Docking Processes Research Articles

The Structure Based Combination Drug Design and RAGE for Diabetic Retinopathy

BackgroundDiabetic Retinopathy (DR) is the leading cause of blindness, stimulating the rise in 20,000 cases in the United States per year in patients with Type II Diabetes. Hyperglycemia is a major risk factor of Type II diabetes leads to the formation of plasma proteins known as Advanced Glycation End Products (AGEs) which bind to the receptor of the Advanced Glycation End Products (RAGEs) lead to DR.PurposeThe purpose of the study is to analyze drug targets (single drugs and combination of drugs) that are currently marketed for heart disease, diabetes, and hypertension and how they interact with RAGE to inhibit the ligand‐receptor interaction.MethodsThis study used molecular docking processes to analyze single and combination drug interactions with RAGE. By using AutoDock Tools and AutoDock Vina, docking simulations can identify the drug targets that can potentially decrease the severity of Diabetic Retinopathy. Molecular drug targets selected for this study were chosen from therapeutics that currently exists in the market. Selection of drugs focused on those which were commonly used for treating inflammatory conditions, such as cardiovascular disease, hyperglycemia, and cholesterol lowering drugs. The Receptor Antagonist Peptide (RAP) is a novel ligand inhibitor that has been shown to inhibit binding interaction between RAGE and many of its ligands for instance AGEs. RAP will represent the control ligand that will be able to bind to the RAGE receptor, and its binding affinity and visual orientation will be observed using a molecular docking software. The study included six (6) single drugs such as Atorvastatin, Enalapril, Fenofibrate, Lisinopril, Lovastatin and Metformin were selected to interact with the RAGE receptor. The Centers for Medicaid and Medicare Services (CMS) Prescription Drug Data Part D was used to determine which drug combinations currently exist within the market. Eight (8) drug combinations such as Canagliflozin + Metformin; Ezetimibe + Atorvastatin; Ezetimibe+ Simvastatin; Hydrochlorothiazide+ Losartan; Lisinopril +Hydrochlorothiazide; Lovastatin+ Niacin; Metformin+ Repaglinide; Telmisartan +Amlodipine were selected to test interaction with the RAGE receptor.. The affinity results were analyzed using the Lamarck's Algorithm. Based on these results the drug target and receptor files were input into AutoDock Tools to create a 3D model of the binding between the drug and receptor. AutoDock Tools was used to label the residue sequences of the binding area to create a visual orientation of the drug and receptor.ResultsBased on a single drug interaction with RAGE, cholesterol lowering drugs such as Atorvastatin and Fenofibrate were able to bind to RAGE with a high hydrophobicity and great negative affinity. In combination of Hypertensive drugs such as Telmisartan and Amlodipine could bind to the RAGE receptor, and had a high hydrophobicity and high negative affinity. These results suggest combination drugs may inhibit RAGE and may reduce the risk of diabetic retinopathy.ConclusionOverall, combination of hypertensive drugs and cholesterol lowering drugs alone may decrease the progression of DR in type II diabetes.Support or Funding InformationNoneThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

European Proximity Operations Simulator 2.0 (EPOS) - A Robotic-Based Rendezvous and Docking Simulator

The European Proximity Operations Simulator (EPOS) 2.0 located at the German Space Operations Center (GSOC) in Oberpfaffenhofen, Germany, is a robotic based test facility of the German Aerospace Center (DLR) used for simulation of rendezvous and docking (RvD) processes. Hardware such as rendezvous sensors (cameras, laser scanners) or docking tools, as well as software (e.g. for navigation and control) can be tested and verified. The facility consists of two robotic manipulators with each six degrees of freedom, a linear slide of 25m length on which one robot can be moved in the laboratory, and a computer-based monitoring and control system. EPOS 2.0 allows for real-time simulations of the rendezvous and docking process during the most critical phase (separation from 25m to 0m) of proximity and docking/berthing operations.

Breakwater control system and structural analysis: physical and numerical modelling (Port of Funchal, Madeira Island, Portugal)

The objective of this work was to analyze the behavior of the Funchal Port (Madeira Island, Portugal) breakwater using physical and numerical models in order to analyze the evolution of the underwater layers, combining information between the two models as well as simulating storm conditions and atypical docking/undocking impacts of cruise ships. Regarding the physical model using a wave generation system, the data collected and the test conditions were addressed considering different scenarios (return periods of 20, 50 and 100 years). It was found that for return periods of 20 years, the behavior of the infrastructure as a whole was stable. For return periods of 50 years, wave overtopping became more frequent and intense, and began to impact the superstructure. For the 100-year return period the behavior of the infrastructure was found to be unstable, with persistent wave overtopping. The morphology of the submerged layers also changed. The main objectives of the numerical model approach were to estimate the natural frequencies of vibration (when materials suffer a considerable modification, a change in their frequencies of vibration can be detected), and to calculate the expected displacements corresponding to cruise ship docking processes (agitation in a protected area) and the associated maximum maritime (agitation in an exposed area) forces. The maximum displacements obtained for the two cases were about 0.03 m, which is a high value. Considering the results obtained from this work, the Port authorities decided to maintain the monitoring program and perform an underwater video inspection.

Characterization of the copper-containing amine oxidase from Trifolium pratense seedlings

We studied the kinetic characterizations of the Trifolium pratense seedlings copper-containing amine oxidase (TPAO) by using various amine-containing substrates. The catalyzing rate for all of amine-containing substrates can be ordered as diamines > polyamines > aromatic monoamines, and it shows an apparent trend in each category of substrates such as the longer the carbon chain, the lower the Vmax is, so does the Vmax/Km values but is opposite for the Km value of TPAO. The distinct differences between the kinetic parameters for different amine-containing substrates indicated that the rate-determining step of the catalytic reaction strongly depends on the substrate’s chemical structure. It is concluded that both pH and ionic strength can affect the catalytic activity of TPAO via influencing the coulomb interaction-mediated enzyme–substrate docking processes, which can be attributed to the potential of charged groups from both substrates and the activity sites of TPAO by the regulation of ionic strength.

Comparative sequence- and structure-inspired drug design for PilF protein of Neisseria meningitidis.

Serogroup A of Neisseria meningitidis is the organism responsible for causing epidemic diseases in developing countries by a pilus-mediated adhesion to human brain endothelial cells. Type IV pilus assembly protein (PilF) associated with bacterial adhesion, aggregation, invasion, host cell signaling, surface motility, and natural transformation can be considered as a candidate for effective anti-meningococcal drug development. Since the crystal structure of PilF was not available, in the present study, it was modeled after the Z2491 strain (CAM09255.1) using crystal structure of chain A of Vibrio cholerae putative Ntpase EpsE (Protein Data Bank (PDB) ID: 1P9R) and then we based this analysis on sequence comparisons and structural similarity using in silico methods and docking processes, to design a suitable inhibitor molecule. The ligand 3-{(4S)-5-{[(1R)-1-cyclohexylethyl]amino}-4-[(5S)-5-(prop-2-en-1-yl) cyclopent-1-en-1-yl]-1,4-dihydro-7H-pyrrolo[2,3-d] pyrimidin-7-yl}-1,2-dideoxy-b-L-erythro-hex-1-en-3-ulofuranosyl binds to the protein with a binding energy of −8.10 kcal and showed a drug likeness of 0.952 with no predicted health hazard. It can be utilized as a potent inhibitor of N. meningitidis pilus-mediated adhesion to human brain endothelial cells preventing meningeal colonization.

Analysis of Amino Acid Properties in Interaction Surfaces of Decoys Generated by Re-Docking Scheme

Rigid-body docking processes generate many protein complexes (decoys) for searching near-native decoys (NN-decoys) in post-docking processes with analyzing various pairs of surfaces on two input proteins, using properties of electrostatics and desolvations between two molecules or proteins. Many powerful docking computer softwares were developed and were used for resolving various categories of problems, for example, analysis of protein interaction networks or drug design. Rigid-body docking process is popular and useful in such works. However, there are some unfortunate cases, which could not obtain NN-decoys. Then, we developed Re-docking scheme using interaction fingerprints (IFPs). Re-docking scheme is the process of iterating rigid-body docking for generating more NN-decoys. After initial-docking, we classify decoys into several interaction surfaces. Thereafter, other docking processes are performed with more fine searching limited in every interaction surfaces classified. We could obtain NN-decoys even if no NN-decoys in initial-docking process [Uchikoga et. al. (2013) PLOS ONE 8:e69365]. Then, we approach a problem of prediction of protein-protein interactions by using IFPs, which gives us properties of physico-chemical interactions because IFPs are composed of interacting amino acid pairs [Uchikoga & Hirokawa (2010) BMC Bioinform. 11:236]. By using IFPs, we can obtain these properties easily and trace interaction surfaces in docking processes. In this work, docking search spaces become to be seen by using amino acid properties involved in molecular surfaces of many decoys, generating by Re-docking scheme. Then, we would like to discuss about understanding interaction mechanisms.

Revealing Non‐covalent Interactions in Molecular Crystals through Their Experimental Electron Densities

Non-covalent interactions (NCI) define the rules underlying crystallisation, self-assembly and drug-receptor docking processes. A novel NCI descriptor, based on the reduced electron density gradient (RDG), that enables easy visualisation of the zones of the electron density (ED) involved in either the supposedly attractive (dispersive, hydrogen bonding) or allegedly repulsive (steric) intermolecular interactions, was recently developed by Johnson et al. Here, it is applied for the first time to EDs derived from single-crystal X-ray diffraction data. A computer code handling both experimental and ab initio EDs in the RDG-NCI perspective was purposely written. Three cases spanning a wide range of NCI classes were analysed: 1) benzene, as the prototype of stacking and weak CH···π interactions; 2) austdiol, a heavily functionalised fungal metabolite with a complex hydrogen-bonding network; 3) two polymorphs of the heteroatom-rich anti-ulcer drug famotidine, with van der Waals and hydrogen-bond contacts between N- and S-containing groups. Even when applied to experimental EDs, the RDG index is a valuable NCI descriptor that can highlight their different nature and strength and provide results of comparable quality to ab initio approaches. Combining the RDG-NCI study with Bader's ED approach was a key step forward, as the RDG index can depict inherently delocalised interactions in terms of extended and flat RDG isosurfaces, in contrast to the bond path analysis, which is often bounded to a too localised and possibly discontinuous (yes/no) description. Conversely, the topological tool can provide quantitative insight into the simple, qualitative NCI picture offered by the RDG index. Hopefully, this study may pave the way to a deeper analysis of weak interactions in proteins using structural and ED information from experiment.

Molecular docking study on the α3β2 neuronal nicotinic acetylcholine receptor complexed with α-Conotoxin GIC

Nicotinic acetylcholine receptors (nAChRs) are a diverse family of homo- or heteropentameric ligand-gated ion channels. Understanding the physiological role of each nAChR subtype and the key residues responsible for normal and pathological states is important. α-Conotoxin neuropeptides are highly selective probes capable of discriminating different subtypes of nAChRs. In this study, we performed homology modeling to generate the neuronal α3, β2 and β4 subunits using the x-ray structure of the α1 subunit as a template. The structures of the extracellular domains containing ligand binding sites in the α3β2 and α3β4 nAChR subtypes were constructed using MD simulations and ligand docking processes in their free and ligand-bound states using α-conotoxin GIC, which exhibited the highest α3β2 vs. α3β4 discrimination ratio. The results provide a reasonable structural basis for such a discriminatory ability, supporting the idea that the present strategy can be used for future investigations on nAChR-ligand complexes.

TBC1D1 and TBC1D4 (AS160) RabGAP Domains are Characterized as Monomers in Solution by Analytical Ultracentrifugation

Upon food intake, the glucose level in circulating blood increases by trans-cellular transport of glucose from the intestinal lumen into the blood. However, the insulin secreted from the pancreatic β-cells directs the individual cells to absorb glucose for energy source to result in the maintenance of the normal blood glucose level. Elevated insulin in blood is sensed by the insulin receptor of the individual cells, and the propagation of the insulin signaling pathway drives the glucose uptake. The final cellular glucose uptake from the blood into the cells is performed by trafficking of vesicles containing the glucose transporter protein (e.g. GLUT4) from the intracellular pools to the cell surfaces. As in most of the vesicle budding, uncoating, docking and fusion processes, this translocation of GLUT4 vesicles are tightly regulated by Rab·GTPases (Rabs) and Rab·GTPase-activating proteins (RabGAPs). Rabs are GTPases with intrinsic activity, however RabGAPs enhance the low hydrolysis rate of Rab-bound GTP. The two major cell types that absorb glucose, which are the adipocytes and the skeletal myocytes, respectively encode TBC1D4 (also known as AS160) and TBC1D1 which are integral RabGAPs in the insulin-mediated GLUT4 vesicle trafficking event. TBC1D4 and TBC1D1 are multi-domain RabGAP proteins which consist of two putative N-termini phosphotyrosine binding (PTB) domains and a C-terminal catalytic RabGAP domain that are linked by regions containing several Akt(PKB)-mediating serine and threonine phosphorylation sites. In the basal state, TBC1D4 (or TBC1D1)-catalyzed hydrolysis of Rab-bound GTP (active) to GDP (inactive) leaves GLUT4 vesicles sequestered in an intracellular compartment. However, insulin signal results in the activation of Akt (PKB) and the consequent phosphorylation of TBC1D4 (or TBC1D1). The Akt phosphorylation of TBC1D4 (or TBC1D1) deactivates the Rab·GTP hydrolysis activity by an unknown mechanism, and concomitantly results in the increase of Rab·GTP concentrations. The active Rab·GTP in turn promotes GLUT4 vesicle translocation to the cell membrane and finally stimulates the glucose uptake from blood into the cells. X-ray crystal structures of the human TBC1D1 RabGAP domain and TBC1D4 RabGAP domain (PDB code: 3QYE & 3QYB, respectively) have been recently reported. Since, TBC1D1 and TBC1D4 share 76% sequence identity over their RabGAP domains, the secondary structure elements mostly overlapped in the two structures which were predominately α-helical with no β-sheet elements. The 17 αhelices in TBC1D1 RabGAP domain were sequentially named from α1 to α16 ending with α16'. However, the oligomeric association states of the two TBC1D1 and TBC1D4 RabGAP domains in the crystal asymmetric unit were different resulting from the presence (or absence) of a single helix α16'. TBC1D1 RabGAP domain containing the C-terminal α16' forms an asymmetric dimer with α16' of one molecule interacting with α16 of the other molecule (Figure 1). However in TBC1D4 RabGAP domain, α16' in the protein construct had to be deleted for crystallization and enhanced X-ray diffraction (Figure 2), and this results in TBC1D4 RabGAP domain being a monomer in the crystal (Figure 1). Certain is the fact that α16' mediates the resulting differences in the oligomeric states, the biological relevance of this asymmetric dimer cannot be concluded only from the association modes within the crystal. Since only two αhelices (α16' and α16) of TBC1D1 mediate the dimerization, it was suspected that the dimer is a result of crystal packing artifacts, and is non-relevant outside the context of the crystal. However in this study, we have compared the oligomeric association states of TBC1D1 and TBC1D4 RabGAP domains (Figure 2) using analytical ultracentrifugation (AUC) techniques to address the state of interaction in solution. Sedimentation equilibrium experiments were performed in a Beckman-Coulter ProteomeLab XL-A analytical ultracentrifuge using 12 mm carbon-filled epoxy double-sector centerpieces and quartz windows at optical density of 0.25 at 280 nm (6.67 μM and 6.69 μM for TBC1D1 and TBC1D4 RabGAP domains, respectively). The measurements were performed at two different rotor speeds of 13,000 and 18,000 RPMs at 20 C. The proteins were in 50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, and 1.5 mM TCEP. The sedimentation equilibrium data were analyzed using MLAB software (Civilized Software, Inc., Silver Spring, MD) using the following equation: C(r) = cb*exp (Ap* Mp* (r − rb) + e (1)

Rab and actomyosin-dependent fission of transport vesicles at the Golgi complex

Trafficking between membrane compartments is a characteristic of eukaryotic cells and relies on transport carriers that bud and fission from a donor membrane, before being transported and fusing with the correct acceptor compartment. Rab GTPases ensure specificity and directionality of trafficking steps by regulating the movement of transport carriers along cytoskeletal tracks, and the recruitment of tethering factors required for the docking and fusion processes. Here we show that Rab6, a Golgi-associated Rab, forms a complex with myosin II, contributes to its localization at the Golgi complex and, unexpectedly, controls the fission of Rab6 vesicles. Inhibition of either Rab6 or myosin II function impairs both the fission of Rab6 transport carriers from Golgi membranes and the trafficking of anterograde and retrograde cargo from the Golgi. These effects are consistent with myosin II being an effector of Rab6 in these processes. Our results provide evidence that the actomyosin system is required in vesicle biogenesis at the Golgi, and uncover a function for Rab GTPases in vesicle fission.

Fluorescent Bioprobes: Structural Matching in the Docking Processes of Aggregation‐Induced Emission Fluorogens on DNA Surfaces

Whereas most conventional DNA probes are flat disklike aromatic molecules, we explored the possibility of developing quadruplex sensors with nonplanar conformations, in particular, the propeller-shaped tetraphenylethene (TPE) salts with aggregation-induced emission (AIE) characteristics. 1,1,2,2-Tetrakis[4-(2-triethylammonioethoxy)phenyl]ethene tetrabromide (TPE-1) was found to show a specific affinity to a particular quadruplex structure formed by a human telomeric DNA strand in the presence of K(+) ions, as indicated by the enhanced and bathochromically shifted emission of the AIE fluorogen. Steady-state and time-resolved spectral analyses revealed that the specific binding stems from a structural matching between the AIE fluorogen and the DNA strand in the folding process. Computational modeling suggests that the AIE molecule docks on the grooves of the quadruplex surface with the aid of electrostatic attraction. The binding preference of TPE-1 enables it to serve as a bioprobe for direct monitoring of cation-driven conformational transitions between the quadruplexes of various conformations, a job unachievable by the traditional G-quadruplex biosensors. Methyl thiazolyl tetrazolium (MTT) assays reveal that TPE-1 is cytocompatible, posing no toxicity to living cells.

Virtual Screening for Experimentalists _ a Xgrid-Powered Web Server

Virtual-screening has emerged as an attractive strategy for discovering inhibitors of biological systems, especially for systems in which appropriate high-throughput assays are absent. Despite the potential benefits, challenges such as insufficient computational resources and lack of experiences often present a barrier that consequently limit a wider use of the technology among experimentalists. A virtual-screen web server aimed at lowering this barrier and providing a practical tool to the general scientific community has been developed (http://omg.phy.umassd.edu/xvHTS/). The server offers “experience” and resources by incorporating a validated screening protocol, and utilizing distributed computing resources, a concept that mirrors the Folding@Home project. The user-friendly interface of the server provides graphical supports that enables novice users to start a virtual screening project by simply providing a PDB code, and optionally customize by a few mouse-clicks. The back-end is driven by xgrid technology, a built-in capability in the Macintosh operating platform that efficiently utilizes idle computing resources over the grid-network. When in execution, parallel docking processes are dispatched onto the grid, the results are analyzed, and returned to the users in an easy-to-interpret fashion. This xgrid-powered virtual-screening web server lays out a foundation for large-scale screening with great growth potential. The “embarrassingly parallel” nature of virtual-screening allows virtually unlimited scalability as more idle CPUs become available over the grid-network. Joining the grid is trivial, and does not require any installation. This website is beneficial to the general scientific community, and thus deserves exposures to both potential users and donors. A successful application of the screening protocol is presented in the discovery of novel inhibitors of the Epidermal Growth Factor Receptors (EGFR). Without the availability of high-throughput assays, two anti-EGFR inhibitors are discovered by virtual-screening a compound library, and confirmed by subsequent low-throughput biochemical assays.

DOC2B: A Novel Syntaxin-4 Binding Protein Mediating Insulin-Regulated GLUT4 Vesicle Fusion in Adipocytes

OBJECTIVE— Insulin stimulates glucose uptake in skeletal muscle and adipose tissues primarily by stimulating the translocation of vesicles containing a facilitative glucose transporter, GLUT4, from intracellular compartments to the plasma membrane. The formation of stable soluble N-ethyl-maleimide–sensitive fusion protein [NSF] attachment protein receptor (SNARE) complexes between vesicle-associated membrane protein-2 (VAMP-2) and syntaxin-4 initiates GLUT4 vesicle docking and fusion processes. Additional factors such as munc18c and tomosyn were reported to be negative regulators of the SNARE complex assembly involved in GLUT4 vesicle fusion. However, despite numerous investigations, the positive regulators have not been adequately clarified.RESEARCH DESIGN AND METHODS— We determined the intracellular localization of DOC2b by confocal immunoflorescent microscopy in 3T3-L1 adipocytes. Interaction between DOC2b and syntaxin-4 was assessed by the yeast two-hybrid screening system, immunoprecipitation, and in vitro glutathione S-transferase (GST) pull-down experiments. Cell surface externalization of GLUT4 and glucose uptake were measured in the cells expressing DOC2b constructs or silencing DOC2b.RESULTS— Herein, we show that DOC2b, a SNARE-related protein containing double C2 domains but lacking a transmembrane region, is translocated to the plasma membrane upon insulin stimulation and directly associates with syntaxin-4 in an intracellular Ca2+-dependent manner. Furthermore, this process is essential for triggering GLUT4 vesicle fusion. Expression of DOC2b in cultured adipocytes enhanced, while expression of the Ca2+-interacting domain mutant DCO2b or knockdown of DOC2b inhibited, insulin-stimulated glucose uptake.CONCLUSIONS— These findings indicate that DOC2b is a positive SNARE regulator for GLUT4 vesicle fusion and mediates insulin-stimulated glucose transport in adipocytes.

Effect of purines on calcium-independent acetylcholine release at the mouse neuromuscular junction

At the mouse neuromuscular junction, activation of adenosine A1 and P2Y receptors inhibits acetylcholine release by an effect on voltage dependent calcium channels related to spontaneous and evoked secretion. However, an effect of purines upon the neurotransmitter-releasing machinery downstream of Ca2+ influx cannot be ruled out. An excellent tool to study neurotransmitter exocytosis in a Ca2+-independent step is the hypertonic response. Intracellular recordings were performed on diaphragm fibers of CF1 mice to determine the action of the specific adenosine A1 receptor agonist 2-chloro-N6-cyclopentyl-adenosine (CCPA) and the P2Y12-13 agonist 2-methylthio-adenosine 5′-diphosphate (2-MeSADP) on the hypertonic response. Both purines significantly decreased such response (peak and area under the curve), and their effect was prevented by specific antagonists of A1 and P2Y12-13 receptors, 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) and N-[2-(methylthioethyl)]-2-[3,3,3-trifluoropropyl]thio-5′-adenylic acid, monoanhydride with dichloromethylenebiphosphonic acid, tetrasodium salt (AR-C69931MX), respectively. Moreover, incubation of preparations only with the antagonists induced a higher response compared with controls, suggesting that endogenous ATP/ADP and adenosine are able to modulate the hypertonic response by activating their specific receptors. To search for the intracellular pathways involved in this effect, we studied the action of CCPA and 2-MeSADP in hypertonicity in the presence of inhibitors of several pathways. We found that the effect of CPPA was prevented by the calmodulin antagonist N-(6-aminohexil)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) while that of 2-MeSADP was occluded by the protein kinase C antagonist chelerythrine and W-7. On the other hand, the inhibitors of protein kinase A (N-(2[pbromocinnamylamino]-ethyl)-5-isoquinolinesulfonamide, H-89) and phosphoinositide-3 kinase (PI3K) (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one hydrochloride, LY-294002) did not modify the modulatory action in hypertonicity of both purines. Our results provide evidence that activation of A1 and P2Y12-13 receptors by CCPA and 2-MeSADP inhibits ACh release from mammalian motor nerve terminals through an effect on a Ca2+-independent step in the cascade of the exocytotic process. Since presynaptic calcium channels are intimately associated with components of the synaptic vesicle docking and fusion processes, further experiments could clarify if the actions of purines on calcium channels and on secretory machinery are related.

Structural Biology, Protein Conformations and Drug Designing

Structure based drug designing is now a popular technique used for increasing the speed of drug designing process. This was made possible by the availability of many protein structures which helped in developing tools to understand the structure function relationships, automated docking and virtual screening. Knowledge of structure based functional properties of a drug target is very essential for a successful in silico designing of drugs. However, some problems associated with the structure determination process and lack of knowledge of conformational freedom associated with available protein structures are the hurdles involved in structure based drug designing. Docking and virtual screening processes depend on the active site structure of the receptor molecule and subtle differences in the conformations of these molecules due to flexibility pose a serious threat to the drug designing process. In this review problems associated with the conformations of proteins and homology models was reviewed.

RNA folding and the origins of catalytic activity in the hairpin ribozyme

The nucleolytic ribozymes catalyse site-specific phosphodiester cleavage and ligation transesterification reactions in RNA. The hairpin ribozyme folds to generate an intimate loop–loop interaction to create the local environment in which catalysis can proceed. We have studied the ion-induced folding using single-molecule FRET experiments, showing that the four-way helical junction accelerates the folding 500-fold by introducing a discrete intermediate that juxtaposes the loops. Using FRET we can observe individual hairpin ribozyme molecules as they undergo multiple cycles of cleavage and ligation, and measure the rates of the internal reactions, free of uncertainties in the contributions of docking and substrate dissociation processes. On average, the cleaved ribozyme undergoes several docking–undocking events before a ligation reaction occurs. On the basis of these experiments, we have explored the role of the nucleobases G 8 and A 38 in the catalysis. Both cleavage and ligation reactions are pH dependent, corresponding to the titration of a group with p K a = 6.2. We have used a novel ribonucleoside in which these bases are replaced by imidazole to investigate the role of acid–base catalysis in this ribozyme. We observe significant rates of cleavage and ligation, and a bell-shaped pH dependence for both.

Characterization of the Substrate Specificity of PhlD, a Type III Polyketide Synthase from Pseudomonas fluorescens

Characterization of the Substrate Specificity of PhlD, a Type III Polyketide Synthase from Pseudomonas fluorescens

Receptor Flexibility in de Novo Ligand Design and Docking

One of the major problems in computational drug design is incorporation of the intrinsic flexibility of protein binding sites. This is particularly crucial in ligand binding events, when induced fit can lead to protein structure rearrangements. As a consequence of the huge conformational space available to protein structures, receptor flexibility is rarely considered in ligand design procedures. In this work, we present an algorithm for integrating protein binding-site flexibility into de novo ligand design and docking processes. The approach allows dynamic rearrangement of amino acid side chains during the docking and design simulations. The impact of protein conformational flexibility is investigated in the docking of highly active inhibitors in the binding sites of acetylcholinesterase and human collagenase (matrix metalloproteinase-1) and in the design of ligands in the S1' pocket of MMP-1. The results of corresponding simulations for both rigid and flexible binding sites are compared in order to gauge the influence of receptor flexibility in drug discovery protocols.

CAMM Techniques for the Prediction of the Mechanical Properties of Tendons and Ligaments Nanostructures

Theoretical prediction of the mechanical properties of soft tissues usually relies on a top-down approach; that is analysis is gradually refined to observe smaller structures and properties until technical limits are reached. Computer-Assisted Molecular Modeling (CAMM) allows for the reversal of this approach and the performance of bottom-up modeling instead. The wealth of available sequences and structures provides an enormous database for computational efforts to predict structures, simulate docking and folding processes, simulate molecular interactions, and understand them in quantitative energetic terms. Tendons and ligaments can be considered an ideal arena due to their well defined and highly organized architecture which involves not only the main structural constituent, the collagen molecule, but also other important molecular “actors” such as proteoglycans and glycosaminoglycans. In this ideal arena each structure is well organized and recognizable, and using the molecular modeling tool it is possible to evaluate their mutual interactions and to characterize their mechanical function. Knowledge of these relationships can be useful in understanding connective tissue performance as a result of the cooperation and mutual interaction between different biological structures at the nanoscale.

Classical force field parameters for the heme prosthetic group of cytochrome c.

Accurate force fields are essential for describing biological systems in a molecular dynamics simulation. To analyze the docking of the small redox protein cytochrome c (cyt c) requires simulation parameters for the heme in both the reduced and oxidized states. This work presents parameters for the partial charges and geometries for the heme in both redox states with ligands appropriate to cyt c. The parameters are based on both protein X-ray structures and ab initio density functional theory (DFT) geometry optimizations at the B3LYP/6-31G* level. The simulations with the new parameter set reproduce the geometries of the X-ray structures and the interaction energies between water and heme prosthetic group obtained from B3LYP/6-31G* calculations. The parameter set developed here will provide new insights into docking processes of heme containing redox proteins.