Protein-inhibitor Complexes Research Articles

Could the "Janus-like" properties of the halobenzene CX bond (X=Cl, Br) be leveraged to enhance molecular recognition?

The CX bond in halobenzenes (XCl, Br) exhibits a dual character, being electron-deficient along the CX direction, and electron-rich on its flanks. We sought to amplify both features by resorting to electron-withdrawing and electron-donating substituents, respectively. This was done by quantum chemistry (QC) computations in the recognition sites of three protein targets: farnesyl transferase, coagulation factor Xa, and the HIV-1 integrase. In this context, some substituents, notably fluorine, CF3 , and NHCH3 , afforded significant overall gains in the binding energies as compared to the parent halobenzene, in the 2-5 kcal/mol range. In fact, we found that some di- and up to tetra-substitutions enabled even larger gains than those they contribute separately owing to many-body effects. Moreover, desolvation was also found to be a key contributor to the energy balances. As a consequence, some particular substituents, contributing to reduce the halobenzene dipole moment, accordingly reduced solvation: this factor acted in synergy with their enhancement of the intermolecular interaction energies along and around the CX bond. We could thus leverage the "Janus-like" properties of such a bond and the fact that it can be tuned and possibly amplified by well-chosen substituents. We propose a simple yet rigorous computational strategy resorting to QC to prescreen novel substituted halobenzenes. The QC results on the recognition sites then set benchmarks to validate polarizable molecular mechanics/dynamics approaches used to handle the entirety of the inhibitor-protein complex.

In silico 3D structure modeling and inhibitor binding studies of human male germ cell-associated kinase

Human male germ cell-associated kinase (hMAK) is an androgen-inducible gene in prostate epithelial cells, and it acts as a coactivator of androgen receptor signaling in prostate cancer. The 3D structure of the hMAK kinase was modeled based on the crystal structure of CDK2 kinase using comparative modeling methods, and the ATP-binding site was characterized. We have collected five inhibitors of hMAK from the literature and docked into the ATP-binding site of the kinase domain. Solvated interaction energies (SIE) of inhibitor binding are calculated from the molecular dynamics simulations trajectories of protein–inhibitor complexes. The contribution from each active site residue in hMAK toward inhibitor binding revealed the nature and extent of interactions between inhibitors and individual residues. The main chain atoms of Met79 invariably form hydrogen bonds with all five inhibitors. The amino acids Leu7, Val15, and Leu129 stabilize the inhibitors via CH–pi interactions. The Asp140 in the active site and Glu77 in hinge region show characteristic hydrogen bonding interactions with inhibitors. From SIE, the residue-wise interactions revealed the nature of non-bonding contacts and modifications required to increase the inhibitor activity. Our work provides 3D model structure of hMAK and molecular basis for the mechanisms of hMAK inhibition at atomic level that aid in designing new potent inhibitors.

Chemical specificity and conformational flexibility in proteinase–inhibitor interaction: Scaffolds for promiscuous binding

One of the most important roles of proteins in cellular milieu is recognition of other biomolecules including other proteins. Protein–protein complexes are involved in many essential cellular processes. Interfaces of protein–protein complexes are traditionally known to be conserved in evolution and less flexible than other solvent interacting tertiary structural surface. But many examples are emerging where these features do not hold good. An understanding of inter-play between flexibility and sequence conservation is emerging, providing a fresh dimension to the paradigm of sequence–structure–function relationship. The functional manifestation of the inter-relation between sequence conservation and flexibility of interface is exemplified in this review using proteinase–inhibitor protein complexes.

Structural study of the Schiff base copper(II) chelates as thrombin inhibitor

Thrombin is a trypsin-like serine protease that plays a critical role in the blood coagulation cascade. Thus, studies on thrombin-specific inhibitor are useful for the design of clinical useful compounds. We have previously reported of the Schiff base metal chelates carrying benzamidine and amino acid moieties. More than 50 kinds of Schiff base copper(II) chelates were synthesized fromp- orm-amidinosalicylaldehyde and various L- or D-amino acids. These chelates have effective inhibitory activity for trypsin and thrombin (Ki= 10-5~ 10-6M). In this series of inhibitors, the copper(II) chelate derived fromp-amidinosalicylaldehyde and D-tryptophan (chelate1g'in original paper) have shown exceptionally potent inhibitory activity against thrombin (Ki= 2.7×10-8M). To elucidate the structural basis of this high efficient inhibition, we were planning to determine and compare with four protein-inhibitor complex structures, chelate1g'or2g'bound to trypsin or thrombin (chelate2g'is derived fromm-amidinosalicylaldehyde and D-Trp). The crystals of trypsin binding the chelate2g'was obtained by sitting-drop vapor diffusion method by mixing 2.5 μL of 20 mg/mL protein-chelate complex solution with 2.5 μL of 0.1 M HEPES, pH 7.4, containing 0.1 M Li2(SO4), 25.0% PEG 3350. In the crystal structure, the imidazole nitrogen of His57 is coordinated with the copper(II) ion (1.94 Å). This close contact is made possible by conformation change of the His57. The indole ring of the chelate2g'simultaneously interacts with the copper(II) and the His57 by cation-π and π-π stacking interaction, respectively. In addition to trypsin-chelate2g'complex structure, we will report on the other three complex structures of trypsin-chelate1g', thrombin-chelate1g'and thrombin-chelate2g'.

Silver(I)‐Catalyzed Route to Pyrroles: Synthesis of Halogenated Pseudilins as Allosteric Inhibitors for Myosin ATPase and X‐ray Crystal Structures of the Protein–Inhibitor Complexes (Eur. J. Org. Chem. 21/2014)

Silver(I)‐Catalyzed Route to Pyrroles: Synthesis of Halogenated Pseudilins as Allosteric Inhibitors for Myosin ATPase and X‐ray Crystal Structures of the Protein–Inhibitor Complexes (Eur. J. Org. Chem. 21/2014)

Silver(I)‐Catalyzed Route to Pyrroles: Synthesis of Halogenated Pseudilins as Allosteric Inhibitors for Myosin ATPase and X‐ray Crystal Structures of the Protein–Inhibitor Complexes

AbstractThe pentahalogenated 2‐arylpyrrole‐type alkaloids pentabromopseudilin and pentachloropseudilin represent a new class of isoform‐specific allosteric inhibitors of myosin ATPase. Herein, we describe an application of the silver(I)‐catalyzed cycloisomerization of N‐(homopropargyl)toluenesulfonamides to the total syntheses of these natural products and several non‐natural analogues. Moreover, we examine the inhibitiory effect of pentahalogenated pseudilins on myosin ATPase activity.

Quantum mechanics/molecular mechanics studies of the mechanism of falcipain-2 inhibition by the epoxysuccinate E64.

Because of the increasing resistance of malaria parasites to antimalarial drugs, the lack of highly effective vaccines, and an inadequate control of mosquito vectors, the problem is growing, especially in the developing world. New approaches to drug development are consequently required. One of the proteases involved in the degradation of human hemoglobin is named falcipain-2 (FP2), which has emerged as a promising target for the development of novel antimalarial drugs. However, very little is known about the inhibition of FP2. In this paper, the inhibition of FP2 by the epoxysuccinate E64 has been studied by molecular dynamics (MD) simulations using hybrid AM1d/MM and M06-2X/MM potentials to obtain a complete picture of the possible free energy reaction paths. A thorough analysis of the reaction mechanism has been conducted to understand the inhibition of FP2 by E64. According to our results, the irreversible attack of Cys42 on E64 can take place on both carbon atoms of the epoxy ring because both processes present similar barriers. While the attack on the C2 atom presents a slightly smaller barrier (12.3 vs 13.6 kcal mol(-1)), the inhibitor-protein complex derived from the attack on C3 appears to be much more stabilized. In contrast to previous hypotheses, our results suggest that residues such as Gln171, Asp170, Gln36, Trp43, Asn81, and even His174 would be anchoring the inhibitor in a proper orientation for the reaction to take place. These results may be useful for the rational design of new compounds with higher inhibitory activity.

Antigen levels of coagulation factor XII, coagulation factor XI and prekallikrein, and the risk of myocardial infarction and ischemic stroke in young women

High levels of activated protein–inhibitor complexes of the intrinsic coagulation proteins are associated with ischemic stroke (IS) but not with myocardial infarction (MI). This study was aimed at determining whether the antigen levels of coagulation factors(factor XII, FXII, and FXI and prekallikrein (PK)are associated with MI and IS, and whether this association is independent of levels of activated protein–inhibitor complexes. The RATIO study included young women (< 50 years) with MI (N = 205)and IS (N = 175), and 638 healthy controls. Antigen levels of FXII, FXI and PK were measured and expressed as percentages of of those in pooled normal plasmas. Odds ratios (ORs) and corresponding 99% confidence intervals (CIs) were calculated for high levels (i.e. ≥ 90th percentile of controls) as measures of rate ratios. After adjustment for potential confounders, high levels of FXII antigen were not associated with MI risk or IS risk(OR(MI) 1.18, 99% CI 0.51–2.74; ORIS 1.03, 9% CI 0.41–2.55). High levels of FXI antigen were slightly associated with an increase in MI risk (OR(MI) 1.55, 9% CI 0.74–3.21), whereas there was a substantial association with IS risk (ORIS 2.65, 9% CI 1.27–5.56). PK antigen was slightly associated with MI risk but not with IS risk(ORMI 1.54, 9% CI 0.67–3.52; ORIS 0.90, 9% CI 0.35–2.33). All associations remained similar after adjustment for levels of protein–inhibitor complexes. Increased levels of FXI antigen were associated with an increase in IS risk, whereas they showed only a marginal association with MI risk. FXII antigen and PK antigen levels were not substantially associated with MI risk and IS risk.

The identification of AF38469: An orally bioavailable inhibitor of the VPS10P family sorting receptor Sortilin

The identification of the novel, selective, orally bioavailable Sortilin inhibitor AF38469 is described. Structure–activity relationships and syntheses are reported, along with an X-ray crystal structure of the sortilin-AF38469 protein-inhibitor complex.

Modulation of the Fibrillogenesis Inhibition Properties of Two Transthyretin Ligands by Halogenation

The amyloidogenic protein transthyretin (TTR) is thought to aggregate into amyloid fibrils by tetramer dissociation which can be inhibited by a number of small molecule compounds. Our analysis of a series of crystallographic protein-inhibitor complexes has shown no clear correlation between the observed molecular interactions and the in vitro activity of the inhibitors. From this analysis, it emerged that halogen bonding (XB) could be mediating some key interactions. Analysis of the halogenated derivatives of two well-known TTR inhibitors has shown that while flufenamic acid affinity for TTR was unchanged by halogenation, diflunisal gradually improves binding up to 1 order of magnitude after iodination through interactions that can be interpreted as a suboptimal XB (carbonyl Thr106: I...O distance 3.96-4.05 Å; C-I...O angle 152-156°) or as rather optimized van der Waals contacts or as a mixture of both. These results illustrate the potential of halogenation strategies in designing and optimizing TTR fibrillogenesis inhibitors.

Activation State-Selective Kinase Inhibitor Assay Based on Ion Mobility-Mass Spectrometry

The discovery of activation state dependent kinase inhibitors, which bind specifically to the inactive conformation of the protein, is considered to be a promising pathway to improved cancer treatments. Identifying such inhibitors is challenging, however, because they can have Kd values similar to molecules known to inhibit kinase function by interacting with the active form. Further, while inhibitor induced changes within the kinase tertiary structure are significant, few technologies are able to correctly assign inhibitor binding modes in a high-throughput fashion based exclusively on protein-inhibitor complex formation and changes in local protein structure. We have developed a new assay, using ion mobility-mass spectrometry, capable of both rapidly detecting inhibitor binding and classifying the resultant kinase binding modes. Here, we demonstrate the ability of our approach to classify a broad set of kinase inhibitors, using micrograms of protein, without the need for protein modification or tagging.

Molecular docking vs structure optimization

There are many in silico studies of molecular systems whose tools help search for new molecules with biological activity. The aim of this work is to present a comparative study made using two different modeling tools in the study of interactions and complex formation between Cathepsin G and morelloflavone. The first m odeling tool used was molecular docking using the genetic algorithm methodology. The second tool was the semi-empirical method for structure optimizations in vacuum and in the presence of a solvent. The results shown that the optimized Cathepsin G-morellofla vone complex had a better affinity (30%) than the complex obta ined from the docking process. This demonstrates the importance of using methods that consider the electronic structure and solvent effects on the formation of protein-inhibitor complexes.

Ligand-based drug design for human endothelin converting enzyme-1 inhibitors

The endothelin converting enzyme-1 (ECE-1) is a zinc binding protein involved in the generation of a 21 amino acid endothelin-1 (ET-1) peptide. The mitogenic action of ET-1 has been shown in several cell types and produced by several human cancer cell lines. We have used the ligand-based drug design methods to understand the structure–activity relationship for inhibitor design of this important multi-disease target. The molecular docking of thiol-based inhibitors into the active site of ECE-1 identified several nonbonding interactions that stabilize the protein-inhibitor complex. The S1 and S1′ sub-sites could accommodate larger hydrophobic substitutions on the inhibitor with highest activity (molecule 16). From the 3D-QSAR studies, the electrostatic and steric descriptors that correlate with the activity of the inhibitors were identified. In the best pharmacophore, the hydrogen bond donor feature at the electronegative sulfur atom of inhibitor indicates that chelation with the Zn-ion is essential in the S1 sub-site, besides the other features. Together, the results from these diverse methods are complementary to each other and provide guidelines for the design of better ECE-1 inhibitors.

Assessment of new anti-HER2 ligands using combined docking, QM/MM scoring and MD simulation

In the development of new anti-cancer drugs to tackle the problem of resistance to current chemotherapeutic agents, a new series of anti-HER2 (human epidermal growth factor receptors 2) agents has been synthesized and investigated using different computational methods. Although non-selective, the most active inhibitor in the new series shows higher activity toward HER2 than EGFR. The induced fit docking protocol (IFD) is performed to find possible binding poses of the new inhibitors in the active site of the HER2 receptor. Molecular dynamic simulations of the inhibitor–protein complexes for the two most active compounds from the new series are carried out. Simulations stability is checked using different stability parameters. Different scoring functions are employed.

Binding Modes of Peptidomimetics Designed to Inhibit STAT3

STAT3 is a transcription factor that has been found to be constitutively activated in a number of human cancers. Dimerization of STAT3 via its SH2 domain and the subsequent translocation of the dimer to the nucleus leads to transcription of anti-apoptotic genes. Prevention of the dimerization is thus an attractive strategy for inhibiting the activity of STAT3. Phosphotyrosine-based peptidomimetic inhibitors, which mimic pTyr-Xaa-Yaa-Gln motif and have strong to weak binding affinities, have been previously investigated. It is well-known that structures of protein-inhibitor complexes are important for understanding the binding interactions and designing stronger inhibitors. Experimental structures of inhibitors bound to the SH2 domain of STAT3 are, however, unavailable. In this paper we describe a computational study that combined molecular docking and molecular dynamics to model structures of 12 peptidomimetic inhibitors bound to the SH2 domain of STAT3. A detailed analysis of the modeled structures was performed to evaluate the characteristics of the binding interactions. We also estimated the binding affinities of the inhibitors by combining MMPB/GBSA-based energies and entropic cost of binding. The estimated affinities correlate strongly with the experimentally obtained affinities. Modeling results show binding modes that are consistent with limited previous modeling studies on binding interactions involving the SH2 domain and phosphotyrosine(pTyr)-based inhibitors. We also discovered a stable novel binding mode that involves deformation of two loops of the SH2 domain that subsequently bury the C-terminal end of one of the stronger inhibitors. The novel binding mode could prove useful for developing more potent inhibitors aimed at preventing dimerization of cancer target protein STAT3.

Evaluation of Adamantane Derivatives as Inhibitors of Dengue Virus mRNA Cap Methyltransferase by Docking and Molecular Dynamics Simulations.

Binding of the Dengue virus S-adenosyl-L-methionine (AdoMet)-dependent mRNA cap methyltransferase (NS5MTaseDV ) with adamantane derivatives was explored using molecular modeling methods and (nucleoside-2'O)-methyltransferase bioassay. The studied compounds include urea derivatives of adamantane and the antiviral drugs amantadine and rimantadine. The urea derivatives of adamantanes had previously been identified as inhibitors of NS5MTaseDV . The docking simulations using GOLD, Glide, and Dock give consistent binding modes and binding affinities of adamantanes in the AdoMet-binding site of NS5MTaseDV and, in particular, yield similar positions for the previously found inhibitors. Combined, they perfectly correspond to the bioassay measurements of nucleoside-2'O-methyltransferase activity of NS5TaseDV , which confirmed inhibitory properties of the active urea adamantane but did not show inhibitory activity for amantadine and rimantadine. We also employed microscopic molecular dynamics (MD) simulations and a linear interaction energy (LIE) method to verify the docking results. The MD/LIE binding free energies of selected protein-inhibitor complexes agree overall with the binding affinities from docking and demonstrate that amantadine and rimantadine only weakly bind at the explored site. The MD simulations also demonstrated the flexible character of a protein loop that is located between the β2 and β3 strands and is part of the AdoMet-binding pocket of NS5MTaseDV .

Simple Structure-Based Approach for Predicting the Activity of Inhibitors of Beta-Secretase (BACE1) Associated with Alzheimer’s Disease

Beta-site amyloid precursor protein cleaving enzyme-1 (BACE1) is a target of interest for treating patients with Alzheimer's disease (AD). Inhibition of BACE1 may prevent amyloid-ß (Aß) plaque formation and the development or progression of Alzheimer's disease. Known BACE1 inhibitors were analyzed using computational chemistry and cheminformatics techniques to search for quantitative structure-activity relationships (QSAR). A remarkable relationship was found with only two simple descriptors. The square of the linear correlation coefficient r(2) is 0.75. The main descriptor is the number of hydrophobic contacts in the range 4-5 Å between the atoms of the ligand and active site. The other descriptor is the number of short (<2.8 Å) hydrogen bonds. Our approach uses readily available structural data on protein-inhibitor complexes in the Protein Data Bank (PDB) but would be equally applicable to proprietary structural biology data. The findings can aid structure-based design of improved BACE-1 inhibitors. If an inhibitor has less observed activity than predicted by our correlation, the compound should be retested because the first assay may have underestimated the compound's true activity.

Multiple virtual screening approaches for finding new Hepatitis c virus RNA-dependent RNA polymerase inhibitors: Structure-based screens and molecular dynamics for the pursue of new poly pharmacological inhibitors

The RNA polymerase NS5B of Hepatitis C virus (HCV) is a well-characterised drug target with an active site and four allosteric binding sites. This work presents a workflow for virtual screening and its application to Drug Bank screening targeting the Hepatitis C Virus (HCV) RNA polymerase non-nucleoside binding sites. Potential polypharmacological drugs are sought with predicted active inhibition on viral replication, and with proven positive pharmaco-clinical profiles. The approach adopted was receptor-based. Docking screens, guided with contact pharmacophores and neural-network activity prediction models on all allosteric binding sites and MD simulations, constituted our analysis workflow for identification of potential hits. Steps included: 1) using a two-phase docking screen with Surflex and Glide Xp. 2) Ranking based on scores, and important H interactions. 3) a machine-learning target-trained artificial neural network PIC prediction model used for ranking. This provided a better correlation of IC50 values of the training sets for each site with different docking scores and sub-scores. 4) interaction pharmacophores-through retrospective analysis of protein-inhibitor complex X-ray structures for the interaction pharmacophore (common interaction modes) of inhibitors for the five non-nucleoside binding sites were constructed. These were used for filtering the hits according to the critical binding feature of formerly reported inhibitors. This filtration process resulted in identification of potential new inhibitors as well as formerly reported ones for the thumb II and Palm I sites (HCV-81) NS5B binding sites. Eventually molecular dynamics simulations were carried out, confirming the binding hypothesis and resulting in 4 hits.

Characterization of human carbonic anhydrase XII stability and inhibitor binding

Human carbonic anhydrase isozyme XII is a transmembrane protein that is overexpressed in many human cancers. Therefore CA XII is an anticancer drug target. However, there are few compounds that specifically target CA XII. The design of specific inhibitors against CA XII relies on the detailed understanding of the thermodynamics of inhibitor binding and the structural features of the protein–inhibitor complex. To characterize the thermodynamic parameters of the binding of known sulfonamides, namely ethoxzolamide, acetazolamide and trifluoromethanesulfonamide, we used isothermal titration calorimetry and fluorescent thermal shift assay. The binding of these sulfonamides to CA XII was buffer and pH-dependent. Dissection of protonation–deprotonation reactions of both the water molecule bound to the CA XII active site and the sulfonamide group of the inhibitor yielded the intrinsic thermodynamic parameters of binding, such as binding enthalpy, entropy and Gibbs free energy. Thermal shift assay was also used to determine CA XII stabilities at various pH and in the presence of buffers and salts.

Modeling the Binding Affinity of p38α MAP Kinase Inhibitors by Partial Least Squares Regression

The p38 mitogen-activated protein kinase is activated by environmental stress and cytokines and plays a role in transcriptional regulation and inflammatory responses. Factors influencing the activity and selectivity of the p38α mitogen-activated protein kinase inhibitors have been investigated in this paper by inspecting the binding orientation and the possible residue-inhibitor interactions in the binding site. The binding pattern of a set of 45 different inhibitors against p38α mitogen-activated protein kinase was studied through Molecular Dynamic Simulations of the protein-inhibitor complexes. Further, Partial Least Squares regression was used to develop a Quantitative Structure Activity Relationship model to predict the binding affinities of ligands. The selected model successfully predicted the test set with a Root Mean Square Error of Prediction of 1.36. The regression coefficients and the Variable Importance in Projection plots highlighted the residue-inhibitor interactions which exhibited the largest absolute effect on the ligand binding, such as the van der Waals interaction with LYS50, ILE81, ASP165; electrostatic interactions with SER29, LEU164; hydrogen bonds with MET106; and total energy interaction with SER29 and LEU83.