A method of active conformation search based on active and inactive analogues and its application to allylamine antimycotics

A new program ACSBAIA (Active Conformation Search Based on Active and Inactive Analogues) for determination of the active conformations was developed based on the rationales that specific functional groups of active analogues could reach and interact with the active site of target receptor by means of the change of conformations, but that of inactive analogues could not interact with the active site owing to conformational restriction. The program consisted of 4 sub-programs: conformation sampling system, active conformation constraint system, inactive conformation exclusion system, and activity prediction system. Pharmacophoric conformation of allylamine antimycotics was studied by this method. Activities of 2 analogues were predicted and tested. The results suggested that the method was scientific and practical. The application of this method was not restricted by the three-dimensional structural knowledge of target receptor. In the absence of structural information about the receptor, the method was particularly applicable.     

Structure of an inactive conformation of GTP-bound RhoA GTPase

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Structural insight into epothilones antitumor activity based on the conformational preferences and tubulin binding modes of epothilones A and B obtained from molecular dynamics simulations

Molecular dynamics simulations were employed to analyze the conformational preferences and binding modes of epothilones A and B as a source of structural information regarding the antitumor properties of these species. Our results suggest that the conformation of free and tubulin-bound epothilones is strongly influenced by the presence of a methyl group at C12 and that epothilones A and B exploit the binding cavity in a unique and different way. The binding sites of epothilones A and B share a common region of association (Leu215, Leu217, His227, Leu228, Ala231, Phe270, Gly360, and Leu361), but lead to different ligand–residue interactions. Average interaction energies predict a larger stabilization for the epothilone B–tubulin complex, which is mainly driven by the enhancement of the electrostatic component of ligand–residue interactions compared to the epothilone A–tubulin complex. These structural and energetic results can be useful to account for the activity difference between epothilones A and B, and to design more active and potent analogs that resemble the mechanism of action of epothilones against cancer cells.     

Long‐range molecular dynamics show that inactive forms of Protein Kinase A are more dynamic than active forms

Many protein kinases are characterized by at least two structural forms corresponding to the highest level of activity (active) and low or no activity, (inactive). Further, protein dynamics is an important consideration in understanding the molecular and mechanistic basis of enzyme function. In this work, we use protein kinase A (PKA) as the model system and perform microsecond range molecular dynamics (MD) simulations on six variants which differ from one another in terms of active and inactive form, with or without bound ligands, C‐terminal tail and phosphorylation at the activation loop. We find that the root mean square fluctuations in the MD simulations are generally higher for the inactive forms than the active forms. This difference is statistically significant. The higher dynamics of inactive states has significant contributions from ATP binding loop, catalytic loop, and αG helix. Simulations with and without C‐terminal tail show this differential dynamics as well, with lower dynamics both in the active and inactive forms if C‐terminal tail is present. Similarly, the dynamics associated with the inactive form is higher irrespective of the phosphorylation status of Thr 197. A relatively stable stature of active kinases may be better suited for binding of substrates and detachment of the product. Also, phosphoryl group transfer from ATP to the phosphosite on the substrate requires precise transient coordination of chemical entities from three different molecules, which may be facilitated by the higher stability of the active state.     

Tetrazole analogues of cyclolinopeptide A: synthesis, conformation, and biology

Linear and cyclic analogues of cyclolinopeptide A (CLA) with two dipeptide segments (Val(5)-Pro(6) and Pro(6)-Pro(7)) replaced by their tetrazole derivatives were synthesized by the SPPS technique and cyclized using TBTU (O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate) reagent. The conformational properties of the c(Leu(1)-Ile(2)-Ile(3)-Leu(4)-Val(5)-Pro(6)-psi[CN(4)]-Ala(7)-Phe(8)-Phe(9)) were investigated by NMR and computational techniques. The overall solution structure of this cyclic peptide resembles that observed for the CLA in the solid state. These studies of cyclic tetrazole CLA analogue confirm that the 1,5-disubstituted tetrazole ring functions as an effective, well-tolerated cis-amide bond mimic in solution. The peptides were examined for their immunosuppressive activity in the humoral response test. For cyclic analogues the immunosuppressive activity, at low doses, is equal in magnitude to the activity presented by cyclosporin A and native CLA. The conformational and biological data seem indicate that the Pro-Pro-Phe-Phe moiety and the preservation of the CLA backbone conformation are important for immunosuppressive activity.     

Solution conformation of a pectin fragment disaccharide using molecular modelling and nuclear magnetic resonance

In the present study, the conformational behaviour of methylated pectic disaccharide 4-O-alpha-D-galactopyranurosyl 1-O-methyl-alpha-D-galactopyranuronic 6,6'-dimethyl diester 1 has been completely characterized through combined n.m.r. and molecular modelling studies. The 1H-1H n.O.e. across the glycosidic bond was measured by both steady-state and transient 1D and 2D experiments. In parallel, the complete conformational analysis of the disaccharide has been achieved with the MM3 molecular mechanics method. The conformation of the pyranose ring is confirmed by the excellent agreement between the experimental and calculated intracyclic scalar coupling constants. The iso-energy contours displayed on the 'relaxed' map indicate an important flexibility about the glycosidic linkage. There is no significant influence of the methoxyl group on the conformational behaviour of the disaccharide. The theoretical n.m.r. data were calculated taking into account all the accessible conformations and using the averaging methods appropriate for slow internal motions. 3JC-H coupling constants were calculated using an equation suitable for C-O-C-H segments. The agreement between experimental and theoretical data is excellent. Within the potential energy surface calculated for the disaccharide, several conformers can be identified. When these conformations are extrapolated to a regular polymer structure, they generate pectins with right- and left-handed chirality along with a two-fold helix. These different types of helical structure are the result of small changes in conformation, without any drastic variation of the fibre repeat.     

A shift of dynamic equilibrium between the KIT active and inactive states causes drug resistance

Tyrosine phosphorylation, a highly regulated post-translational modification, is carried out by the enzyme tyrosine kinase (TK). TKs are important mediators in signaling cascades, facilitating diverse biological processes in response to stimuli. TKs may acquire mutations leading to malignancy and are viable targets for anti-cancer drugs. Mast/stem cell growth factor receptor KIT is a TK involved in cell differentiation, whose dysregulation leads to various types of cancer, including gastrointestinal stromal tumors, leukemia, and melanoma. KIT can be targeted by a range of inhibitors that predominantly bind to the inactive state of the enzyme. A mutation Y823D in the activation loop of KIT is known to be responsible for the loss of sensitivity to some drugs in metastatic tumors. We used all-atom molecular dynamics simulations to study the impact of Y823D on the KIT conformation and dynamics and compared it to the effect of phosphorylation of Y823. We simulated in total 6.4 μs of wild-type, mutant and phosphorylated KIT in the active- and inactive-state conformations. We found that Y823D affects the protein dynamics differently: in the active state, the mutation increases the protein stability, whereas in the inactive state it induces local destabilization, thus shifting the dynamic equilibrium towards the active state, altering the communication between distant regulatory regions. The observed dynamics of the Y823D mutant is similar to the dynamics of KIT phosphorylated at position Y823, thus we hypothesize that this mutation mimics a constitutively active kinase, which is not responsive to inhibitors that bind its inactive conformation.     

Modeling an active conformation for linear peptides and design of a competitive inhibitor for HMG-CoA reductase

This study presents an approach that can be used to search for lead peptide candidates, including unconstrained structures in a recognized sequence. This approach was performed using the design of a competitive inhibitor for 3-hydroxy-3-methylglutaryl CoA reductase (HMGR). In a previous design for constrained peptides, a head-to-tail cyclic structure of peptide was used as a model of linear analog in searches for lead peptides with a structure close to an active conformation. Analysis of the conformational space occupied by the peptides suggests that an analogical approach can be applied for finding a lead peptide with an unconstrained structure in a recognized sequence via modeling a cycle using fixed residues of the peptide backbone. Using the space obtained by an analysis of the bioactive conformations of statins, eight cyclic peptides were selected for a peptide library based on the YVAE sequence as a recognized motif. For each cycle, the four models were assessed according to the design criterion ("V" parameter) applied for constrained peptides. Three cyclic peptides (FGYVAE, FPYVAE, and FFYVAE) were selected as lead cycles from the library. The linear FGYVAE peptide (IC(50) = 0.4 microM) showed a 1200-fold increase the inhibitory activity compared to the first isolated LPYP peptide (IC(50) = 484 microM) from soybean. Experimental analysis of the modeled peptide structures confirms the appropriateness of the proposed approach for the modeling of active conformations of peptides.     

Modulation of p53 N-terminal transactivation domain 2 conformation ensemble and kinetics by phosphorylation

Abstract

Phosphorylation of protein is critical for various cell processes, which preferentially happens in intrinsically disordered proteins (IDPs). How phosphorylation modulates structural ensemble of disordered peptide remains largely unexplored. Here, using replica exchange molecular dynamics (REMD) and Markov state model (MSM), the conformational distribution and kinetics of p53 N-terminal transactivation domain (TAD) 2 as well as its dual-site phosphorylated form (pSer46, pThr55) were simulated. It reveals that the dual phosphorylation does not change overall size and secondary structure element fraction, while a change in the distribution of hydrogen bonds induces slightly more pre-existing bound helical conformations. MSM analysis indicates that the dual phosphorylation accelerates conformation exchange between disordered and order-like states in target-binding region. It suggests that p53 TAD2 after phosphorylation would be more apt to bind to both the human p62 pleckstrin homology (PH) domain and the yeast tfb1?PH domain through different binding mechanism, where experimentally it exhibits an extended and α-helix conformation, respectively, with increased binding strength in both complexes. Our study implies except binding interface, both conformation ensemble and kinetics should be considered for the effects of phosphorylation on IDPs. Abbreviations IDPs intrinsically disordered proteins

REMD replica exchange molecular dynamics

MSM Markov state model

TAD transactivation domain

PH pleckstrin homology

PRR proline-rich region

DBD DNA-binding domain

TET Tetramerization domain

REG regulatory domain

MD molecular dynamics

PME particle-mesh Ewald

TICA time-lagged independent component analysis

CK Chapman–Kolmogorov

GMRQ generalized matrix Rayleigh quotient

SARW self-avoiding random walk

KID kinase-inducible domain

MFPT mean first passage time

DSSP definition of secondary structure of proteins

RMSD root mean square deviation

R_g radius of gyration

R_ee end to end distance

Communicated by Ramaswamy H. Sarma     

Molecular dynamics simulations of conformation and chain length dependent terahertz spectra of alanine polypeptides

Terahertz absorption spectra of alanine polypeptides in water were simulated with classical molecular dynamics at 310 K. Vibrational modes and oscillator strengths were calculated based on a quasi-harmonic approximation. Absorption spectra of Ala_n (n = 5, 15, 30) with different chain lengths and Ala₁₅ in coiled and helical conformations were studied in 10–40 cm^? 1 bandwidth. Simulation results indicated both the chain length and the conformation have significant influences on THz spectra of alanine polypeptides. With the increase of chain length, the average THz absorption intensity increases. Compared with the helical Ala₁₅ polypeptide, the THz spectra of coiled one shows stronger absorption peaks. These results were explained from different numbers of hydrogen bonds formed between polypeptides and the surrounding water molecules.     

2011～2017年新农药活性成分及天然产物药效团衍生法在农药分子设计中的应用

统计了全球范围内首次登记日期位于2011～2017年的102种(不包含生物农药)农药活性成分,并以此为研究对象综述分析了SND(Synthetic natural derived)和NP(Natural product)类农药分子的天然产物来源、作用机制及在农药活性分子中的占比等,综合评估天然产物药效团衍生法在农药分子开发设计中的重要性,并在此基础上提供对未来发展方向。     

Open and closed conformation of the E. coli purine nucleoside phosphorylase active center and implications for the catalytic mechanism.

The crystal structure of the ternary complex of hexameric purine nucleoside phosphorylase (PNP) from Escherichia coli with formycin A derivatives and phosphate or sulphate ions is determined at 2.0 A resolution. The hexamer is found as a trimer of unsymmetric dimers, which are formed by pairs of monomers with active sites in different conformations. The conformational difference stems from a flexible helix (H8: 214-236), which is continuous in one conformer, and segmented in the other. With the continuous helix, the entry into the active site pocket is wide open, and the ligands are bound only loosely ("open" or "loose binding" conformation). By segmentation of the helix (H8: 214-219 and H8': 223-236, separated by a gamma-turn), the entry into the active site is partially closed, the pocket is narrowed and the ligands are bound much more tightly ("closed" or "tight binding" conformation). Furthermore, the side-chain of Arg217 is carried by the moving helix into the active site. This residue, conserved in all homologous PNPs, plays an important role in the proposed catalytic mechanism. In this mechanism, substrate binding takes place in the open, and and the catalytic action occurs in the closed conformation. Catalytic action involves protonation of the purine base at position N7 by the side-chain of Asp204, which is initially in the acid form. The proton transfer is triggered by the Arg217 side-chain which is moved by the conformation change into hydrogen bond distance to Asp204. The mechanism explains the broad specificity of E. coli PNP, which allows 6-amino as well as 6-oxo-nucleosides as substrates. The observation of two kinds of binding sites is fully in line with solution experiments which independently observe strong and weak binding sites for phosphate as well as for the nucleoside inhibitor.     

NMR‐based conformation and dynamics of a tetrasaccharide‐repeating sulfated fucan substituted by different counterions

The sulfated fucan from the sea urchin Lytechinus variegatus is composed of the repetitive sequence [‐3)‐α‐l ‐Fucp‐4( )‐(1‐3)‐α‐l ‐Fucp‐2,4‐di( )‐(1‐3)‐α‐l ‐Fucp‐2( )‐(1‐3)‐α‐l ‐Fucp‐2( )‐(1‐]_n. Conformation (of rings and chains) and dynamics of this tetrasaccharide‐repeating sulfated fucan substituted by Na⁺, Ca²⁺, and Li⁺ as counterions have been examined through experiments of liquid‐state nuclear magnetic resonance spectroscopy. Scalar coupling and nuclear Overhauser effect (NOE)‐based data have confirmed that all composing units occur as ¹C₄ chair conformer regardless of the cation type, unit position within the repeating sequence, and sulfation type. Chain conformation determined by NOE signal pattern assisted by molecular modeling for a theoretical octasaccharide has shown a similar linear 3D structure for the three differently substituted forms. Data derived from spin‐relaxation measurements have indicated a contribution of counterion type to dynamics. The calcium‐based preparation has shown the highest mobility while the sodiated one showed the lowest mobility. The set of results from this work suggests that counterion type can affect the physicochemical properties of the structurally well‐defined sulfated fucan. The counterion effect seems to impact more on the structural mobility than on average conformation of the studied sulfated glycan in solution.     

Conformational dynamics of active site loop in Escherichia coli phytase

Phytases catalyze the release of phosphate by stepwise hydrolysis of phytate, a major source of phosphate in cereal grains, legumes, and oilseeds. Phytase improves, as a feed supplement, the nutritional quality of phytate rich diets and eventually reduce environmental pollution. Recently, phytases from enterobacteriaceae family have attracted industrial interest due to their high specific activity (2500–4000 U/mg). However, only limited information is available concerning structural dynamics of this class of enzymes. In this study, 50 nanosecond molecular dynamics simulation was performed on two Escherichia coli phytase structures (closed and open active site loop) to investigate conformational dynamics of the active site loop. Cluster analysis and principal component analysis (PCA) reveal significant difference in the conformational dynamics of active site compared to reported crystal structure. Molecular dynamic studies indicated that the movement in the active site of E. coli phytase is mainly confined by the active site loop resulted in wider opening of the loop in absence of phytate. The molecular dynamics studies highlight the possible role of loop residues as prerequisite for highly active phytases. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 994–1002, 2010.     

Molecular dynamics characterization of the active cavity of carboxypeptidase A and some of its inhibitor adducts.

Molecular dynamics (MD) calculations have been performed on carboxypeptidase A and on its adducts with inhibitors, such as d-phenylalanine (dPhe) and acetate. The catalytically essential zinc ion present in the protein was explicitly included in all the simulations. The simulation was carried out over a sphere of 15 A centered on the zinc ion. The crystallographic water molecules were explicitly taken into account; then the protein was solvated with a 18 A sphere of water molecules. MD calculations were carried out for 45-60 ps. There is no large deviation from the available X-ray structures of native and the dPhe adduct for the MD structures. Average MD structures were calculated starting from the X-ray structure of the dPhe adduct, and, from a structure obtained by docking the inhibitor in the native structure. Comparison between these two structures and with that of the native protein shows that some of the key variations produced by inhibitor binding are reproduced by MD calculations. Addition of acetate induces structural changes relevant for the understanding of the interaction network in the active cavity. The structural variations induced by different inhibitors are examined. The effects of these interactions on the catalytic mechanism and on the binding of substrate are discussed.     

The retinal conformation and its environment in rhodopsin in light of a new 2.2 A crystal structure

A new high-resolution structure is reported for bovine rhodopsin, the visual pigment in rod photoreceptor cells. Substantial improvement of the resolution limit to 2.2 A has been achieved by new crystallization conditions, which also reduce significantly the probability of merohedral twinning in the crystals. The new structure completely resolves the polypeptide chain and provides further details of the chromophore binding site including the configuration about the C6-C7 single bond of the 11-cis-retinal Schiff base. Based on both an earlier structure and the new improved model of the protein, a theoretical study of the chromophore geometry has been carried out using combined quantum mechanics/force field molecular dynamics. The consistency between the experimental and calculated chromophore structures is found to be significantly improved for the 2.2 A model, including the angle of the negatively twisted 6-s-cis-bond. Importantly, the new crystal structure refinement reveals significant negative pre-twist of the C11-C12 double bond and this is also supported by the theoretical calculation although the latter converges to a smaller value. Bond alternation along the unsaturated chain is significant, but weaker in the calculated structure than the one obtained from the X-ray data. Other differences between the experimental and theoretical structures in the chromophore binding site are discussed with respect to the unique spectral properties and excited state reactivity of the chromophore.     

Designing of enzyme inhibitors based on active site specificity: lessons from methyl gallate and its lipoxygenase inhibitory profile

Methyl gallate was purified, by lipoxygenase (LOX) inhibitory activity-guided method since its alleged anti-inflammatory property, from Bergenia ligulata (Wall), a plant used in the traditional, Ayurvedic system of medicine extensively. The LOX inhibitory property of methyl gallate was studied by enzyme kinetics, isothermal titration calorimetry and molecular docking followed by molecular simulation studies. The wet-laboratory experiments and in silico studies showed complete agreement, and promise of methyl gallate as a drug-lead molecular scaffold for anti-inflammatory therapy, based on LOX inhibition. The expressed work shows the need of nonactive site binding parameters to be considered while designing of inhibitors based on the specificities toward active sites of enzymes.