[Show abstract][Hide abstract] ABSTRACT: Experimental evidence indicates that the primary structure of the β2-α2 loop region (residues 165-175) in mammalian prion proteins (PrP) influences the conversion from the cellular species (PrPC ) to the β-sheet-rich aggregate. Here, we captured the transition of the β2-α2 loop from 310-helical turn to β turn by unbiased molecular dynamics simulations of the single-point mutant Y169G. Multiple conformations along the spontaneous transition of the mutant were then used as starting point for sampling of the free-energy surface of the wild type and other single-point mutants. Using two different methods for the determination of free energy profiles, we found that the barrier for the 310 -helical turn to β turn transition of the wild type is higher by about 2.5 kcal/mol than for the Y169G mutant which is due to favorable stacking of the aromatic rings of Y169 and F175, and a stable hydrogen bond between the side chains of Y169 and D178. The transition of the β2-α2 loop to β turn increases the solvent-exposure of the hydrophobic stretch 169-YSNQNNF-175. Thus, the simulations indicate that the strictly conserved Y169 in mammalian prion proteins stabilizes the 310 -helical turn in the β2-α2 loop, thus hindering the conversion to an aggregation-prone conformation.
Journal of the American Chemical Society 02/2015; 137(8). DOI:10.1021/ja511568m · 11.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have solved the crystal structures of the EphA3 tyrosine kinase in complex with nine small-molecule inhibitors, which represent five different chemotypes and three main binding modes, i.e., types I and I1/2 (DFG in) and type II (DFG out). The three structures with type I1/2 inhibitors show that the higher affinity with respect to type I is due to an additional polar group (hydroxyl or pyrazole ring of indazole) which is fully buried and is involved in the same hydrogen bonds as the (urea or amide) linker of the type II inhibitors. Overall, the type I and type II binding modes belong to the lock-and-key and induced fit mechanism, respectively. In the type II binding, the scaffold in contact with the hinge region influences the position of the Phe765 side chain of the DFG motif and the orientation of the Gly-rich loop. The binding mode of Birb796 in the EphA3 kinase does not involve any hydrogen bond with the hinge region, which is different from the Birb796/p38 MAP kinase complex. Our structural analysis emphasizes the importance of accounting for structural plasticity of the ATP binding site in the design of type II inhibitors of tyrosine kinases.
[Show abstract][Hide abstract] ABSTRACT: Bromodomains are α-helical bundles of approximately 110 residues that recognize acetylated lysine side chains mainly on histone tails. Bromodomains are known to play an important role in cancer and inflammation, and as such, significant efforts are being made to identify small-molecule inhibitors of these epigenetic reader proteins. Here, explicit solvent molecular dynamics (MD) simulations of two bromodomains (BAZ2B and CREBBP) are used to analyze the water molecules that seem to be conserved at the bottom of the acetyl-lysine binding site in most crystal structures of bromodomains. The MD runs suggest that the occupancy of the structured water molecules is influenced by conformational transitions of the loop that connects helices Z and A. Additional simulations in the presence of 50 molecules of cosolvent (i.e., 440 mM of dimethylsulfoxide, methanol, or ethanol) indicate that some of the structured water molecules can be displaced transiently. The residence time in the acetyl-lysine binding site is calculated to be about 1 ns, 2-5 ns, and 10-30 ns for methanol, ethanol, and dimethylsulfoxide, respectively, while the affinity of the three cosolvents for BAZ2B and CREBBP is in the range of 50-500 mM. The results described have implications for ligand design, suggesting that only structured water molecules that do not exchange with cosolvent should be maintained in crystal structures used for docking campaigns, and that hydroxy substituents should be incorporated in the ligand so as to map the structured water molecules replaced by (m)ethanol.
[Show abstract][Hide abstract] ABSTRACT: Bromodomains are protein modules that selectively recognize histones by binding to acetylated lysines. Here, we have carried out multiple molecular dynamics simulations of 20 human bromodomains to investigate the flexibility of their binding site. Some bromodomains show alternative side chain orientations of three evolutionarily conserved residues: the Asn involved in acetyl-lysine binding and two conserved aromatic residues. Furthermore, for the BAZ2B and CREBBP bromodomains we observe occlusion of the binding site which is coupled to the displacement of the two aromatic residues. In contrast to available structures, the simulations reveal large variability of the binding site accessibility. The simulations suggest that the flexibility of the bromodomain binding site and presence of self-occluded metastable states influence the recognition of acetyl-lysine on histone tails.
[Show abstract][Hide abstract] ABSTRACT: Inhibition of the tyrosine kinase erythropoietin-producing human hepatocellular carcinoma receptor B4 (EphB4) is an effective strategy for the treatment of solid tumors. We have previously reported a low nanomolar ATP-competitive inhibitor of EphB4 discovered in silico by fragment-based high-throughput docking combined with explicit solvent molecular dynamics simulations. Here we present a second generation of EphB4 inhibitors that show high inhibitory potency in both enzymatic and cell-based assays while preserving the appealing selectivity profile exhibited by the parent compound. In addition, respectable levels of antiproliferative activity for these compounds have been obtained. Finally, the binding mode predicted by docking and molecular dynamics simulations is validated by solving the crystal structures of three members of this chemical class in complex with the EphA3 tyrosine kinase whose ATP-binding site is essentially identical to that of EphB4.
[Show abstract][Hide abstract] ABSTRACT: Several small molecules that bind to the inactive DFG-out conformation of tyrosine kinases (called type II inhibitors) have shown a good selectivity profile over other kinase targets. To obtain a set of DFG-out structures, we performed an explicit solvent molecular dynamics (MD) simulation of the complex of the catalytic domain of a tyrosine kinase receptor, ephrin type-A receptor 3 (EphA3), and a manually docked type II inhibitor. Automatic docking of four previously reported type II inhibitors was used to select a single snapshot from the MD trajectory for virtual screening. High-throughput docking of a pharmacophore-tailored library of 175 000 molecules resulted in about 4 million poses, which were further filtered by van der Waals efficiency and ranked according to a force-field-based energy function. Notably, around 20 % of the compounds with predicted binding energy smaller than -10 kcal mol(-1) are known type II inhibitors. Moreover, a series of 5-(piperazine-1-yl)isoquinoline derivatives was identified as a novel class of low-micromolar inhibitors of EphA3 and unphosphorylated Abelson tyrosine kinase (Abl1). The in silico predicted binding mode of the new inhibitors suggested a similar affinity to the gatekeeper mutant T315I of Abl1, which was verified in vitro by using a competition binding assay. Additional evidence for the type II binding mode was obtained by two 300 ns MD simulations of the complex between N-(3-chloro-4-(difluoromethoxy)phenyl)-2-(4-(8-nitroisoquinolin-5-yl)piperazin-1-yl)acetamide and EphA3.
[Show abstract][Hide abstract] ABSTRACT: The drug Darunavir (DRV) is a potent inhibitor of HIV-1 protease (PR), a homodimeric essential enzyme of the AIDS virus. Recent experimental data suggest that DRV is able to prevent dimerization of HIV-1 PR, which, together with its high affinity for the mature enzyme, has been linked to the high genetic barrier to the development of viral resistance. The mechanism of dimerization inhibition and the binding mode(s) of DRV to monomeric HIV-1 PR are unknown. Here, multiple molecular dynamics simulations with explicit solvent (for a total of 11 μs with the CHARMM force field and 1 μs with the Amber force field) show that the monomer of HIV-1 PR is structurally stable and reveal a major binding mode of DRV. This binding mode is stabilized by favorable interactions between the apolar groups of DRV and the hydrophobic residues Ile32, Ile47, Ile50, Ile54, Pro79, Val82, and Ile84. The binding mode to monomeric HIV-1 PR identified by molecular dynamics is different from the two binding modes observed in the crystal structure of the complex with dimeric HIV-1 PR. As an example, there are no interactions between DRV and the catalytic Asp25 in the binding mode to monomeric HIV-1 PR revelead by the simulations. In contrast, the simulations show extensive and stable interactions between DRV and the flap (residues 46–55), which are likely to sterically hinder the formation of the flap interface as observed in the dimeric structure. Which of the two mechanisms of inhibition (dimerization inhibition by association with the flap or binding to the active site of the mature enzyme) dominates might depend on the HIV-1 PR mutations, and it is likely that dimerization inhibition is predominant for multiple mutations at the active site in multidrug resistant strains.
Journal of Chemical Theory and Computation 04/2012; 8(5):1786–1794. DOI:10.1021/ct300032r · 5.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The non-structural 3 protease is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus infections. In this chapter, we discuss in detail the computational methods used in the previous two docking campaigns which lead to the discovery of non-peptidic low micromolar inhibitors. Not only an X-ray structure but also an alternative conformation generated from molecular dynamic simulations is used in the in silico screening. Moreover, unique scoring schemes are developed based on the properties of the binding site of the protein.
[Show abstract][Hide abstract] ABSTRACT: A dynamic situation! Molecular dynamics simulations at equilibrium are shown to correctly identify the binding mode of dimethyl sulfoxide in the rotamase FKBP without using any experimental information or bias. Furthermore, both the binding and unbinding kinetics were found to have a double-exponential time dependence.
[Show abstract][Hide abstract] ABSTRACT: Ligand binding involves breakage of hydrogen bonds with water molecules and formation of new hydrogen bonds between protein and ligand. In this work, the change of hydrogen bonding energy in the binding process, namely hydrogen bonding penalty, is evaluated with a new method. The hydrogen bonding penalty can not only be used to filter unrealistic poses in docking, but also improve the accuracy of binding energy calculation. A new model integrated with hydrogen bonding penalty for free energy calculation gives a root mean square error of 0.7 kcal/mol on 74 inhibitors in the training set and of 1.1 kcal/mol on 64 inhibitors in the test set. Moreover, an application of hydrogen bonding penalty into a high throughput docking campaign for EphB4 inhibitors is presented, and remarkably, three novel scaffolds are discovered out of seven tested. The binding affinity and ligand efficiency of the most potent compound is about 300 nM and 0.35 kcal/mol per non-hydrogen atom, respectively.
PLoS ONE 06/2011; 6(6):e19923. DOI:10.1371/journal.pone.0019923 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The spontaneous dissociation of six small ligands from the active site of FKBP (the FK506 binding protein) is investigated by explicit water molecular dynamics simulations and network analysis. The ligands have between four (dimethylsulphoxide) and eleven (5-diethylamino-2-pentanone) non-hydrogen atoms, and an affinity for FKBP ranging from 20 to 0.2 mM. The conformations of the FKBP/ligand complex saved along multiple trajectories (50 runs at 310 K for each ligand) are grouped according to a set of intermolecular distances into nodes of a network, and the direct transitions between them are the links. The network analysis reveals that the bound state consists of several subbasins, i.e., binding modes characterized by distinct intermolecular hydrogen bonds and hydrophobic contacts. The dissociation kinetics show a simple (i.e., single-exponential) time dependence because the unbinding barrier is much higher than the barriers between subbasins in the bound state. The unbinding transition state is made up of heterogeneous positions and orientations of the ligand in the FKBP active site, which correspond to multiple pathways of dissociation. For the six small ligands of FKBP, the weaker the binding affinity the closer to the bound state (along the intermolecular distance) are the transition state structures, which is a new manifestation of Hammond behavior. Experimental approaches to the study of fragment binding to proteins have limitations in temporal and spatial resolution. Our network analysis of the unbinding simulations of small inhibitors from an enzyme paints a clear picture of the free energy landscape (both thermodynamics and kinetics) of ligand unbinding.
[Show abstract][Hide abstract] ABSTRACT: The chromatin-associated enzyme PARP1 has previously been suggested to ADP-ribosylate histones, but the specific ADP-ribose acceptor sites have remained enigmatic. Here, we show that PARP1 covalently ADP-ribosylates the amino-terminal histone tails of all core histones. Using biochemical tools and novel electron transfer dissociation mass spectrometric protocols, we identify for the first time K13 of H2A, K30 of H2B, K27 and K37 of H3, as well as K16 of H4 as ADP-ribose acceptor sites. Multiple explicit water molecular dynamics simulations of the H4 tail peptide into the catalytic cleft of PARP1 indicate that two stable intermolecular salt bridges hold the peptide in an orientation that allows K16 ADP-ribosylation. Consistent with a functional cross-talk between ADP-ribosylation and other histone tail modifications, acetylation of H4K16 inhibits ADP-ribosylation by PARP1. Taken together, our computational and experimental results provide strong evidence that PARP1 modifies important regulatory lysines of the core histone tails.
Nucleic Acids Research 10/2010; 38(19):6350-62. DOI:10.1093/nar/gkq463 · 9.11 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Quantum mechanical (QM) methods are becoming popular in computational drug design and development mainly because high accuracy is required to estimate (relative) binding affinities. For low-to medium-throughput in silico screening, (e.g., scoring and prioritizing a series of inhibitors sharing the same molecular scaffold) efficient approximations have been developed in the past decade, like linear scaling QM in which the computation time scales almost linearly with the number of basis functions. Furthermore, QM-based procedures have been used recently for determining protonation states of ionizable groups, evaluating energies, and optimizing molecular structures. For high-throughput in silico screening QM approaches have been employed to derive robust quantitative structure-activity relationship models. It is expected that the use of QM methods will keep growing in all phases of computer-aided drug design and development. However, extensive sampling of conformational space and treatment of solution of macromolecules are still limiting factors for the broad application of QM in drug design.
Current topics in medicinal chemistry 11/2009; 10(1):33-45. DOI:10.2174/156802610790232242 · 3.45 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: MOTIVATION AND METHOD: Small-molecule inhibitors targeting the adenosine triphosphate (ATP) binding pocket of the catalytic domain of protein kinases have potential to become drugs devoid of (major) side effects, particularly if they bind selectively. Here, the sequences of the 518 human kinases are first mapped onto the structural alignment of 116 kinases of known three-dimensional structure. The multiple structure alignment is then used to encode the known strategies for developing selective inhibitors into a fingerprint. Finally, a network analysis is used to partition the kinases into clusters according to similarity of their fingerprints, i.e. physico-chemical characteristics of the residues responsible for selective binding. RESULTS: For each kinase the network analysis reveals the likelihood to find selective inhibitors targeting the ATP binding site. Systematic guidelines are proposed to develop selective inhibitors. Importantly, the network analysis suggests that the tyrosine kinase EphB4 has high selectivity potential, which is consistent with the selectivity profile of two novel EphB4 inhibitors. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
[Show abstract][Hide abstract] ABSTRACT: In drug discovery, the occurrence of false positives is a major hurdle in the search for lead compounds that can be developed into drugs. A small-molecular-weight compound that inhibits dengue virus protease at low micromolar levels was identified in a screening campaign. Binding to the enzyme was confirmed by isothermal titration calorimetry (ITC) and nuclear magnetic resonance (NMR). However, a structure-activity relationship study that ensued did not yield more potent leads. To further characterize the parental compound and its analogues, we developed a high-speed, low-cost, quantitative fluorescence quenching assay. We observed that specific analogues quenched dengue protease fluorescence and showed variation in IC(50) values. In contrast, nonspecifically binding compounds did not quench its fluorescence and showed similar IC(50) values with steep dose-response curves. We validated the assay using single Trp-to-Ala protease mutants and the competitive protease inhibitor aprotinin. Specific compounds detected in the binding assay were further analyzed by competitive ITC, NMR, and surface plasmon resonance, and the assay's utility in comparison with these biophysical methods is discussed. The sensitivity of this assay makes it highly useful for hit finding and validation in drug discovery. Furthermore, the technique can be readily adapted for studying other protein-ligand interactions.
[Show abstract][Hide abstract] ABSTRACT: The two-component NS2B-NS3 protease of West Nile virus is essential for its replication and presents an attractive target for drug development. Here, we describe protocols for the high-yield expression of stable isotope-labelled samples in vivo and in vitro. We also describe the use of NMR spectroscopy to determine the binding mode of new low molecular mass inhibitors of the West Nile virus NS2B-NS3 protease which were discovered using high-throughput in vitro screening. Binding to the substrate-binding sites S1 and S3 is confirmed by intermolecular NOEs and comparison with the binding mode of a previously identified low molecular mass inhibitor. Our results show that all these inhibitors act by occupying the substrate-binding site of the protease rather than by an allosteric mechanism. In addition, the NS2B polypeptide chain was found to be positioned near the substrate-binding site, as observed previously in crystal structures of the protease in complex with peptide inhibitors or bovine pancreatic trypsin inhibitor. This indicates that the new low molecular mass compounds, although inhibiting the protease, also promote the proteolytically active conformation of NS2B, which is very different from the crystal structure of the protein without inhibitor.
[Show abstract][Hide abstract] ABSTRACT: The tyrosine kinase EphB4 is an attractive target for drug design because of its recognized role in cancer-related angiogenesis. Recently, a series of commercially available xanthine derivatives were identified as micromolar inhibitors of EphB4 by high-throughput fragment-based docking into the ATP-binding site of the kinase domain. Here, we have exploited the binding mode obtained by automatic docking for the optimization of these EphB4 inhibitors by chemical synthesis. Addition of only two heavy atoms, methyl and hydroxyl groups, to compound 4 has yielded the single-digit nanomolar inhibitor 66, with a remarkable improvement of the ligand efficiency from 0.26 to 0.37 kcal/(mol per non-hydrogen atom). Compound 66 shows very high affinity for a few other tyrosine kinases with threonine as gatekeeper residue (Abl, Lck, and Src). On the other hand, it is selective against kinases with a larger gatekeeper. A 45 ns molecular dynamics (MD) simulation of the complex of EphB4 and compound 66 provides further validation of the binding mode obtained by fragment-based docking.
[Show abstract][Hide abstract] ABSTRACT: Fragment-based docking was used to select a conformation for virtual screening from a molecular dynamics trajectory of the West Nile virus nonstructural 3 protease. This conformation was chosen from an ensemble of 100 molecular dynamics snapshots because it optimally accommodates benzene, the most common ring in known drugs, and two positively charged fragments (methylguanidinium and 2-phenylimidazoline). The latter fragments were used as probes because of the large number of hydrogen bond acceptors in the substrate binding site of the protease. Upon high-throughput docking of a diversity set of 18,694 molecules and pose filtering, only five compounds were chosen for experimental validation, and two of them are active in the low micromolar range in an enzymatic assay and a tryptophan fluorescence quenching assay. Evidence for specific binding to the protease active site is provided by nuclear magnetic resonance spectroscopy. The two inhibitors have different scaffolds (diphenylurea and diphenyl ester) and are promising lead candidates because they have a molecular weight of about 300 Da.
[Show abstract][Hide abstract] ABSTRACT: The non-structural 3 protease (NS3pro) is an essential flaviviral enzyme and therefore one of the most promising targets for drug development against West Nile virus (WNV) and dengue infections.
In this work, a small-molecule inhibitor of the WNV NS3pro has been identified by automatic fragment-based docking of about 12000 compounds and testing by nuclear magnetic resonance (NMR) spectroscopy of only 22 molecules. Specific binding of the inhibitor into the active site of NS3pro and its binding mode are confirmed by 15N-HSQC NMR spectra. The inhibitory activity is further validated by an enzymatic assay and a tryptophan fluorescence quenching assay.
The inhibitor [4-(carbamimidoylsulfanylmethyl)-2,5-dimethylphenyl]-methylsulfanylmethanimidamide has a good ratio of binding affinity versus molecular weight (ligand efficiency of 0.33 kcal/mol per non-hydrogen atom), and thus has good potential as lead compound for further development to combat West Nile virus infections.