MCDOCK: A Monte Carlo simulation approach to the molecular docking problem

Prediction of the binding mode of a ligand (a drug molecule) to its macromolecular receptor, or molecular docking, is an important problem in rational drug design. We have developed a new docking method in which a non-conventional Monte Carlo (MC) simulation technique is employed. A computer program, MCDOCK, was developed to carry out the molecular docking operation automatically. The current version of the MCDOCK program (version 1.0) allows for the full flexibility of ligands in the docking calculations. The scoring function used in MCDOCK is the sum of the interaction energy between the ligand and its receptor, and the conformational energy of the ligand. To validate the MCDOCK method, 19 small ligands, the binding modes of which had been determined experimentally using X-ray diffraction, were docked into their receptor binding sites. To produce statistically significant results, 20 MCDOCK runs were performed for each protein–ligand complex. It was found that a significant percentage of these MCDOCK runs converge to the experimentally observed binding mode. The root-mean-square (rms) of all non-hydrogen atoms of the ligand between the predicted and experimental binding modes ranges from 0.25 to 1.84 Å for these 19 cases. The computational time for each run on an SGI Indigo2/R10000 varies from less than 1 min to 15 min, depending upon the size and the flexibility of the ligands. Thus MCDOCK may be used to predict the precise binding mode of ligands in lead optimization and to discover novel lead compounds through structure-based database searching.     

Novel biosensor for the rapid measurement of estrogen based on a ligand-receptor interaction.

A bioaffinity sensor was developed aiming at the detection of estrogen. This biosensor system is based on the specific binding of estrogen to its receptor immobilized on a gold disk electrode. The recombinant DNA encoding human estrogen receptor ligand-binding domain was expressed in bacteria using the histidine-tag fusion system. The expression of the fusion protein was under control of a bacteriophage T7 promoter, and the protein was purified under native conditions by affinity chromatography, which is based on a specific interaction between a histidine-tag, located in the N-terminus of the protein, and the Ni(II) chelate adsorbent. The protein was immobilized on an Au-electrode with Ni(II)-mediated chemisorption using a histidine tag and thiol-modified iminodiacetic acid. Cyclic voltammetric measurements showed that the reversible electrochemical reaction of a ferrocyanide/ferricyanide redox couple was suppressed by the presence of estrogen in a concentration-dependent manner. It seems reasonable to suppose that the electrostatic property of the protein layer on the electrode surface was altered by complexation with estrogen. These data suggest that this biosensor is applicable to the evaluation binding activities of the chemicals toward the human estrogen receptor.     

An acetylcholine receptor-based biosensor for the detection of cholinergic agents

A practical, acetylcholine receptor-based prototype biosensor is reported. The biosensor utilizes a polymeric film containing the receptor and appropriate stabilizers to coat an interdigitated electrode transducer. Upon binding of specific cholinergic ligands to the immobilized receptor, changes occur in the electrical field of the electrodes proportional to the amount of cholinergic agent present. The biosensor can detect micro- to nano-gram quantities of cholinergic ligands per ml within 1–5 s. The biosensor can be recycled with acetylcholine and is stable for at least 72 h during use and over 6 months in storage at normal temperatures. The cholinergic biosensor represents the first instance of a biosensor based on a physiologically active receptor for the detection of a class of chemical compounds.     

Role of Nanomechanics in Canonical and Noncanonical Pro-angiogenic Ligand/VEGF Receptor-2 Activation

Vascular endothelial growth factor receptor-2 (VEGFR2) is an endothelial cell receptor that plays a pivotal role in physiologic and pathologic angiogenesis and is a therapeutic target for angiogenesis-dependent diseases, including cancer. By leveraging on a dedicated nanomechanical biosensor, we investigated the nanoscale mechanical phenomena intertwined with VEGFR2 surface recognition by its prototypic ligand VEGF-A and its noncanonical ligand gremlin. We found that the two ligands bind the immobilized extracellular domain of VEGFR2 (sVEGFR2) with comparable binding affinity. Nevertheless, they interact with sVEGFR2 with different binding kinetics and drive different in-plane piconewton intermolecular forces, suggesting that the binding of VEGF-A or gremlin induces different conformational changes in sVEGFR2. These behaviors can be effectively described in terms of a different "nanomechanical affinity" of the two ligands for sVEGFR2, about 16-fold higher for VEGF-A with respect to gremlin. Such nanomechanical differences affect the biological activity driven by the two angiogenic factors in endothelial cells, as evidenced by a more rapid VEGFR2 clustering and a more potent mitogenic response triggered by VEGF-A in respect to gremlin. Together, these data point to surface intermolecular interactions on cell membrane between activated receptors as a key modulator of the intracellular signaling cascade.     

Architectural and structural optimization of the protective polymer layer for enhanced targeting

Using Monte Carlo simulations we study the influence of ligand architecture (valence, branching length) and structure (polydispersity) of a flat protective polymer layer on the accessibility of its functional groups and efficiency of receptor targeting. Two types of receptor surfaces were considered: the surface homogeneously covered with receptors and the surface containing a finite number of receptor sites. We found that multivalent ligands provide a larger density of targeting groups on the periphery of the layer compared to monovalent ligands for the same overall number of targeting groups per polymer layer. Because of their cooperativity in binding, multivalent ligands were also considerably more efficient in binding to both types of receptor surfaces. With an increase of ligand valence the number of functional groups attached to receptors noticeably increases. Short-branched divalent ligands show an especially high cooperativity in binding to closely packed receptors. However, in the case of immobile receptors separated by a finite distance from each other, the average distance between the functional groups belonging to the same short divalent ligand is too small to reach different receptors simultaneously and the receptor binding is less efficient than in the monovalent ligand case. Using a bidisperse protective polymer layer formed by short nonfunctional polymers and long functionalized polymers considerably increases the fraction of functional groups on the periphery of the layer. Simulations of receptor binding confirm the high efficiency of receptor targeting by bidisperse polymer layers, which is achieved by means of larger compressibility and higher capability of the ligands to reach out compared to the corresponding monodisperse layers. The concepts of multivalent ligands and a bidisperse protective polymer layer each have their own advantages which can be combined for an enhanced targeting effect.     

Structure-based discovery of novel inhibitors of Mycobacterium tuberculosis CYP121 from Indonesian natural products

Tuberculosis (TB) continues to be a serious global health threat with the emergence of multidrug-resistant tuberculosis (MDR-TB) and extremely drug-resistant tuberculosis (XDR-TB). There is an urgent need to discover new drugs to deal with the advent of drug-resistant TB variants. This study aims to find new M. tuberculosis CYP121 inhibitors by the screening of Indonesian natural products using the principle of structure-based drug design and discovery. In this work, eight natural compounds isolated from Rhoeo spathacea and Pluchea indica were selected based on their antimycobacterial activity. Derivatives compound were virtually designed from these natural molecules to improve the interaction of ligands with CYP121. Virtual screening of ligands was carried out using AutoDock Vina followed by 50 ns molecular dynamics simulation using YASARA to study the inhibition mechanism of the ligands. Two ligands, i.e., kaempferol (KAE) and its benzyl derivative (KAE3), are identified as the best CYP121 inhibitors based on their binding affinities and adherence to the Lipinski’s rule. Results of molecular dynamics simulation indicate that KAE and KAE3 possess a unique inhibitory mechanism against CYP121 that is different from GGJ (control ligand). The control ligand alters the overall dynamics of the receptor, which is indicated by changes in residue flexibility away from CYP121 binding site. Meanwhile, the dynamic changes caused by the binding of KAE and KAE3 are isolated around the binding site of CYP121. These ligands can be developed for further potential biological activities.     

Computer‐aided design of a novel ligand for retinoic acid receptor in cancer chemotherapy

The isotypes of RAR and RXR are retinoic acid and retinoid X acid receptors, respectively, whose ligand‐binding domain contains the ligand‐dependent activation function, with distinct pharmacological targets for retinoids, involved in the treatment of various cancers and skin diseases. Due to the major challenge which cancer treatment and cure still imposes after many decades to the international scientific community, there is actually considerable interest in new ligands with increased bioactivity. We have focused on the retinoid acid receptor, which is considered an interesting target for drug design. In this work, we carried out density functional geometry optimizations and different docking procedures. We performed screening in a large database (hundreds of thousands of molecules which we optimized at the AM1 level) yielding a set of potential bioactive ligands. A new ligand was selected and optimized at the B3LYP/6‐31G* level. A flexible docking program was used to investigate the interactions between the receptor and the new ligand. The result of this work is compared with several crystallographic ligands of RAR. Our theoretically more bioactive new ligand indicates stronger and more hydrogen bonds as well as hydrophobic interactions with the receptor. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2005     

Electronic Olfactory Sensor Based on A. mellifera Odorant‐Binding Protein 14 on a Reduced Graphene Oxide Field‐Effect Transistor

An olfactory biosensor based on a reduced graphene oxide (rGO) field‐effect transistor (FET), functionalized by the odorant‐binding protein 14 (OBP14) from the honey bee (Apis mellifera) has been designed for the in situ and real‐time monitoring of a broad spectrum of odorants in aqueous solutions known to be attractants for bees. The electrical measurements of the binding of all tested odorants are shown to follow the Langmuir model for ligand–receptor interactions. The results demonstrate that OBP14 is able to bind odorants even after immobilization on rGO and can discriminate between ligands binding within a range of dissociation constants from K_d=4 μM to K_d=3.3 mM . The strongest ligands, such as homovanillic acid, eugenol, and methyl vanillate all contain a hydroxy group which is apparently important for the strong interaction with the protein.     

Improving the Sensitivity of a Quartz Crystal Microbalance for Biosensing by Using Porous Gold

 A quartz crystal microbalance can be used as a biosensor if biological receptor molecules (ligands) are attached to the crystal. To improve the sensitivity, the surface area of the gold electrodes on the crystal was increased by rendering the electrodes porous. This increased the response up to a factor of 3. A protein model system with human anti-myoglobin as ligand and sheep skeletal myoglobin at different concentrations as analyte was used. The quartz crystal was mounted in a flow-cell, where immobilisation, binding and regeneration of the surface were carried out. The kinetics of the model system was monitored under various experimental conditions. Received February 2, 2000. Revision June 30, 2000.     

Effect of cobratoxin binding on the normal mode vibration within acetylcholine binding protein

Recent crystal structures of the acetylcholine binding protein (AChBP) have revealed surprisingly small structural alterations upon ligand binding. Here we investigate the extent to which ligand binding may affect receptor dynamics. AChBP is a homologue of the extracellular component of ligand-gated ion channels (LGICs). We have previously used an elastic network normal-mode analysis to propose a gating mechanism for the LGICs and to suggest the effects of various ligands on such motions. However, the difficulties with elastic network methods lie in their inability to account for the modest effects of a small ligand or mutation on ion channel motion. Here, we report the successful application of an elastic network normal mode technique to measure the effects of large ligand binding on receptor dynamics. The present calculations demonstrate a clear alteration in the native symmetric motions of a protein due to the presence of large protein cobratoxin ligands. In particular, normal-mode analysis revealed that cobratoxin binding to this protein significantly dampened the axially symmetric motion of the AChBP that may be associated with channel gating in the full nAChR. The results suggest that alterations in receptor dynamics could be a general feature of ligand binding.     

Effect of the axial cysteine ligand on the electronic structure and reactivity of high-valent iron(IV) oxo-porphyrins (Compound I): a theoretical study

The effect of axial ligands on the reactivity of high-valent iron(IV) oxo-porphyrins (Compound I) was investigated using the B3LYP hybrid density functional method. We studied alkane hydroxylation using four models: Compound I with thiolate, imidazole, phenolate, and chloride anions as axial ligands. The first three ligands were employed as models for cysteinate, histidine, and tyrosinate, respectively. Our calculations show that anionic ligands and neutral ligands favor different electronic states for stationary points in the reaction coordinate, and the calculated energy barrier and energy of several reaction intermediates show similar values. A remarkable effect of axial ligands was found in the final product release step. Our calculations show that the thiolate ligand weakens a bond between heme and an alcohol. In contrast, the imidazole ligand significantly increases the interaction between heme and an alcohol, which causes the catalytic cycle to be less efficient.     

Influencing receptor-ligand binding mechanisms with multivalent ligand architecture

Multivalent ligands can function as inhibitors or effectors of biological processes. Potent inhibitory activity can arise from the high functional affinities of multivalent ligand-receptor interactions. Effector functions, however, are influenced not only by apparent affinities but also by alternate factors, including the ability of a ligand to cluster receptors. Little is known about the molecular features of a multivalent ligand that determine whether it will function as an inhibitor or effector. We envisioned that, by altering multivalent ligand architecture, ligands with preferences for different binding mechanisms would be generated. To this end, a series of 28 ligands possessing structural diversity was synthesized. This series provides the means to explore the effects of ligand architecture on the inhibition and clustering of a model protein, the lectin concanavalin A (Con A). The structural parameters that were varied include scaffold shape, size, valency, and density of binding elements. We found that ligands with certain architectures are effective inhibitors, but others mediate receptor clustering. Specifically, high molecular weight, polydisperse polyvalent ligands are effective inhibitors of Con A binding, whereas linear oligomeric ligands generated by the ring-opening metathesis polymerization have structural properties that favor clustering. The shape of a multivalent ligand also influences specific aspects of receptor clustering. These include the rate at which the receptor is clustered, the number of receptors in the clusters, and the average interreceptor distance. Our results indicate that the architecture of a multivalent ligand is a key parameter in determining its activity as an inhibitor or effector. Diversity-oriented syntheses of multivalent ligands coupled with effective assays that can be used to compare the contributions of different binding parameters may afford ligands that function by specific mechanisms.     

Assessment of programs for ligand binding affinity prediction

The prediction of the binding free energy between a ligand and a protein is an important component in the virtual screening and lead optimization of ligands for drug discovery. To determine the quality of current binding free energy estimation programs, we examined FlexX, X-Score, AutoDock, and BLEEP for their performance in binding free energy prediction in various situations including cocrystallized complex structures, cross docking of ligands to their non-cocrystallized receptors, docking of thermally unfolded receptor decoys to their ligands, and complex structures with "randomized" ligand decoys. In no case was there a satisfactory correlation between the experimental and estimated binding free energies over all the datasets tested. Meanwhile, a strong correlation between ligand molecular weight-binding affinity correlation and experimental predicted binding affinity correlation was found. Sometimes the programs also correctly ranked ligands' binding affinities even though native interactions between the ligands and their receptors were essentially lost because of receptor deformation or ligand randomization, and the programs could not decisively discriminate randomized ligand decoys from their native ligands; this suggested that the tested programs miss important components for the accurate capture of specific ligand binding interactions.     

Development of a unique 3D interaction model of endogenous and synthetic peripheral benzodiazepine receptor ligands

Different classes of Peripheral-type Benzodiazepine Receptor (PBR) ligands were examined and common structural elements were detected and used to develop a rational binding model based on energetically allowed ligand conformations. Two lipophilic regions and one electrostatic interaction site are essential features for high affinity ligand binding, while a further lipophilic region plays an important modulator role. A comparative molecular field analysis, performed over 130 PBR ligands by means of the GRID/GOLPE methodology, led to a PLS model with both high fitting and predictive values (r² = 0.898, Q² = 0.761). The outcome from the 3D QSAR model and the GRID interaction fields computed on the putative endogenous PBR ligands DBI (Diazepam Binding Inhibitor) and TTN (Tetracontatetraneuropeptide) was used to identify the amino acids most probably involved in PBR binding. Three amino acids, bearing lipophilic side chains, were detected in DBI (Phe49, Leu47 and Met46) and in TTN (Phe33, Leu31 and Met30) as likely residues underlying receptor binding. Moreover, a qualitative comparison of the molecular electrostatic potentials of DBI, TTN and selected synthetic ligands indicated also similar electronic properties. Convergent results from the modeling studies of synthetic and endogenous ligands suggest a common binding mode to PBRs. This may help the rational design of new high affinity PBR ligands.     

What Can We Learn from Molecular Recognition in Protein–Ligand Complexes for the Design of New Drugs?

The understanding of noncovalent interactions in protein–ligand complexes is essential in modern biochemistry and should contribute toward the discovery of new drugs. In the present review, we summarize recent work aimed at a better understanding of the physical nature of molecular recognition in protein–ligand complexes and also at the development and application of new computational tools that exploit our current knowledge on structural and energetic aspects of protein–ligand interactions in the design of novel ligands. These approaches are based on the exponentially growing amount of information about the geometry of protein structures and the properties of small organic molecules exposed to a structured molecular environment. The various contributions that determine the binding affinity of ligands toward a particular receptor are discussed. Their putative binding site conformations are analyzed, and some predictions are attempted. The similarity of ligands is examined with respect to their recognition properties. This information is used to understand and propose binding modes. In addition, an overview of the existing methods for the design and selection of novel protein ligands is given.     

基于美洲蜚蠊下颚须的整合受体生物传感器测定抗坏血酸

报道了以美洲蜚蠊的下颚须作为分子识别和转换元件研制的一种新型生物组织 传感器,发现该传感器对抗坏血酸（AA）有选择性的响应,响应脉冲的发放频率与 AA的浓度对数成正比,在5 * 10~(-5)至5 * 10~(-2)mol/L范围内呈线性关系,检 测下限低于10~(-6)mol/L AA。该传感器响应快,并具有较好的重现性。     

Structural evaluation of an alternative Protein A biomimetic ligand for antibody purification

Affinity chromatography is one of the most common techniques employed at the industrial-scale for antibody purification. In particular, the purification of human immunoglobulin G (hIgG) has gained relevance with the immobilization of its natural binding counterpart—Staphylococcus aureus Protein A (SpA) or with the recent development of biomimetic affinity ligands, namely triazine-based ligands. These ligands have been developed in order to overcome economic and leaching issues associated to SpA. The most recent triazine-based ligand—TPN-BM, came up as an analogue of 2-(3-amino-phenol)-6-(4-amino-1-naphthol)-4-chloro-sym-triazine ligand also known as ligand 22/8 with improved physico-chemical properties and a greener synthetic route. This work intends to evaluate the potential of TPN-BM as an alternative affinity ligand towards antibody recognition and binding, namely IgG, at an atomic level, since it has already been tested, after immobilization onto chitosan-based monoliths and demonstrated interesting affinity behaviour for this purpose. Herein, combining automated molecular docking and molecular dynamics simulations it was predicted that TPN-BM has high propensity to bind IgG through the same binding site found in the crystallographic structure of SpA_IgG complex, as well as theoretically predicted for ligand 22/8_IgG complex. Furthermore, it was found that TPN-BM established preferential interactions with aromatic residues at the Fab domain (Trp 50, Tyr 53, Tyr 98 and Trp 100), while in the Fc domain the main interactions are based on hydrogen bonds with pH sensitive residues at operational regime for binding and elution like histidines (His 460, His 464, His 466). Moreover, the pH dependence of TPN-BM_IgG complex formation was more evident for the Fc domain, where at pH 3 the protonation state and consequently the charge alteration of histidine residues located at the IgG binding site induced ligand detachment which explains the optimal elution condition at this pH observed experimentally.     

An advanced biosensor for the prediction of estrogenic effects of endocrine-disrupting chemicals on the estrogen receptor alpha

A label-free and time-resolved biosensor based on reflectometric interference spectroscopy (RIfS) has been developed to evaluate the agonistic or antagonistic effects of potential ligands with unknown behavior. The biosensor utilizes the specific interaction between the estrogen receptor alpha (ER alpha) and short specific peptides. The unique feature of these peptides allows the investigation of the behavior of ligands and the discrimination between the agonistic and antagonistic effects caused by conformational changes of the receptor. Thus, this developed biosensor allows not only the differentiation between ligands and nonligands of a receptor, but also the potential of these ligands to influence conformational changes in the receptor, leading to activation or inhibition of the receptor-dependent pathways. Owing to the robustness of the direct optical detection principle used, the biosensor is applicable to complex biological matrices, even crude cell extracts. Moreover, the reliability of the biosensor, including regeneration steps when performing subsequent measurements, has been verified.