Tuning the Binding Affinity and Selectivity of Perfluoroaryl-Stapled Peptides by Cysteine-Editing

A growing number of approaches to “staple” α-helical peptides into a bioactive conformation using cysteine cross-linking are emerging. Here, the replacement of l -cysteine with “cysteine analogues” in combinations of different stereochemistry, side chain length and beta-carbon substitution, is explored to examine the influence that the thiol-containing residue(s) has on target protein binding affinity in a well-explored model system, p53–MDM2/MDMX, which is constituted by the interaction of the tumour suppressor protein p53 and proteins MDM2 and MDMX, which regulate p53 activity. In some cases, replacement of one or more l -cysteine residues afforded significant changes in the measured binding affinity and target selectivity of the peptide. Computationally constructed homology models indicate that some modifications, such as incorporating two d -cysteine residues, favourably alter the positions of key functional amino acid side chains, which is likely to cause changes in binding affinity, in agreement with measured surface plasmon resonance data.     

In silico study directed towards identification of the key structural features of GyrB inhibitors targeting MTB DNA gyrase: HQSAR,CoMSIA and molecular dynamics simulations

ABSTRACT

Mycobacterium tuberculosis DNA gyrase subunit B (GyrB) has been identified as a promising target for rational drug design against fluoroquinolone drug-resistant tuberculosis. In this study, we attempted to identify the key structural feature for highly potent GyrB inhibitors through 2D-QSAR using HQSAR, 3D-QSAR using CoMSIA and molecular dynamics (MD) simulations approaches on a series of thiazole urea core derivatives. The best HQSAR and CoMSIA models based on IC₅₀ and MIC displayed the structural basis required for good activity against both GyrB enzyme and mycobacterial cell. MD simulations and binding free energy analysis using MM-GBSA and waterswap calculations revealed that the urea core of inhibitors has the strongest interaction with Asp79 via hydrogen bond interactions. In addition, cation-pi interaction and hydrophobic interactions of the R₂ substituent with Arg82 and Arg141 help to enhance the binding affinity in the GyrB ATPase binding site. Thus, the present study provides crucial structural features and a structural concept for rational design of novel DNA gyrase inhibitors with improved biological activities against both enzyme and mycobacterial cell, and with good pharmacokinetic properties and drug safety profiles.     

A robust force field based method for calculating conformational energies of charged drug-like molecules

The binding affinity of a drug-like molecule depends among other things on the availability of the bioactive conformation. If the bioactive conformation has a significantly higher energy than the global minimum energy conformation, then the molecule is unlikely to bind to its target. Determination of the global minimum energy conformation and calculation of conformational penalties of binding is a prerequisite for prediction of reliable binding affinities. Here, we present a simple and computationally efficient procedure to estimate the global energy minimum for a wide variety of structurally diverse molecules, including polar and charged compounds. Identifying global energy minimum conformations of such compounds with force field methods is problematic due to the exaggeration of intramolecular electrostatic interactions. We demonstrate that the global energy minimum conformations of zwitterionic compounds generated by conformational analysis with modified electrostatics are good approximations of the conformational distributions predicted by experimental data and with molecular dynamics performed in explicit solvent. Finally the method is used to calculate conformational penalties for zwitterionic GluA2 agonists and to filter false positives from a docking study.     

Interchain doubly-bridged α-helical peptides for the development of protein binders

This work reported the design and synthesis of interchain doubly-bridged α-helical peptides, involving mutual stabilization of two α -helical peptides crosslinked by two interchain bisthioether crosslinkers.     

Design of multi-binding-site inhibitors, ligand efficiency, and consensus screening of avian influenza H5N1 wild-type neuraminidase and of the oseltamivir-resistant H274Y variant

The binding sites of wild-type avian influenza A H5N1 neuraminidase, as well as those of the Tamiflu (oseltamivir)-resistant H274Y variant, were explored computationally to design inhibitors that target simultaneously several adjacent binding sites of the open conformation of the virus protein. The compounds with the best computed free energies of binding, in agreement by two docking methods, consensus scoring, and ligand efficiency values, suggest that mimicking a polysaccharide, beta-lactam, and other structures, including known drugs, could be routes for multibinding site inhibitor design. This new virtual screening method based on consensus scoring and ligand efficiency indices is introduced, which allows the combination of pharmacodynamic and pharmacokinetic properties into unique measures.     

Inhibition Mechanism of Hydroxyproline-like Small Inhibitors to Disorder HIF-VHL Interaction by Molecular Dynamic Simulations and Binding Free Energy Calculations

Protein-protein interactions are vital for a wide range of biological processes. The interactions between the hypoxia-inducible factor and von Hippel Lindau (VHL) are attractive drug targets for ischemic heart disease. In order to disrupt this interaction, the strategy to target VHL binding site using a hydroxyproline-like (pro-like) small molecule has been reported. In this study, we focused on the inhibition mechanism between the pro-like inhibitors and the VHL protein, which were investigated via molecular dynamics simulations and binding free energy calculations. It was found that pro-like inhibitors showed a strong binding affinity toward VHL. Binding free energy calculations and free energy decompositions suggested that the modification of various regions of pro-like inhibitors may provide useful information for future drug design.     

Z_(Aβ3)和Aβ_(16–40)亲和作用的分子机理解析

淀粉样多肽(amyloid-βpeptide,Aβ)聚集是引起阿尔兹海默症(Alzheimer's disease,AD)的主要原因。开发Aβ聚集抑制剂是治疗AD的最有效手段之一。利用噬菌体展示技术筛选出来的Z_(Aβ3)蛋白质能够有效抑制Aβ聚集,但Z_(Aβ3)和Aβ之间的作用区域和关键氨基酸残基尚不清楚。针对此问题,本研究利用分子动力学模拟、MM-PBSA自由能计算和分解方法研究了Z_(Aβ3)-Aβ_(16–40)复合物之间的相互作用机制。结果表明,Z_(Aβ3)的β-股和Aβ_(16–40)之间的亲和作用占主导,而Z_(Aβ3)的α-螺旋贡献很小。利用分子力学-帕松波尔茨曼溶剂可及化表面积方法(MM-PBSA)自由能分解发现Z_(Aβ3)的热点残基为E15、I16、V17、Y18、L19、P20、N21和L22,而Aβ_(16–40)的热点残基为F19、F20、A21、E22、D23、K28、I31、I32、G33、L34、M35、V36、G38和V40。Z_(Aβ3)通过将发夹型Aβ单体包埋在α-螺旋围成的疏水性腔体内来阻碍Aβ聚集。这种结合模式为设计高效的Aβ蛋白质类抑制剂提供了三个基本要素:高亲和性的结合片段(β-股)、附属结构(α-螺旋)和通过二硫键形成的稳定构象。高亲和性结合片段能竞争性地与Aβ单体结合,附属结构α-螺旋可以阻碍其它Aβ单体靠近,而稳定的构象是上述两种要素发挥作用的基础,三者协同作用可以有效地抑制Aβ聚集。     

Predicting and harnessing protein flexibility in the design of species-specific inhibitors of thymidylate synthase

BACKGROUND: Protein plasticity in response to ligand binding abrogates the notion of a rigid receptor site. Thus, computational docking alone misses important prospective drug design leads. Bacterial-specific inhibitors of an essential enzyme, thymidylate synthase (TS), were developed using a combination of computer-based screening followed by in-parallel synthetic elaboration and enzyme assay [Tondi et al. (1999) Chem. Biol. 6, 319-331]. Specificity was achieved through protein plasticity and despite the very high sequence conservation of the enzyme between species. RESULTS: The most potent of the inhibitors synthesized, N,O-didansyl-L-tyrosine (DDT), binds to Lactobacillus casei TS (LcTS) with 35-fold higher affinity and to Escherichia coli TS (EcTS) with 24-fold higher affinity than to human TS (hTS). To reveal the molecular basis for this specificity, we have determined the crystal structure of EcTS complexed with DDT and 2'-deoxyuridine-5'-monophosphate (dUMP). The 2.0 A structure shows that DDT binds to EcTS in a conformation not predicted by molecular docking studies and substantially differently than other TS inhibitors. Binding of DDT is accompanied by large rearrangements of the protein both near and distal to the enzyme's active site with movement of C alpha carbons up to 6 A relative to other ternary complexes. This protein plasticity results in novel interactions with DDT including the formation of hydrogen bonds and van der Waals interactions to residues conserved in bacterial TS but not hTS and which are hypothesized to account for DDT's specificity. The conformation DDT adopts when bound to EcTS explains the activity of several other LcTS inhibitors synthesized in-parallel with DDT suggesting that DDT binds to the two enzymes in similar orientations. CONCLUSIONS: Dramatic protein rearrangements involving both main and side chain atoms play an important role in the recognition of DDT by EcTS and highlight the importance of incorporating protein plasticity in drug design. The crystal structure of the EcTS/dUMP/DDT complex is a model system to develop more selective TS inhibitors aimed at pathogenic bacterial species. The crystal structure also suggests a general formula for identifying regions of TS and other enzymes that may be treated as flexible to aid in computational methods of drug discovery.     

Molecular recognition: minimizing the acid–base interaction of a tunable host–guest system changes the selectivity of binding

Two new receptors incorporating a 4-n-butyl aniline moiety has been designed, synthesized and evaluated for their binding properties towards a series of ureido-glycine derivatives. The host design is based on an urea adamantyl host motif known from large generations of poly(propylene imine) dendrimers functionalized with urea adamantyl moieties on the periphery. The design of the host molecules was directed towards a study of the effects of basicity of an amine function versus the effect of molecular recognition on the binding strength as seen from comparing the results obtained in the present work with previously guest–host studies. The guest–host interaction features an electrostatic interaction and multiple hydrogen binding interactions, where the main difference between the hosts described here and previously described is a substitution from an amine to aniline. Anilines are weaker bases than aliphatic amines and they generally give lower binding constants when treated with acidic guest molecules. The association constants have been measured using NMR titrations and the nature of the guest–host system is discussed based on these results. A general decrease in binding affinities is observed upon changing from the trialkyl amine hosts to the dialkyl aniline based hosts. One exception was observed where the weaker base host had stronger affinity to one of the guests. Thus, when the basicity of the host is decreased other factors influence the binding such as a better geometric fit. A crystal structure of one of the receptors has been solved and it shows no intramolecular hydrogen bonding.     

Discovery of Novel Acetaldehyde Dehydrogenase 1A1(ALDH1A1) Inhibitors by Utilizing 3D-QSAR,Molecular Docking and Molecular Dynamics Simulation

Acetaldehyde dehydrogenase 1A1 is a hopeful therapeutic target to ovarian cancer. In this present work, 3D-QSAR, molecular docking and molecular dynamics(MD) simulations were implemented on a series of quinoline-based ALDH1A1 inhibitors to investigate novel acetaldehyde dehydrogenase 1A1 inhibitors as anticancer adjuvant drugs for ovarian cancer. Two reliable CoMFA(Q~2 = 0.583, R~2 = 0.967) and CoMSIA(Q~2 = 0.640, R~2 = 0.977) models of ALDH1A1 inhibitors were established. Novel ALDH1A1 inhibitors were predicted by the 3D-QSAR models. Molecular docking reveals important residues for protein-compound interactions, and the results revealed ALDH1A1 inhibitors had stronger electrostatic interaction and binding affinity with key residues of protein, such as Phe171, Val174 and Cys303. Molecular dynamics simulations further verified the results of molecular docking. The above information provided significant guidance for the design of novel ALDH1A1 inhibitors.     

Design, synthesis and binding studies of a novel quadruple ADDA hydrogen-bond array

The design and synthesis of a novel ADDA hydrogen-bond array is described. The ureidodiimidazole motif (UDIM) 2 engages in interactions with complementary diamidonaphthyridine (DAN) 3 motifs with an association constant K(a) = 825 ± 16 M(-1) in chloroform. (1)H NMR and molecular modelling studies were carried out in order to explain the unexpected behaviour of this new supramolecular motif. These revealed that a combination of effects including; an energetic bias for the folded conformer, subtle differences in shape complementarity between the two components and the potential for self-association of UDIM 2 disfavour higher affinity interactions between the two components.     

Structure‐Based Design of Inhibitors of Protein–Protein Interactions: Mimicking Peptide Binding Epitopes

Protein–protein interactions (PPIs) are involved at all levels of cellular organization, thus making the development of PPI inhibitors extremely valuable. The identification of selective inhibitors is challenging because of the shallow and extended nature of PPI interfaces. Inhibitors can be obtained by mimicking peptide binding epitopes in their bioactive conformation. For this purpose, several strategies have been evolved to enable a projection of side chain functionalities in analogy to peptide secondary structures, thereby yielding molecules that are generally referred to as peptidomimetics. Herein, we introduce a new classification of peptidomimetics (classes A–D) that enables a clear assignment of available approaches. Based on this classification, the Review summarizes strategies that have been applied for the structure‐based design of PPI inhibitors through stabilizing or mimicking turns, β‐sheets, and helices.     

Deactivation of Mcl‐1 by Dual‐Function Small‐Molecule Inhibitors Targeting the Bcl‐2 Homology 3 Domain and Facilitating Mcl‐1 Ubiquitination

By means of limited proteolysis assay, three‐dimensional NMR, X‐ray crystallography and alanine mutations, a dynamic region at the Q221R222N223 motif in the Bcl‐2 homology 3 (BH3) domain of Mcl‐1 has been identified as a conformational switch which controls Mcl‐1 ubiquitination. Noxa^BH3 binding biases the QRN motif toward a helical conformation, thus leading to an enhanced in vitro ubiquitination of Mcl‐1. In contrast, Bim^BH3 binding biases the QRN motif toward a nonhelical conformation, thus leading to the inhibition of ubiquitination. A dual function Mcl‐1 inhibitor, which locates at the BH3 domain of Mcl‐1 and forms hydrogen bond with His224 to drive a helical QRN conformation, so that it not only interferes with the pro‐apoptotic partners, but also facilitates Mcl‐1 ubiquitination in living cells, is described. As a result, this inhibitor manifests a more effective apoptosis induction in Mcl‐1‐dependent cancer cells than other inhibitors exhibiting a similar binding affinity with it.     

N,N'-linked oligoureas as foldamers: chain length requirements for helix formation in protic solvent investigated by circular dichroism, NMR spectroscopy, and molecular dynamics

N,N'-linked oligoureas with proteinogenic side chains are peptide backbone mimetics belonging to the gamma-peptide lineage. In pyridine, heptamer 4 adopts a stable helical fold reminiscent of the 2.6(14) helical structure proposed for gamma-peptide foldamers. In the present study, we have used a combination of CD and NMR spectroscopies to correlate far-UV chiroptical properties and conformational preferences of oligoureas as a function of chain length from tetramer to nonamer. Both the intensity of the CD spectra and NMR chemical shift differences between alphaCH2 diastereotopic protons experienced a marked increase for oligomers between four and seven residues. No major change in CD spectra occurred between seven and nine residues, thus suggesting that seven residues could be the minimum length required for stabilizing a dominant conformation. Unexpectedly, in-depth NMR conformational investigation of heptamer 4 in CD3OH revealed that the 2.5 helix probably coexists with partially (un)folded conformations and that Z-E urea isomerization occurs, to some degree, along the backbone. Removing unfavorable electrostatic interactions at the amino terminal end of 4 and adding one H-bond acceptor by acylation with alkyl isocyanate (4 --> 7) was found to reinforce the 2.5 helical population. The stability of the 2.5 helical fold in MeOH is further discussed in light of unrestrained molecular dynamics (MD) simulation. Taken together, these new data provide additional insight into the folding propensity of oligoureas in protic solvent and should be of practical value for the design of helical bioactive oligoureas.     

Paralog-selective ligands for bcl-2 proteins

There is considerable current interest in molecules that bind intra- or extracellular protein surfaces and inhibit protein-protein interactions. Previously we have reported that miniature proteins based on pancreatic-fold polypeptides can recognize even shallow alpha-helix binding clefts with high affinity and selectivity against unrelated proteins. One such miniature protein, PPBH3-1, binds the anti-apoptotic protein paralogs Bcl-2 and Bcl-XL with nanomolar affinity and a DeltaDeltaG = 1.2 kcal.mol-1 preference for Bcl-XL. Here we describe the directed evolution of PPBH3-1 into two new miniature proteins, PPBH3-5 and PPBH3-6, whose paralog specificity is reversed relative to PPBH3-1. PPBH3-5 and PPBH3-6 bind Bcl-2 with nanomolar affinity and a DeltaDeltaG = 0.9-1.3 kcal.mol-1 preference for Bcl-2 over Bcl-XL. Experiments with Bcl-XL variants suggest that PPBH3-5 and PPBH3-6 achieve high paralog specificity by exploiting subtle structural or electrostatic differences in the Bcl-2 and Bcl-XL molecular landscapes. PPBH3-5 and PPBH3-6 may have unique applications as early examples of nonnatural ligands that interact selectively with Bcl-2 proteins.     

Molecular simulation studies to reveal the binding mechanisms of shikonin derivatives inhibiting VEGFR-2 kinase

Traditional vascular endothelial growth factor receptor 2 (VEGFR-2) inhibitors can manage angiogenesis; however, severe toxicity and resistance limit their long-term applications in clinical therapy. Shikonin (SHK) and its derivatives could be promising to inhibit the VEGFR-2 mediated angiogenesis, as they are reported to bind in the catalytic kinase domain with low affinity. However, the detailed molecular insights and binding dynamics of these natural inhibitors are unknown, which is crucial for potential SHK based lead design. Therefore, the present study employed molecular modeling and simulations techniques to get insight into the binding behaviors of SHK and its two derivates, β-hydroxyisovalerylshikonin (β-HIVS) and acetylshikonin (ACS). Here the intermolecular interactions between protein and ligands were studied by induced fit docking approach, which were further evaluated by treating QM/MM (quantum mechanics/molecular mechanics) and molecular dynamics (MD) simulation. The result showed that the naphthazarin ring of the SHK derivates is vital for strong binding to the catalytic domain; however, the binding stability can be modulated by the side chain modification. Because of having electrostatic potential, this ring makes essential interactions with the DFG (Asp1046 and Phe1047) motif and also allows interacting with the allosteric binding site. Taken together, the studies will advance our knowledge and scope for the development of new selective VEGFR-2 inhibitors based on SHK and its analogs.     

Design of inhibitors of the MurF enzyme of Streptococcus pneumoniae using docking, 3D-QSAR, and de Novo design

The biosynthetic pathway for formation of the bacterial cell wall (peptidoglycan) presents an attractive target for intervention. This is exploited by many of the clinically useful antibiotics, which inhibit enzymes involved in the later stages of peptidoglycan synthesis. MurF is one of the four amide bond-forming enzymes (d-alanyl-d-alanine ligating enzyme) that catalyzes the ATP-dependent formation of UDP-MurNAc-tripeptide. In the present study, several MurF inhibitors were docked into the active site of MurF to explore their binding modes and also to gain an insight into the crucial ligand-receptor interactions at the molecular level. The final selection of the "bioactive" conformation of every ligand was influenced by consensus scoring in which various independent scoring functions such as GoldScore, ChemScore, HINT score and X-CScore were employed. Subsequently, 3D-QSAR studies using comparative molecular field analysis (CoMFA) and the new approach comparative residue interaction analysis (CoRIA) have been carried out on the enzyme-inhibitor complexes obtained by docking and postscoring analysis. Finally, new inhibitors have been designed using the de novo approach of Ludi, and the activities of the most promising hits have been predicted with the CoMFA and CoRIA models.     

Exponential repulsion improves structural predictability of molecular docking

Molecular docking is a powerful tool for theoretical prediction of the preferred conformation and orientation of small molecules within protein active sites. The obtained poses can be used for estimation of binding energies, which indicate the inhibition effect of designed inhibitors, and therefore might be used for in silico drug design. However, the evaluation of ligand binding affinity critically depends on successful prediction of the native binding mode. Contemporary docking methods are often based on scoring functions derived from molecular mechanical potentials. In such potentials, nonbonded interactions are typically represented by electrostatic interactions between atom‐centered partial charges and standard 6–12 Lennard–Jones potential. Here, we present implementation and testing of a scoring function based on more physically justified exponential repulsion instead of the standard Lennard–Jones potential. We found that this scoring function significantly improved prediction of the native binding modes in proteins bearing narrow active sites such as serine proteases and kinases. © 2016 Wiley Periodicals, Inc.     

葡萄糖苷酶抑制剂作用机理的分子动力学模拟和自由能计算

含有锍离子的葡萄糖苷酶抑制剂如kotalanol (SK)和它除去磺酸基团后的衍生物(DSK), 是潜在的毒副作用较小的治疗II 型糖尿病的候选药物. α-葡萄糖苷酶抑制活性实验显示, DSK活性比SK略高, 而将二者环上的S原子替换成NH后(分别称为DSN和SN), DSN的活性要比SN高1500倍左右. 本文用分子动力学模拟, 结合自由能计算和自由能分解的方法对上述四个抑制剂的作用机理进行了研究. 研究结果表明活性的巨大差异是由NH基团取代效应和磺酸基团立体效应共同作用的结果, 由于N―C键长比S―C键长短, NH基团取代导致烷基链的翻转, 同时, 磺酸基团限制了链的翻转, 因此改变了抑制剂的结合模式. 计算结果与实验基本一致.本文的研究结果有助于进一步理解含锍离子的葡萄糖苷酶抑制剂的结合机理, 并为设计更有潜力的葡萄糖苷酶抑制剂提供了有价值的信息.     

A molecular mechanism of P-loop pliability of Rho-kinase investigated by molecular dynamic simulation

Rho-kinase is a leading player in the regulation of cytoskeletal events involving smooth muscle contraction and neurite growth-cone collapse and retraction, and is a promising drug target in the treatment of both vascular and neurological disorders. Recent crystal structure of Rho-kinase complexed with a small-molecule inhibitor fasudil has revealed structural details of the ATP-binding site, which represents the target site for the inhibitor, and showed that the conserved phenylalanine on the P-loop occupies the pocket, resulting in an increase of protein–ligand contacts. Thus, the P-loop pliability is considered to play an important role in inhibitor binding affinity and specificity. In this study, we carried out a molecular dynamic simulation for Rho-kinase–fasudil complexes with two different P-loop conformations, i.e., the extended and folded conformations, in order to understand the P-loop pliability and dynamics at atomic level. A PKA–fasudil complex was also used for comparison. In the MD simulation, the flip-flop movement of the P-loop conformation starting either from the extended or folded conformation was not able to be observed. However, a significant conformational change in a long loop region covering over the P-loop, and also alteration of ionic interaction-manner of fasudil with acidic residues in the ATP binding site were shown only in the Rho-kinase–fasudil complex with the extended P-loop conformation, while Rho-kinase with the folded P-loop conformation and PKA complexes did not show large fluctuations, suggesting that the Rho-kinase–fasudil complex with the extended P-loop conformation represents a meta-stable state. The information of the P-loop pliability at atomic level obtained in this study could provide valuable clues to designing potent and/or selective inhibitors for Rho-kinase. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.