Study of the Differential Activity of Thrombin Inhibitors Using Docking, QSAR, Molecular Dynamics, and MM-GBSA

Indexación: Web of Science; Scopus.Non-peptidic thrombin inhibitors (TIs; 177 compounds) with diverse groups at motifs P1 (such as oxyguanidine, amidinohydrazone, amidine, amidinopiperidine), P2 (such as cyano-fluorophenylacetamide, 2-(2-chloro-6-fluorophenyl)acetamide), and P3 (such as phenylethyl, arylsulfonate groups) were studied using molecular modeling to analyze their interactions with S1, S2, and S3 subsites of the thrombin binding site. Firstly, a protocol combining docking and three dimensional quantitative structure-activity relationship was performed. We described the orientations and preferred active conformations of the studied inhibitors, and derived a predictive CoMSIA model including steric, donor hydrogen bond, and acceptor hydrogen bond fields. Secondly, the dynamic behaviors of some selected TIs (compounds 26, 133, 147, 149, 162, and 177 in this manuscript) that contain different molecular features and different activities were analyzed by creating the solvated models and using molecular dynamics (MD) simulations.We used the conformational structures derived from MD to accomplish binding free energetic calculations using MM-GBSA. With this analysis, we theorized about the effect of van der Waals contacts, electrostatic interactions and solvation in the potency of TIs. In general, the contents reported in this article help to understand the physical and chemical characteristics of thrombin-inhibitor complexes. © 2015 Mena-Ulecia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.http://journals.plos.org/plosone/article?id=10.1371/journal.pone.014277

Characterization of Binding Pocket Flexibility of Aldose Reductase

Aldose Reductase (AR) is the first enzyme of the 'sorbitol pathway'. It is an NADPH dependent enzyme and catalyzes the reduction of various aldehydes to the corresponding alcohols. Its binding pocket shows intrinsic flexibility which is mainly mediated by a small loop region. A specificity pocket can be opened or closed via this loop stretch. In the first part of this work the relevance of considering protein flexibility in structure-based drug design was highlighted. Docking experiments were carried out for two inhibitors which were originally designed to mimic a certain binding mode. By carrying out separate docking simulations for each known major binding pocket conformation, it was shown that the intended binding mode was not predicted to be the most favorable. This hypothesis could later be verified by X-ray crystallography. However, the predictions were by no means perfect. One of the compounds induced a new conformation to the binding pocket. Thus, no appropriate crystal structure was available as template for the docking experiments. For the second molecule the importance of considering water during docking was emphasized. In the second part of this thesis a new method to simplify the tedious process of docking to multiple targets was evaluated in the context of protein flexibility: in-situ cross-docking. With this method, instead of performing sequential docking experiments of multiple ligands into multiple protein structures, several protein conformations can be addressed at once. In the next part of this thesis the flexibility of the AR binding pocket was examined in detail. It was shown that with respect to the binding pocket, flexibility is limited to only a handful amino acids close to the specificity pocket. It was elucidated how the enzyme performs its 'induced-fit' binding mechanism. To further explore the conformational space available to the AR binding pocket, multiple MD simulations were carried out. Good overall agreement between the results from the MD simulations and the crystal structure analysis was found. Residues which exhibited elevated levels of flexibilities in the MD simulations showed in most cases also differences between the single crystal structures. However, a few residues showed unexpected behavior in the MD simulations: Phe 122, Trp 219, and Tyr 309. The behavior of these residues were examined in great detail. In a further project, the generated MD trajectories were used to energetically analyze the process of 'induced-fit' adaptation. The method MM-PBSA was chosen for this purpose. Considering the enormous amount of computational power required to perform the necessary calculations and the remarkable time needed to analyze the results, MM-PBSA did not turn out to be a cost-effective method to predict binding free energies for the dataset of AR inhibitors used in this study. In the final section of this thesis a study was presented where in the first part aspects of flexibility of the C-terminal loop of AR were examined. In a combined study using MD simulations and multiple crystal structures, it was shown that there are clear differences between individual crystal structures of the same protein-ligand complex in this region of the enzyme. A nice agreement between the observations made in the MD and multiple crystal structures derived from different experimental crystallization conditions was found. In the second part of this section the unexpected occurrence of multiple ligands in and close to the binding pocket of AR was described. In summary, this study has dealt with many aspects of protein flexibility using AR as a test case. AR has proves to be a valuable test system to investigate protein flexibility with different methods

CARATTERIZZAZIONE ED ATTIVITÀ DI ESTRATTI VEGETALI AD ELEVATO CONTENUTO DI POLIFENOLI

Polyphenols are a class of natural chemical substances that can be found in many\ud vegetables. Their main chemical characteristic is the presence of multiple phenolic groups.\ud Among them there are other several subclasses like flavonoids.\ud Flavonoids are important secondary metabolites that can be also found in plants. Flavonoids\ud are based around a phenylbenzopyrone structure.\ud In general plants alone possess the biosynthetic ability. Flavonoid were found also in\ud primitive plant species like green algae and they survived through evolution.\ud Flavonoids can be divided in several subclasses depending on their sustituents. Moreover\ud flavonoids have other substitution patterns with addition hydroxyl, methyl, methoxyl and\ud glycosil groups

Poisson-Boltzmann Calculations of Nonspecific Salt Effects on Protein-Protein Binding Free Energies

The salt dependence of the binding free energy of five protein-protein hetero-dimers and two homo-dimers/tetramers was calculated from numerical solutions to the Poisson-Boltzmann equation. Overall, the agreement with experimental values is very good. In all cases except one involving the highly charged lactoglobulin homo-dimer, increasing the salt concentration is found both experimentally and theoretically to decrease the binding affinity. To clarify the source of salt effects, the salt-dependent free energy of binding is partitioned into screening terms and to self-energy terms that involve the interaction of the charge distribution of a monomer with its own ion atmosphere. In six of the seven complexes studied, screening makes the largest contribution but self-energy effects can also be significant. The calculated salt effects are found to be insensitive to force-field parameters and to the internal dielectric constant assigned to the monomers. Nonlinearities due to high charge densities, which are extremely important in the binding of proteins to negatively charged membrane surfaces and to nucleic acids, make much smaller contributions to the protein-protein complexes studied here, with the exception of highly charged lactoglobulin dimers. Our results indicate that the Poisson-Boltzmann equation captures much of the physical basis of the nonspecific salt dependence of protein-protein complexation

Structure of a Bovine Thrombin-Hirudin\u3csub\u3e51-65\u3c/sub\u3e Complex Determined by a Combination of Molecular Replacement and Graphics. Incorporation of Known Structural Information in Molecular Replacement

Crystals of the bovine thrombin-hirudins51-65 complex have space group P6122 with cell constants a = 116.4, and c = 200.6 Å and two thrombin molecules in the asymmetric unit. Only one thrombin molecule could be located by generalized molecular replacement; the second was fit visually as a rigid body to an improved electron-density difference map. The structure was refined to R = 0.192 with two B values per residue (main chain and side chain) at 3.2 Å. The polar interactions of the peptides with the exosite of thrombin show differences consistent with the known flexibility in the interactions of the C-terminal peptide of hirudin with thrombin. The hirudin peptide in complex 2 has a higher temperature factor as compared with peptide 1 which may be correlated partly with a larger number of short-range electrostatic interactions between peptide 1 and thrombin and partly with the fact that thrombin 2 is -thrombin which is cleaved at Thr149A near the peptide binding site. Later, using this structure as a test case, it was shown that the position for the second thrombin could also be determined by a novel modification of the molecular-replacement method in which the contribution of the known molecule is subtracted from the structure factors. This approach is facile and applicable to any crystal containing two or more macromolecules in the asymmetric unit in which some but not all of the molecules can be determined by molecular replacement

Expanding the Toolbox for Computational Analysis in Rational Drug Discovery: Using Biomolecular Solvation to Predict Thermodynamic, Kinetic and Structural Properties of Protein-Ligand Complexes

Most biomolecular interactions occur in aqueous environment. Therefore, one must consider the interactions between proteins and water molecules when developing a drug molecule against a target protein. The study of these interactions is challenging using experimental techniques alone, therefore computer simulations are commonly used to study the molecular details of protein-water or ligand-water interactions. In the first study presented in this doctoral dissertation (Chapter 2), the development, parameterization and testing of an approach is presented that can be used to calculate the solvation contribution in protein-ligand binding thermodynamics. The approach uses an extensive amount of molecular dynamics trajectories in conjunction with GIST calculations in order to obtain models that can predict relative protein-ligand solvation thermodynamics. In order to validate the approach, the model system thrombin is investigated using a set of 53 ligands with experimentally characterized protein-ligand structures and ITC profiles. We found that the binding thermodynamics of 186 congeneric pairs of ligands can be accurately described using our solvation-based models. The relative free energy of binding for these 186 pairs can be calculated from the desolvation free energy of the ligand molecules alone. Furthermore, complete thermodynamic profiles for protein-ligand binding reactions (i.e. free energy, enthalpy and entropy of binding) are accurately predicted by incorporating GIST solvent data from the unbound ligand as well as the protein-ligand complex. In Chapter 3, the aforementioned approach is applied to develop a strategy that enables to equip drug molecules with a desired set of solvation thermodynamics properties. For this purpose, the thrombin ligands (same ligand series as in previous Chapter 2) and the corresponding GIST integrals are decomposed into smaller building block molecules. In the next step, the solvation thermodynamics for the building blocks in the ligand molecule as well as the solvation thermodynamics for the isolated building block in aqueous solution are calculated. We found greatly varying solvation thermodynamics for the different building blocks, demonstrating their potential to design ligands with a wide range of solvation characteristics. Also, we found that the building block decomposition of ligand molecules and the corresponding GIST integrals can be readily used to understand remote solvent structuring effects. These effects occur in the unbound ligand molecule and describe the enhanced solvent structuring on a building block in the ligand molecule due to the presence of another building block at a distal site of the ligand. Furthermore, we demonstrated that the fluorination of building blocks leads to an increased unfavorable desolvation free energy and thus disfavors binding for the presented dataset. The research presented in Chapter 2 and Chapter 3 was accomplished with the computer program Gips that was developed as part of this doctoral dissertation. In the following Chapter 4, the mechanism and time scale of desolvation is being analyzed for the protein-ligand dissociation reaction of trypsin and thrombin in complex with benzamidine and N amidinopiperidine. The analysis is carried out using umbrella sampling free energy calculations and LoCorA calculations. The LoCorA approach is a method for the analysis of residence times of water molecules on the surface of amino acids. It was found that water molecules reside approximately 1.3 ns in the binding pocket of thrombin, whereas in trypsin they are residing one order of magnitude shorter (0.3 ns). This difference is explained with special solvent channels that connect the interior of the binding pocket to bulk solvent environment. The solvent channels are present in thrombin but not in trypsin. Furthermore, the selectivity profiles of benzamidine and N amidinopiperidine are related to a solvent-mediated free energy barrier that is present in thrombin but not trypsin. Also due to the presence of the solvent channels, the water molecules show similar residence time for both complexes in the case of thrombin but differing residence times in the case of the two trypsin complexes. The LoCorA approach is implemented in the computer program LoCorA (same name as the approach itself), which was developed as part of this doctoral dissertation. In the course of this doctoral dissertation, further computational studies were carried out in combination with experimental ones. These can be found in chapter 5 of this dissertation. Each of these studies is preceded by a separate abstract and a statement concerning the author contribution

Virtual compound screening and SAR analysis: method development and practical applications in the design of new serine and cysteine protease inhibitors

Virtual screening is an important tool in drug discovery that uses different computational methods to screen chemical databases for the identification of possible drug candidates. Most virtual screening methodologies are knowledge driven where the availability of information on either the nature of the target binding pocket or the type of ligand that is expect to bind is essential. In this regard, the information contained in X-ray crystal structures of protein-ligand complexes provides a detailed insight into the interactions between the protein and the ligand and opens the opportunity for further understanding of drug action and structure activity relationships at molecular level. Protein-ligand interaction information can be utilized to introduce target-specific interaction-based constraints in the design of focused combinatorial libraries. It can also be directly transformed into structural interaction fingerprints and can be applied in virtual screening to analyze docking studies or filter compounds. However, the integration of protein-ligand interaction information into two-dimensional compound similarity searching is not fully explored. Therefore, novel methods are still required to efficiently utilize protein-ligand interaction information in two-dimensional ligand similarity searching. Furthermore, application of protein-ligand interaction information in the interpretation of SARs at the ligand level needs further exploration. Thus, utilization of three-dimensional protein ligand interaction information in virtual screening and SAR analysis was the major aim of this thesis. The thesis is presented in two major parts. In the first part, utilization of three-dimensional protein-ligand interaction information for the development of a new hybrid virtual screening method and analysis of the nature of SARs in analog series at molecular level is presented. The second part of the thesis is focused on the application of different virtual screening methods for the identification of new cysteine and membrane-bound serine proteases inhibitors. In addition, molecular modeling studies were also applied to analyze the binding mode of structurally complex cyclic peptide inhibitors

Biophysical exploration of conformational environments in zymogen prothrombin and blood coagulant thrombin.

The serine protease thrombin plays important roles in coagulation, anticoagulation, and platelet activation. Thrombin is initially expressed as the inactive zymogen prothrombin (ProT). The final cleaved and activated form of thrombin has mature anion binding exosites (ABEs) and several regulatory loops. Engagement with these exosites helps define the fate of thrombin as a procoagulant or an anticoagulant. Researchers previously reported that zymogen ProT may already bind exosite ligands. Little is known about conformational changes associated with ProT maturation, resultant ligand binding affinities, individual ligand-protein contacts, and long-range communication between thrombin exosites. Protease Activated Receptors (PARs) play critical roles in controlling platelet activation. Using solution NMR methods, we demonstrated that PAR3 (44-56) and weaker binding version PAR3G (44-56, P51G) can already bind to immature pro-ABE I. 1D and 2D 1H-15N HSQC titrations revealed that PAR3G 15N-E48 and 15N-D54 both entered into higher affinity, intermediate exchange regimes as ProT was converted into thrombin. The high affinity of PAR3G D54 suggests that the thrombin R77a region is better oriented for binding than thrombin R75. PAR3G 15N-F47 and 15N-L52 experienced significant changes in chemical shift and thus chemical environment upon ABE I maturation. However, the ProT 30s loop made better contacts with PAR3 than the ProT hydrophobic cluster (F34, L65, and I82). The project was extended to PAR1 (49-62). Both PAR1P and weaker version PAR1G (P54G) bound to ProT and proton NMR line broadening increased with thrombin. 1D and 2D 1H-15N HSQC titrations revealed that unlike PAR3G (44-56), PAR1G (49-62) 15N- K51, E53, F55, D58, and E60 exhibited little interactions with ProT. Affinities increased with mature thrombin ABE I. NMR titrations could probe PAR1 (58DEEKN62), a region previously unresolved by X-ray crystallography. Interestingly, the ABE II ligand GpIbα (269-282, 3YP) influenced the NMR binding affinities of PAR1G and PAR3G supporting long-range communication between the ABE II and ABE I exosites. Finally, our studies shifted toward the thrombin active site region. Kinetic assays and 2D tr-NOESY studies provided clues on why Fibrinogen Bβ (5-16) is such a weak thrombin substrate. The Fibrinogen Bβ 10FFSAR14 sequence contributes towards hindering binding (Km) and product turnover (kcat)