simple mesoscopic model of a synthetic ligand-containing copolymer is proposed. Proteinlike copolymers—copolymers containing special sequences that can form globules with a dense solvophobic core and a loose solvophilic corona in solutions—are considered as ligand carriers. It is assumed that solvophilic units contain groups that can coordinate ligands from solution. The binding ability of such copolymers at various chain conformations is studied. It is found that the globular conformation promotes the coordination of ligands. On average, the amount of ligands bound by the copolymer in this conformation is a factor of 3 greater than that in the coil conformation. Apparently, this finding may be explained by the presence of the loose corona, which, on the one hand, provides the increased concentration of solvophilic units and, on the other hand, does not prevent the free diffusion of ligands in it.     

Detection of unwanted protein-bound ligands by capillary zone electrophoresis: the case of hidden ligands that stabilize cholinesterase conformation

Detection, identification and characterization of compounds present in purified proteins and biopharmaceuticals are of central interest. As well as chemical remedies, proteins of pharmacological interest have to exhibit their nakedness to become therapeutic drugs. Cholinesterases (ChE) are enzymes of major importance for detoxification of poisonous esters. Likewise, ChE are characterized by the high catalytic efficiency of an active site positioned at the bottom of a deep gorge. The gorge can be partially or fully occupied by ligands, i.e., substrates and inhibitors that are currently used in affinity chromatography purification steps. Accordingly, a suitable method allowing to analyse the presence of unwanted ligands and its influence on the functional conformation and stability of these enzymes was essential. We have developed CZE approaches for that purpose. The factors causing discrepancies between data for thermal unfolding of ChE by electrophoretic and by calorimetric methods were investigated. The presence of unwanted hidden ligands bound to purified enzymes was first demonstrated. The incidence of these ligands was discussed. Altogether, our results raised several questions concerning the real conformation of the native state of enzymes. Finally, CZE was proved to be a pertinent tool to validate the conformity of purified enzymes to a status of biopharmaceutical.     

Drug-resistant molecular mechanism of CRF01_AE HIV-1 protease due to V82F mutation

Human immunodeficiency virus type 1 protease (HIV-1 PR) is one of the major targets of anti-AIDS drug discovery. The circulating recombinant form 01 A/E (CRF01_AE, abbreviated AE) subtype is one of the most common HIV-1 subtypes, which is infecting more humans and is expanding rapidly throughout the world. It is, therefore, necessary to develop inhibitors against subtype AE HIV-1 PR. In this work, we have performed computer simulation of subtype AE HIV-1 PR with the drugs lopinavir (LPV) and nelfinavir (NFV), and examined the mechanism of resistance of the V82F mutation of this protease against LPV both structurally and energetically. The V82F mutation at the active site results in a conformational change of 79′s loop region and displacement of LPV from its proper binding site, and these changes lead to rotation of the side-chains of residues D25 and I50′. Consequently, the conformation of the binding cavity is deformed asymmetrically and some interactions between PR and LPV are destroyed. Additionally, by comparing the interactive mechanisms of LPV and NFV with HIV-1 PR we discovered that the presence of a dodecahydroisoquinoline ring at the P1′ subsite, a [2-(2,6-dimethylphenoxy)acetyl]amino group at the P2′ subsite, and an N2 atom at the P2 subsite could improve the binding affinity of the drug with AE HIV-1 PR. These findings are helpful for promising drug design. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Optimization of high throughput virtual screening by combining shape-matching and docking methods

Receptor flexibility is a critical issue in structure-based virtual screening methods. Although a multiple-receptor conformation docking is an efficient way to account for receptor flexibility, it is still too slow for large molecular libraries. It was reported that a fast ligand-centric, shape-based virtual screening was more consistent for hit enrichment than a typical single-receptor conformation docking. Thus, we designed a "distributed docking" method that improves virtual high throughput screening by combining a shape-matching method with a multiple-receptor conformation docking. Database compounds are classified in advance based on shape similarities to one of the crystal ligands complexed with the target protein. This classification enables us to pick the appropriate receptor conformation for a single-receptor conformation docking of a given compound, thereby avoiding time-consuming multiple docking. In particular, this approach utilizes cross-docking scores of known ligands to all available receptor structures in order to optimize the algorithm. The present virtual screening method was tested for reidentification of known PPARgamma and p38 MAP kinase active compounds. We demonstrate that this method improves the enrichment while maintaining the computation speed of a typical single-receptor conformation docking.     

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.     

A QXP-based multistep docking procedure for accurate prediction of protein-ligand complexes

The two great challenges of the docking process are the prediction of ligand poses in a protein binding site and the scoring of the docked poses. Ligands that are composed of extended chains in their molecular structure display the most difficulties, predominantly because of the torsional flexibility. On the basis of the molecular docking program QXP-Flo+0802, we have developed a procedure particularly for ligands with a high degree of rotational freedom that allows the accurate prediction of the orientation and conformation of ligands in protein binding sites. Starting from an initial full Monte Carlo docking experiment, this was achieved by performing a series of successive multistep docking runs using a local Monte Carlo search with a restricted rotational angle, by which the conformational search space is limited. The method was established by using a highly flexible acetylcholinesterase inhibitor and has been applied to a number of challenging protein-ligand complexes known from the literature.     

Refinement of the conformation of UDP-galactose bound to galactosyltransferase using the STD NMR intensity-restrained CORCEMA optimization

The STD NMR technique has originally been described as a tool for screening large compound libraries to identify the lead compounds that are specific to target proteins of interest. The application of this technique in the qualitative epitope mapping of ligands weakly binding to proteins, virus capsid shells, and nucleic acids has also been described. Here we describe the application of the STD NMR intensity-restrained CORCEMA optimization (SICO) procedure for refining the bound conformation of UDP-galactose in galactosyltransferase complex using STD NMR intensities recorded at 500 MHz as the experimental constraints. A comparison of the SICO structure for the bound UDP-galactose in solution with that in the crystal structure for this complex shows some differences in ligand torsion angles and V253 side-chain orientation in the protein. This work describes the first application of an STD NMR intensity-restrained CORCEMA optimization procedure for refining the torsion angles of a bound ligand structure. This method is likely to be useful in structure-based drug design programs since most initial lead compounds generally exhibit weak affinity (millimolar to micromolar) to target proteins of pharmaceutical interest, and the bound conformation of these lead compounds in the protein binding pocket can be determined by the CORCEMA-ST refinement.     

Micropreparative ligand fishing with a cuvette-based optical mirror resonance biosensor

We have previously demonstrated the role of an optical biosensor (BIAcore 2000) as a specific detector to monitor chromatographic fractions during the purification and characterisation of ligands for orphan biomolecules. We have now extended this application to perform micropreparative ligand fishing directly on the sensor surface using an automated cuvette-based optical biosensor (Iasys Auto+) equipped with a high-capacity carboxymethyldextran surface (surface area 16 mm2). Using a F(ab)2' fragment of the A33 monoclonal antibody as bait, we have recovered microgram quantities of essentially homogeneous A33 ligand from the sensor surface in a form suitable for subsequent sensitive and specific down stream analysis (micropreparative HPLC, sodium dodecyl sulphate-polyacrylamide gel electrophoresis and Western blotting). The design of the cuvette-based system facilitates recovery of desorbed material from the constrained workspace in small volumes at high concentration. The use of on-surface detection allows the surface viability to be continuously monitored and permits direct quantitation of both bound and recovered material.     

Carbohydrate-based DNA ligands: sugar-oligoamides as a tool to study carbohydrate-nucleic acid interactions

Sugar-oligoamides have been designed and synthesized as structurally simple carbohydrate-based ligands to study carbohydrate-DNA interactions. The general design of the ligands 1-3 has been done as to favor the bound conformation of Distamycin-type gamma-linked covalent dimers which is a hairpin conformation. Indeed, NMR analysis of the sugar-oligoamides in the free state has indicated the presence of a percentage of a hairpin conformation in aqueous solution. The DNA binding activity of compounds 1-3 was confirmed by calf thymus DNA (ct-DNA) NMR titration. Interestingly, the binding of the different sugar-oligoamides seems to be modulated by the sugar configuration. Semiquantitative structural information about the DNA ligand complexes has been derived from NMR data. A competition experiment with Netropsin suggested that the sugar-oligoamide 3 bind to DNA in the minor groove. The NMR titrations of 1-3 with poly(dA-dT) and poly(dG-dC) suggested preferential binding to the ATAT sequence. TR-NOE NMR experiments for the sugar-oligoamide 3-ct-DNA complex both in D(2)O and H(2)O have confirmed the complex formation and given information on the conformation of the ligand in the bound state. The data confirmed that the sugar-oligoamide ligand is a hairpin in the bound state. Even more relevant to our goal, structural information on the conformation around the N-glycosidic linkage has been accessed. Thus, the sugar asymmetric centers pointing to the NH-amide and N-methyl rims of the molecule have been characterized.     

NMR elucidation of some ligands derived from the pentacycloundecane skeleton

The complete NMR elucidation of three novel pentacycloundecane (PCU)-derived ligands is reported. 2D NMR techniques are used to overcome the problem of major overlapping of methine signals on the cage skeleton. The compounds were synthesized as potential ligands to be used in asymmetric catalysis. They represent the first instance where aromatic moieties have been attached directly to the cage skeleton using lithiation techniques. The X-ray crystal structure of one of the ligands was obtained. The X-ray structure was helpful in determining the potential NOESY interactions within the set of molecules. For the other ligands a high level Density Functional Theory (DFT) optimization was performed [B3LYP/6-31+g(d)] to visualize possible NOE interactions. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Conformational studies of immunodominant myelin basic protein 1�C11 analogues using NMR and molecular modeling

Τwo dimensional nuclear magnetic resonance studies complimented by molecular dynamics simulations were conducted to investigate the conformation of the immunodominant epitope of acetylated myelin basic protein residues 1-11 (Ac-MBP(1-11)) and its altered peptide ligands, mutated at position 4 to an alanine (Ac-MBP(1-11)[4A]) or a tyrosine residue (Ac-MBP(1-11)[4Y]). Conformational analysis of the three analogues indicated that they adopt an extended conformation in DMSO solution as no long distance NOE connectivities were observed and seem to have a similar conformation when bound to the active site of the major histocompatibility complex (MHC II). The interaction of each peptide with MHC class II I-A(u) was further investigated in order to explore the molecular mechanism of experimental autoimmune encephalomyelitis induction/inhibition in mice. The present findings indicate that the Gln(3) residue, which serves as a T-cell receptor (TCR) contact site in the TCR/peptide/I-A(u) complex, has a different orientation in the mutated analogues especially in the Ac-MBP(1-11)[4A] peptide. In particular the side chain of Gln(3) is not solvent exposed as for the native Ac-MBP(1-11) and it is not available for interaction with the TCR.