Computational modelling of inhibitor binding to human thrombin

Thrombin is an essential protein involved in blood clot formation and an important clinical target, since disturbances of the coagulation process cause serious cardiovascular diseases such as thrombosis. Here we evaluate the performance of a molecular dynamics based method for predicting the binding affinities of different types of human thrombin inhibitors. For a series of eight ligands the method ranks their relative affinities reasonably well. The binding free energy difference between high and low affinity representatives in the test set is quantitatively reproduced, as well as the stereospecificity for a chiral inhibitor. The original parametrisation of this linear interaction energy method requires the addition of a constant energy term in the case of thrombin. This yields a mean unsigned error of 0.68 kcal/mol for the absolute binding free energies. This type of approach is also useful for elucidating three-dimensional structure–activity relationships in terms of microscopic interactions of the ligands with the solvated enzyme.     

The strength of weak interactions: aromatic fluorine in drug design

Selective aromatic fluorine substitution can increase the affinity of a molecule for a macromolecular recognition site through non-covalent interactions. These effects are evaluated most accurately by direct comparison of binding affinities of selectively fluorinated compounds with their corresponding hydrocarbons. In cases where structural data confirm similar binding geometries for the fluorocarbon and hydrocarbon analogues, reliable estimates for the impact of fluorination upon arene-pi...X and C-F...X interaction energies are possible. Existing studies show that fluorination's impact on any individual molecular interaction is quite modest. Upon binding to a protein receptor, cumulative fluorinated aromatic quadrupolar and C-F...X dipolar interaction energies rarely differ from those the corresponding hydrocarbons by more than 1.3 kcal/mol, and most individual interactions appear to be in the 0.1-0.4 kcal/mol range. Similarly, non-ideal selective fluorination is rarely associated with a dramatic decrease in affinity, because the impact of weak repulsive interactions in the bound state is counterbalanced by increased lipophilicity.     

Compensating enthalpic and entropic changes hinder binding affinity optimization

A common strategy to improve the potency of drug candidates is to introduce chemical functionalities, like hydrogen bond donors or acceptors, at positions where they are able to establish strong interactions with the target. However, it is often observed that the added functionalities do not necessarily improve potency even if they form strong hydrogen bonds. Here, we explore the thermodynamic and structural basis for those observations. KNI-10033 is a potent experimental HIV-1 protease inhibitor with picomolar affinity against the wild-type enzyme (K(d) = 13 pm). The potency of the inhibitor is the result of favorable enthalpic (DeltaH = -8.2 kcal/mol) and entropic (-TDeltaS = -6.7 kcal/mol) interactions. The replacement of the thioether group in KNI-10033 by a sulfonyl group (KNI-10075) results in a strong hydrogen bond with the amide of Asp 30B of the HIV-1 protease. This additional hydrogen bond improves the binding enthalpy by 3.9 kcal/mol; however, the enthalpy gain is completely compensated by an entropy loss, resulting in no affinity change. Crystallographic and thermodynamic analysis of the inhibitor/protease complexes indicates that the entropy losses are due to a combination of conformational and solvation effects. These results provide a set of practical guidelines aimed at overcoming enthalpy/entropy compensation and improve binding potency.     

Some insights into the stereochemistry of inhibition of macrophage migration inhibitory factor with 2-fluoro-p-hydroxycinnamate and its analogues from molecular dynamics simulations

Macrophage migration inhibitory factor (MIF) exhibits tautomerase activity on phenylpyruvate and has E-stereochemistry preference. To investigate the binding modes of its competitive inhibitors and evaluate their binding affinities, molecular dynamics simulations together with MM-PBSA (molecular mechanics Poisson-Boltzmann surface area) analysis were performed on MIF complexed with (E)-2-fluoro-p-hydroxycinnamate and five analogues. Pro-1 was discovered to form a bifurcated hydrogen bond between its protonated nitrogen and carboxylate oxygens of E-ligands and Tyr-36. No hydrogen bonds were found between Pro-1 and Z-ligands. This distinct binding characteristic of E- and Z-ligands with Pro-1 may be the main factor for the large difference in their binding affinities, which is consistent with the previous report that Pro-1 is essential for the catalytic activity of MIF. MM-PBSA analysis revealed that energy components including van der Waals, electrostatic, and hydrophobic interactions are in favor of binding, among which electrostatic interactions are predominant to the binding affinity difference.     

Predicted three-dimensional structural models of venom serine protease inhibitors and their interactions with trypsin and chymotrypsin

Three homology models of trypsin and chymotrypsin inhibitor polypeptides from snake venom of Naja naja naja and Leaf-nosed viper in the unbound state and in complex with trypsin and chymotrypsin were built based on homology to bovine pancreatic trypsin inhibitor (BPTI). These venom inhibitors belong to the Kunitz-type inhibitor family, which is characterized by a distinct tertiary fold with three-conserved disulfide bonds. The general folding pattern in these trypsin and chymotrypsin inhibitor homology models is conserved when compared to BPTI. The respective orientations of the inhibitors bound to trypsin/chymotrypsin are similar to that of BPTI bound to bovine trypsin/chymotrypsin. The principal binding loop structure of the inhibitors fills the active site of enzymes in a substrate-like conformation and forms a series of independent main-chain and side-chain interactions with enzymes. In order to provide the possible fingerprints for molecular recognition at the enzyme-inhibitor interface, a detailed theoretical analysis of the interactions between the principal binding loop of these inhibitors and active site of trypsin/chymotrypsin is performed based on available crystal structural, site-directed mutagenetic, kinetic, and sequence analysis studies. Despite the variations present at different positions of the principal binding loop of trypsin and chymotrypsin inhibitor models from Leaf-nosed viper and cobra Naja naja naja, respectively (designated as LnvTI and NCI), there are favorable subsite binding interactions which are expected to exhibit equally potent inhibitory activity as BPTI. On the contrary, significant mutations at several secondary specificity positions in the Naja naja naja trypsin inhibitor (designated as NTI) are likely to affect different inhibitor-enzyme-subsites interactions. This may explain the observed increased inhibitory activity of this polypeptide on a structural basis.     

Application of the lambda-dynamics method to evaluate the relative binding free energies of inhibitors to HCV protease

The lambda-dynamics method was used to calculate the relative binding free energies of inhibitors to the hepatitis C virus (HCV) protease. A total of seven HCV protease p-side product inhibitors were used in this study. The inhibitors are 6-mer peptides spanning P6-P1 (Ac-Asp-d-Glu-Leu-Ile-Cha-P1-CO(2)H). For this protein, S1 is a major hydrophobic pocket for binding. Binding of various residues to this pocket was investigated through free energy simulations and experimental inhibition constants. Several 300 ps lambda-dynamics simulations in explicit solvent were performed. The relative binding free energy was estimated from these simulations. From a single simulation, the inhibitors can be correctly classified into highly potent and weakly potent groups. The multiple simulations give an accurate rank ordering of inhibitor potency; computed and experimental binding free energies agree with 0.6 kcal/mol for five of the seven inhibitors. In addition, free energy perturbation (FEP) calculations were carried out to validate the results from lambda-dynamics. A total of 6 ligand pairs were compared. For each pair, 5-11 windows were used to map one ligand to the other. The cumulative simulation time was over 2 ns for each ligand pair. For four of the six ligand pairs, the lambda-dynamics free energy difference fits better than the FEP difference to the experimental value. The fact that the lambda-dynamics method achieved similar results in only a fraction of the total simulation time for FEP further demonstrates the robustness of the lambda-dynamics method.     

Toward RNase inhibitors: thermodynamics of 2'-CMP/RNase-A binding in multi-ion buffer

Certain ribonucleases (RNases), such as eosinophil-derived neurotoxin, are associated with pathological conditions (e.g. asthma and inflammatory bowel disease) and can even be overtly cyto(neuro)toxic. It has been proposed that small-molecule inhibitors should have therapeutic utility. We used isothermal titration microcalorimetry to characterize reversible inhibitor cytidine 2'-monophosphate (2'-CMP) binding to RNase-A in a multi-ion buffer at 37 degrees as a representative system. The estimated parameters were: K(d)=13.9 microM; DeltaG degrees =-6.90 kcal/mol; DeltaH degrees =-15.7 kcal/mol; and DeltaS degrees =-0.028 kcal/mol-K ('enthalpy-driven' interaction). These data should assist drug design of small-molecule inhibitors of homologous RNase catalytic domains.     

Novel structural features of CDK inhibition revealed by an ab initio computational method combined with dynamic simulations

The rational development of specific inhibitors for the approximately 500 protein kinases encoded in the human genome is impeded by a poor understanding of the structural basis for the activity and selectivity of small molecules that compete for ATP binding. Combining classical dynamic simulations with a novel ab initio computational approach linear-scalable to molecular interactions involving thousands of atoms, we have investigated the binding of five distinct inhibitors to the cyclin-dependent kinase CDK2. We report here that polarization and dynamic hydrogen bonding effects, so far undetected by crystallography, affect both their activity and selectivity. The effects arise from the specific solvation patterns of water molecules in the ATP binding pocket or the intermittent formation of hydrogen bonds during the dynamics of CDK/inhibitor interactions and explain the unexpectedly high potency of certain inhibitors such as 3-(3H-imidazol-4-ylmethylene)-5-methoxy-1,3-dihydro-indol-2-one (SU9516). The Lys89 residue in the ATP-binding pocket of CDK2 is observed to form temporary hydrogen bonds with the three most potent inhibitors. This residue is replaced in CDK4 by Thr89, whose shorter side-chain cannot form similar bonds, explaining the relative selectivity of the inhibitors for CDK2. Our results provide a generally applicable computational method for the analysis of biomolecular structures and reveal hitherto unrecognized features of the interaction between protein kinases and their inhibitors.     

Computer-aided identification of novel protein targets of bisphenol A

The xenoestrogen bisphenol A (2,2-bis-(p-hydroxyphenyl)-2-propane, BPA) is a known endocrine-disrupting chemical used in the fabrication of plastics, resins and flame retardants, that can be found throughout the environment and in numerous every day products. Human exposure to this chemical is extensive and generally occurs via oral route because it leaches from the food and beverage containers that contain it. Although most of the effects related to BPA exposure have been linked to the activation of the estrogen receptor (ER), the mechanisms of the interaction of BPA with protein targets different from ER are still unknown. Therefore, the objective of this work was to use a bioinformatics approach to identify possible new targets for BPA. Docking studies were performed between the optimized structure of BPA and 271 proteins related to different biochemical processes, as selected by text-mining. Refinement docking experiments and conformational analyses were carried out using LigandScout 3.0 for the proteins selected through the affinity ranking (lower than −8.0 kcal/mol). Several proteins including ERR gamma (−9.9 kcal/mol), and dual specificity protein kinases CLK-4 (−9.5 kcal/mol), CLK-1 (−9.1 kcal/mol) and CLK-2 (−9.0 kcal/mol) presented great in silico binding affinities for BPA. The interactions between those proteins and BPA were mostly hydrophobic with the presence of some hydrogen bonds formed by leucine and asparagine residues. Therefore, this study suggests that this endocrine disruptor may have other targets different from the ER.     

Conformations of trypsin-bound amidine inhibitors of blood coagulant factor Xa by double REDOR NMR and MD simulations.

Double rotational-echo double resonance (double REDOR) has been used to investigate the bound conformations of (13)C,(15)N,(19)F-labeled factor Xa inhibitors to bovine trypsin. Carbon-fluorine dipolar couplings were measured by (13)C{(19)F} REDOR with natural-abundance background interferences removed by (13)C{(15)N} REDOR. The conformations of the bound inhibitors were characterized by molecular dynamics (MD) simulations of binding restrained by double REDOR-determined intramolecular C-F distances. A symmetrical bisamidine inhibitor and an asymmetrical monoamidine-monoamine inhibitor of the same general shape had distinctly different conformations in the bound state. According to the MD models, these differences arise from specific interactions of the amidine and amine groups with the active-site residues of trypsin and nearby water molecules.     

Extra precision glide: docking and scoring incorporating a model of hydrophobic enclosure for protein-ligand complexes

A novel scoring function to estimate protein-ligand binding affinities has been developed and implemented as the Glide 4.0 XP scoring function and docking protocol. In addition to unique water desolvation energy terms, protein-ligand structural motifs leading to enhanced binding affinity are included: (1) hydrophobic enclosure where groups of lipophilic ligand atoms are enclosed on opposite faces by lipophilic protein atoms, (2) neutral-neutral single or correlated hydrogen bonds in a hydrophobically enclosed environment, and (3) five categories of charged-charged hydrogen bonds. The XP scoring function and docking protocol have been developed to reproduce experimental binding affinities for a set of 198 complexes (RMSDs of 2.26 and 1.73 kcal/mol over all and well-docked ligands, respectively) and to yield quality enrichments for a set of fifteen screens of pharmaceutical importance. Enrichment results demonstrate the importance of the novel XP molecular recognition and water scoring in separating active and inactive ligands and avoiding false positives.