Common Docking Research Articles

Amplification and cloning of arabidopsis 6xhis-tagged mpk6 fusion encoded gene to characterize biochemical mitogen-activated protein kinase in disease resistance role against Fusarium graminearum

The Mitogen-Activated Protein Kinase (MPK) cascade plays an important role in the intracellular signaling transduction pathway leading to resistance against phytopathogens produced by Fusarium graminearum. In the cascade, there are three prominent kinase protein groups involved, an MPK kinase kinase (MPKKK), MPK kinase (MPKK), and an MPK. Recognitions of pathogen-derived molecules in plants trigger rapid activation of some MPKs including MPK6 which are found in a wide variety of plant species, including in Arabidopsis thaliana. The structure of MPK6 contains kinase domain and common docking (CD) domain. CD domain is phosphorylated by interact with MPKK. Moreover, the MPKK which binds to MPK6 and its phosphorylation mechanism are still unknown, so as initial study is needed to investigate biochemical characterization by prepare MPK6 protein. In this research, mpk 6 was amplified by using a pair primer and subsequently was ligated into pET160/GW/D-TOPO vector which contained with sequence encoded 6xHistidine tag protein for protein purification assay.

Tail Docking of Piglets 2: Effects of Meloxicam on the Stress Response to Tail Docking.

Simple SummaryTail docking is a common industry practice in pork production to reduce tail biting in groups of pigs. Tail biting behaviour involves destructive chewing of the tails of group-mates, which can both compromise pig welfare and cause economic losses. The etiology of tail biting remains poorly understood and potential predisposing factors include crowding, poor ventilation, nutrition and health. This experiment examined the stress response of piglets to two common tail docking procedures: docking with either clippers or cauterisation, surgical castration and a sham handling treatment (handling alone). In comparison to the sham handling treatment, the two tail docking treatments increased the duration of vocalisations and number of escape attempts during treatment, the cortisol response at 15 min post-treatment and the duration of standing with head lowered in the first 60 min post-treatment. However, both these behavioural responses during treatment and the cortisol response at 15 min post-treatment to clippers or cauterisation were lower than that of castration. Piglets in the cauterisation treatment group also had a lower cortisol response at 30 min post-treatment than those in the clipper treatment group. The cortisol and behavioural responses in the tail docking and castration treatments had diminished by 23–24 h post-treatment.This experiment compared the stress responses of piglets to tail docking. Two hundred and eighty-eight piglets were allocated to the following treatments at 2 d post-farrowing: (1) sham handling treatment; (2) surgical castration; (3) tail docking using clippers; (4) tail docking using a cauterising iron. Blood samples were collected at 15 min, 30 min and 24 h post-treatment and analysed for total plasma cortisol. Behaviours indicative of pain, such as escape attempts, vocalisations and standing with head lowered were measured. Cortisol concentrations at 15 min post-treatment were higher (p < 0.001) in the tail docking and castration treatment groups than the sham handling treatment group, but at 30 min post-treatment, only the clipper and castration treatment groups had higher (p < 0.001) cortisol concentrations than the sham handling treatment. Duration of vocalisations and escape attempts were greater (p < 0.0001) during the castration treatment than the sham and tail docking treatments, but these behaviours occurred less (p < 0.05) in tail-docked piglets than those that were castrated. Piglets undergoing the tail-docked treatments and the castration treatment exhibited more behaviours indicative of pain, such as standing longer (p < 0.05) with the head lowered in the 60 min after treatment, than those in the sham handling treatment group. There were no treatment effects on cortisol concentrations and behaviour at 23–24 h post-treatment. The physiological results at 30 min post-treatment indicate that tail docking with cauterisation may be less aversive than tail docking with clippers.

Evaluation of Potential Human Sodium Taurocholate Co‐transporting Polypeptide Inhibitors

ObjectiveChronic hepatitis B virus (HBV) infection is responsible for 50% of the cases of hepatocellular carcinoma. As currently available treatment is not able to eradicate the virus, new therapeutic approaches are highly needed. Sodium taurocholate co‐transporting polypeptide (NTCP), a bile acid transporter at the sinusoidal side of hepatocytes, has been identified as receptor for HBV entry into the cell. Since HBV shares a common docking site with NTCP substrates, inhibition of NTCP‐mediated transport is an attractive strategy to inhibit HBV entry and has already been proven successful (e.g. Myrcludex). The aim of this research was to identify novel NTCP inhibitors in vitro and develop a chemometric model that allows virtual screening.MethodsWild‐type and NTCP‐transfected Chinese hamster ovary cells were cultured and seeded in black 96‐well plates with transparent bottom. They were preincubated for 15 minutes with a potential inhibitor and the reaction was initiated by adding the fluorescent NTCP substrate tauro‐nor‐THCA‐24‐DBD. The final concentration of compound and substrate was 3 μM. Uptake was determined using fluorescence spectroscopy (excitation 440 nm and emission 580 nm). Potency of the hits was determined by incubating at least seven concentrations of each hit in presence of 3 μM substrate. IC50 values were estimated by fitting the sigmoid Emax model in R version 3.5.1. The inhibitors were subsequently docked into the taurocholate binding site of NTCP and a chemometric model was developed using rapid overlay of chemical structures.ResultsWe assessed more than 2500 compounds for their ability to inhibit NTCP. Forty‐one compounds were able to reduce the uptake to less than 50% (of the control, i.e. only substrate present) and were therefore classified as hits. The pIC50 of 30 compounds could be determined and ranged from 3.6 to 6.8. Several rings form the core of the 3D overlay model, surrounded by hydrophobic groups as well as hydrogen bond donor and acceptor groups. While the latter appear to be exposed to the solvent, many of the hydrophobic groups are buried inside the taurocholate binding site.ConclusionsWe identified NTCP inhibitors in vitro and developed a chemometric model that can be used for virtual screening. This model is currently being applied to the ChEMBL database for in silico identification of NTCP inhibitors. Future work also includes in vitro confirmation of the in silico hits.Support or Funding InformationThis research was supported by a grant of the Research Foundation Flanders (FWO grant G089515N).

Insights revealed by the co-crystal structure of the Saccharomyces cerevisiae histidine phosphotransfer protein Ypd1 and the receiver domain of its downstream response regulator Ssk1.

Two‐component signaling systems are the primary means by which bacteria, archaea, and certain plants and fungi react to their environments. The model yeast, Saccharomyces cerevisiae, uses the Sln1 signaling pathway to respond to hyperosmotic stress. This pathway contains a hybrid histidine kinase (Sln1) that autophosphorylates and transfers a phosphoryl group to its own receiver domain (R1). The phosphoryl group is then transferred to a histidine phosphotransfer protein (Ypd1) that finally passes it to the receiver domain (R2) of a downstream response regulator (Ssk1). Under normal conditions, Ssk1 is constitutively and preferentially phosphorylated in the phosphorelay. Upon detecting hyperosmotic stress, Ssk1 rapidly dephosphorylates and activates the high‐osmolarity glycerol (HOG) pathway, initiating a response. Despite their distinct physiological roles, both Sln1 and Ssk1 bind to Ypd1 at a common docking site. Co‐crystal structures of response regulators in complex with their phosphorelay partners are scarce, leaving many mechanistic and structural details uncharacterized for systems like the Sln1 pathway. In this work, we present the co‐crystal structure of Ypd1 and a near wild‐type variant of the receiver domain of Ssk1 (Ssk1‐R2‐W638A) at a resolution of 2.80 Å. Our structural analyses of Ypd1‐receiver domain complexes, biochemical determination of binding affinities for Ssk1‐R2 variants, in silico free energy estimates, and sequence comparisons reveal distinctive electrostatic properties of the Ypd1/Ssk1‐R2‐W638A complex that may provide insight into the regulation of the Sln1 pathway as a function of dynamic osmolyte concentration.

Modulation of TOR complex 2 signaling by the stress-activated MAPK pathway in fission yeast.

Sin1 is a substrate-binding subunit of target of rapamycin complex 2 (TORC2), an evolutionarily conserved protein kinase complex. In fission yeast, Sin1 has also been identified as a protein that interacts with Spc1 (also known as Sty1) in the stress-activated protein kinase (SAPK) pathway. Therefore, this study examined the relationship between TORC2 and Spc1 signaling. We found that the common docking (CD) domain of Spc1 interacts with a cluster of basic amino acid residues in Sin1. Although diminished TORC2 activity in the absence of the functional Spc1 cascade suggests positive regulation of TORC2 by Spc1, such regulation appears to be independent of the Sin1-Spc1 interaction. Hyperosmotic stress transiently inhibits TORC2, and its swift recovery is dependent on Spc1, the transcription factor Atf1, and the glycelrol-3-phosphate dehydrogenase Gpd1, whose expression is induced upon osmostress by the Spc1-Atf1 pathway. Thus, cellular adaptation to osmostress seems important for TORC2 reactivation, though Spc1 and Atf1 contribute to TORC2 activation also in the absence of osmostress. These results indicate coordinated actions of the SAPK and TORC2 pathways, both of which are essential for fission yeast cells to survive environmental stress.

Quantitative Structure Activity Relationship Studies and Molecular Dynamics Simulations of 2-(Aryloxyacetyl)cyclohexane-1,3-Diones Derivatives as 4-Hydroxyphenylpyruvate Dioxygenase Inhibitors.

4-Hydroxyphenylpyruvate dioxygenase (HPPD) is a significant enzyme in the biosynthesis of plastoquinone and tocopherol. Moreover, it is also a potential target to develop new herbicide. The technology of computer-aided drug design (CADD) is a useful tool in the efficient discovery of new HPPD inhibitors. Forty-three compounds with known activities were used to generate comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) models based on common framework and molecular docking. The structural contribution to the activity was determined, which provided further information for the design of novel inhibitors. Molecular docking was used to explain the changes in activity caused by the binding mode between ligand and protein. The molecular dynamics (MD) results indicated that the electrostatic energy was the major driving force for ligand–protein interaction and the Phe403 made the greatest contribution to the binding. The present work has provided useful information for the rational design of novel HPPD inhibitors with improved activity.

A temperature-dependent conformational shift in p38α MAPK substrate–binding region associated with changes in substrate phosphorylation profile

Febrile-range hyperthermia worsens and hypothermia mitigates lung injury, and temperature dependence of lung injury is blunted by inhibitors of p38 mitogen-activated protein kinase (MAPK). Of the two predominant p38 isoforms, p38α is proinflammatory and p38β is cytoprotective. Here, we analyzed the temperature dependence of p38 MAPK activation, substrate interaction, and tertiary structure. Incubating HeLa cells at 39.5 °C stimulated modest p38 activation, but did not alter tumor necrosis factor-α (TNFα)-induced p38 activation. In in vitro kinase assays containing activated p38α and MAPK-activated kinase-2 (MK2), MK2 phosphorylation was 14.5-fold greater at 39.5 °C than at 33 °C. By comparison, we observed only 3.1- and 1.9-fold differences for activating transcription factor-2 (ATF2) and signal transducer and activator of transcription-1α (STAT1α) and a 7.7-fold difference for p38β phosphorylation of MK2. The temperature dependence of p38α:substrate binding affinity, as measured by surface plasmon resonance, paralleled substrate phosphorylation. Hydrogen-deuterium exchange MS (HDX-MS) of p38α performed at 33, 37, and 39.5 °C indicated temperature-dependent conformational changes in an α helix near the common docking and glutamate:aspartate substrate-binding domains at the known binding site for MK2. In contrast, HDX-MS analysis of p38β did not detect significant temperature-dependent conformational changes in this region. We observed no conformational changes in the catalytic domain of either isoform and no corresponding temperature dependence in the C-terminal p38α-interacting region of MK2. Because MK2 participates in the pathogenesis of lung injury, the observed changes in the structure and function of proinflammatory p38α may contribute to the temperature dependence of acute lung injury.

Functional divergence caused by mutations in an energetic hotspot in ERK2

The most frequent extracellular signal-regulated kinase 2 (ERK2) mutation occurring in cancers is E322K (E-K). ERK2 E-K reverses a buried charge in the ERK2 common docking (CD) site, a region that binds activators, inhibitors, and substrates. Little is known about the cellular consequences associated with this mutation, other than apparent increases in tumor resistance to pathway inhibitors. ERK2 E-K, like the mutation of the preceding aspartate (ERK2 D321N [D-N]) known as the sevenmaker mutation, causes increased activity in cells and evades inactivation by dual-specificity phosphatases. As opposed to findings in cancer cells, in developmental assays in Drosophila, only ERK2 D-N displays a significant gain of function, revealing mutation-specific phenotypes. The crystal structure of ERK2 D-N is indistinguishable from that of wild-type protein, yet this mutant displays increased thermal stability. In contrast, the crystal structure of ERK2 E-K reveals profound structural changes, including disorder in the CD site and exposure of the activation loop phosphorylation sites, which likely account for the decreased thermal stability of the protein. These contiguous mutations in the CD site of ERK2 are both required for docking interactions but lead to unpredictably different functional outcomes. Our results suggest that the CD site is in an energetically strained configuration, and this helps drive conformational changes at distal sites on ERK2 during docking interactions.

A Novel Class of Common Docking Domain Inhibitors That Prevent ERK2 Activation and Substrate Phosphorylation.

Extracellular signal-regulated kinases (ERK1/2) are mitogen-activated protein kinases (MAPKs) that play a pro-tumorigenic role in numerous cancers. ERK1/2 possess two protein-docking sites that are distinct from the active site: the D-recruitment site (DRS) and the F-recruitment site. These docking sites facilitate substrate recognition, intracellular localization, signaling specificity, and protein complex assembly. Targeting these sites on ERK in a therapeutic context may overcome many problems associated with traditional ATP-competitive inhibitors. Here, we identified a new class of inhibitors that target the ERK DRS by screening a synthetic combinatorial library of more than 30 million compounds. The screen detects the competitive displacement of a fluorescent peptide from the DRS of ERK2. The top molecular scaffold from the screen was optimized for structure-activity relationship by positional scanning of different functional groups. This resulted in 10 compounds with similar binding affinities and a shared core structure consisting of a tertiary amine hub with three functionalized cyclic guanidino branches. Compound 2507-1 inhibited ERK2 from phosphorylating a DRS-targeting substrate and prevented the phosphorylation of ERK2 by a constitutively active MEK1 (MAPK/ERK kinase 1) mutant. Interaction between an analogue, 2507-8, and the ERK2 DRS was confirmed by nuclear magnetic resonance and X-ray crystallography. 2507-8 forms critical interactions at the common docking domain residue Asp319 via an arginine-like moiety that is shared by all 10 hits, suggesting a common binding mode. The structural and biochemical insights reported here provide the basis for developing new ERK inhibitors that are not ATP-competitive but instead function by disrupting critical protein-protein interactions.

Direct binding of Cdt2 to PCNA is important for targeting the CRL4Cdt2 E3 ligase activity to Cdt1.

The CRL4Cdt2 ubiquitin ligase complex is an essential regulator of cell-cycle progression and genome stability, ubiquitinating substrates such as p21, Set8, and Cdt1, via a display of substrate degrons on proliferating cell nuclear antigens (PCNAs). Here, we examine the hierarchy of the ligase and substrate recruitment kinetics onto PCNA at sites of DNA replication. We demonstrate that the C-terminal end of Cdt2 bears a PCNA interaction protein motif (PIP box, Cdt2PIP), which is necessary and sufficient for the binding of Cdt2 to PCNA. Cdt2PIP binds PCNA directly with high affinity, two orders of magnitude tighter than the PIP box of Cdt1. X-ray crystallographic structures of PCNA bound to Cdt2PIP and Cdt1PIP show that the peptides occupy all three binding sites of the trimeric PCNA ring. Mutating Cdt2PIP weakens the interaction with PCNA, rendering CRL4Cdt2 less effective in Cdt1 ubiquitination and leading to defects in Cdt1 degradation. The molecular mechanism we present suggests a new paradigm for bringing substrates to the CRL4-type ligase, where the substrate receptor and substrates bind to a common multivalent docking platform to enable subsequent ubiquitination.

Is It Reliable to Take the Molecular Docking Top Scoring Position as the Best Solution without Considering Available Structural Data?

Molecular docking is the most frequently used computational method for studying the interactions between organic molecules and biological macromolecules. In this context, docking allows predicting the preferred pose of a ligand inside a receptor binding site. However, the selection of the “best” solution is not a trivial task, despite the widely accepted selection criterion that the best pose corresponds to the best energy score. Here, several rigid-target docking methods were evaluated on the same dataset with respect to their ability to reproduce crystallographic binding orientations, to test if the best energy score is a reliable criterion for selecting the best solution. For this, two experiments were performed: (A) to reconstruct the ligand-receptor complex by performing docking of the ligand in its own crystal structure receptor (defined as self-docking), and (B) to reconstruct the ligand-receptor complex by performing docking of the ligand in a crystal structure receptor that contains other ligand (defined as cross-docking). Root-mean square deviation (RMSD) was used to evaluate how different the obtained docking orientation is from the corresponding co-crystallized pose of the same ligand molecule. We found that docking score function is capable of predicting crystallographic binding orientations, but the best ranked solution according to the docking energy is not always the pose that reproduces the experimental binding orientation. This happened when self-docking was achieved, but it was critical in cross-docking. Taking into account that docking is typically used with predictive purposes, during cross-docking experiments, our results indicate that the best energy score is not a reliable criterion to select the best solution in common docking applications. It is strongly recommended to choose the best docking solution according to the scoring function along with additional structural criteria described for analogue ligands to assure the selection of a correct docking solution.

Molecular basis for potentiation of Cx36 gap junction channel conductance by n-alcohols and general anesthetics

In our recent study, we have demonstrated that short carbon chain n-alcohols (up to octanol) stimulated while long carbon chain n-alcohols inhibited the conductance of connexin (Cx) 36 (Cx36) gap junction (GJ) channels. In contrast, GJ channels composed of other types of Cxs all were inhibited by n-alcohols independent of their carbon chain length. To identify the putative structural domains of Cx36, responsible for the dual effect of n-alcohols, we performed structural modeling of Cx36 protein docking with hexanol and isoflurane that stimulated as well as nonanol and carbenoxolone that inhibited the conductance of Cx36 GJs and revealed their multiple common docking sites and a single pocket accessible only to hexanol and isoflurane. The pocket is located in the vicinity of three unique cysteine residues, namely C264 in the fourth, and C92 and C87 in the second transmembrane domain of the neighboring Cx36 subunits. To examine the hypothesis that disulphide bonding might be involved in the stimulatory effect of hexanol and isoflurane, we generated cysteine substitutions in Cx36 and demonstrated by a dual whole-cell patch-clamp technique that in HeLa (human cervix carcinoma cell line) and N2A (mouse neuroblastoma cell line) cells these mutations reversed the stimulatory effect of hexanol and isoflurane to inhibitory one, typical of other Cxs that lack respective cysteines and a specific docking pocket for these compounds. Our findings suggest that the stimulatory effect of hexanol and isoflurane on Cx36 GJ conductance could be achieved by re-shuffling of the inter-subunit disulphide bond between C264 and C92 to the intra-subunit one between C264 and C87.

Cloning and Characterization of a Mitogen-Activated Protein Kinase Gene 84&amp;lt;i&amp;gt;KMPK&amp;lt;/i&amp;gt;14 in Hybrid Poplar (&amp;lt;i&amp;gt;Populus alba&amp;lt;/i&amp;gt; &amp;#215; &amp;lt;i&amp;gt;P. glandulosa&amp;lt;/i&amp;gt; cv. “84K”)

Mitogen-activated protein kinases (MAPKs) are important components in signal transduction modules which play crucial roles in regulation of many biological processes in plants. Although genome-wide analysis of MAPK and MAPKK family has been carried out in poplar species, few data about the biological function analysis of this gene family are available to date. In this study, a group C MAPK gene 84KMPK14 was cloned from hybrid poplar (Populus alba × P. glandulosa cv. “84K”). It contained a typical protein kinase domain, a conserved TEY-motif and an atypical conserved common docking (CD) domain. Sequence alignment revealed that 84KMPK14 was the most homologous to Populus trichocarpa PtMPK14. Expression analysis indicated that the transcript of 84KMPK14 in roots and young leaves was higher than that in other tissues. The expression of 84KMPK14 was down-regulated by low or high temperature and was induced by H2O2 significantly. It was suppressed by drought and salinity stresses slightly one hour after treatment and then increased quickly three hours after treatment. These results indicated that 84KMPK14 may be involved in environmental stresses, which provides basis for further characterization of the physiological analysis on this gene.

A cellular threshold for active ERK1/2 levels determines Raf/MEK/ERK-mediated growth arrest versus death responses

In addition to its conventional role for cell proliferation and survival, the Raf/MEK/Extracellular signal-regulated kinase (ERK) pathway can also induce growth arrest and death responses, if aberrantly activated. Here, we determined a molecular basis of ERK1/2 signaling that underlies these growth inhibitory physiological outputs. We found that overexpression of ERK1 or ERK2 switches ΔRaf-1:ER-induced growth arrest responses to caspase-dependent apoptotic death responses in different cell types. These death responses, however, were reverted to growth arrest responses upon titration of cellular phospho-ERK1/2 levels by the MEK1/2 inhibitor AZD6244. These data suggest that a cellular threshold for active ERK1/2 levels exists and affects the cell fate between death and growth arrest. We also found that death-mediating ability of ERK2 is abolished by the catalytic site-disabling Lys52Arg replacement or significantly attenuated by the F-site recruitment site-disabling Tyr261Asn replacement, although unaffected by the mutations that disable the common docking groove or the dimerization interface. Therefore, ERK1/2 mediates death signaling dependently of kinase activity and specific physical interactions. Intriguingly, Tyr261Asn-replaced ERK2 could still mediate growth arrest signaling, further contrasting the molecular basis of ERK1/2-mediated growth arrest and death signaling. These data reveal a mechanism underlying the role of ERK1/2 as a focal point of Raf/MEK/ERK-mediated growth arrest and death signaling.

Antidepressive potential of aqueous extract of common vervain (V. officinalis L. Verbenaceae) and molecular docking studies of its main components as potential antidepressive agents

Antidepressive potential of aqueous extract of common vervain (V. officinalis L. Verbenaceae) and molecular docking studies of its main components as potential antidepressive agents

Regulation of atypical MAP kinases ERK3 and ERK4 by the phosphatase DUSP2

The atypical MAP kinases ERK3 and ERK4 are activated by phosphorylation of a serine residue lying within the activation loop signature sequence S-E-G. However, the regulation of ERK3 and ERK4 phosphorylation and activity is poorly understood. Here we report that the inducible nuclear dual-specificity MAP kinase phosphatase (MKP) DUSP2, a known regulator of the ERK and p38 MAPKs, is unique amongst the MKP family in being able to bind to both ERK3 and ERK4. This interaction is mediated by a conserved common docking (CD) domain within the carboxyl-terminal domains of ERK3 and ERK4 and the conserved kinase interaction motif (KIM) located within the non-catalytic amino terminus of DUSP2. This interaction is direct and results in the dephosphorylation of ERK3 and ERK4 and the stabilization of DUSP2. In the case of ERK4 its ability to stabilize DUSP2 requires its kinase activity. Finally, we demonstrate that expression of DUSP2 inhibits ERK3 and ERK4-mediated activation of its downstream substrate MK5. We conclude that the activity of DUSP2 is not restricted to the classical MAPK pathways and that DUSP2 can also regulate the atypical ERK3/4-MK5 signalling pathway in mammalian cells.

Accelerated flexible protein-ligand docking using Hamiltonian replica exchange with a repulsive biasing potential.

A molecular dynamics replica exchange based method has been developed that allows rapid identification of putative ligand binding sites on the surface of biomolecules. The approach employs a set of ambiguity restraints in replica simulations between receptor and ligand that allow close contacts in the reference replica but promotes transient dissociation in higher replicas. This avoids long-lived trapping of the ligand or partner proteins at nonspecific, sticky, sites on the receptor molecule and results in accelerated exploration of the possible binding regions. In contrast to common docking methods that require knowledge of the binding site, exclude solvent and often keep parts of receptor and ligand rigid the approach allows for full flexibility of binding partners. Application to peptide-protein, protein-protein and a drug-receptor system indicate rapid sampling of near-native binding regions even in case of starting far away from the native binding site outperforming continuous MD simulations. An application on a DNA minor groove binding ligand in complex with DNA demonstrates that it can also be used in explicit solvent simulations.

Is It Reliable to Use Common Molecular Docking Methods for Comparing the Binding Affinities of Enantiomer Pairs for Their Protein Target?

Molecular docking is a computational chemistry method which has become essential for the rational drug design process. In this context, it has had great impact as a successful tool for the study of ligand–receptor interaction modes, and for the exploration of large chemical datasets through virtual screening experiments. Despite their unquestionable merits, docking methods are not reliable for predicting binding energies due to the simple scoring functions they use. However, comparisons between two or three complexes using the predicted binding energies as a criterion are commonly found in the literature. In the present work we tested how wise is it to trust the docking energies when two complexes between a target protein and enantiomer pairs are compared. For this purpose, a ligand library composed by 141 enantiomeric pairs was used, including compounds with biological activities reported against seven protein targets. Docking results using the software Glide (considering extra precision (XP), standard precision (SP), and high-throughput virtual screening (HTVS) modes) and AutoDock Vina were compared with the reported biological activities using a classification scheme. Our test failed for all modes and targets, demonstrating that an accurate prediction when binding energies of enantiomers are compared using docking may be due to chance. We also compared pairs of compounds with different molecular weights and found the same results.

Curcuma longa Mitogen-Activated Protein Kinase 6 (ClMPK6) Stimulates the Defense Response Pathway and Enhances the Resistance to Necrotrophic Fungal Infection

Mitogen-activated protein kinase (MAPK) cascades are highly conserved signaling modules that transduce the externally perceived signals and play crucial role in plant defense against pathogen attack. In the present study, a turmeric (Curcuma longa L.) complementary DNA (cDNA) encoding a MAPK gene responsive to Pythium aphanidermatum infection was isolated using rapid amplification of cDNA ends (RACE)-PCR. It was designated as ClMPK6 based on its high homology with Arabidopsis AtMPK6. The full-length cDNA of 1484 bp length carried an open reading frame (ORF) of 1176 bp encoding a 391 amino acid polypeptide. ClMPK6 protein contains Thr-Glu-Tyr (TEY) motif on its activation loop with a common docking (CD) domain at the C-terminal end and belong to subgroup A of MAPK family. Southern hybridization revealed single copy of ClMPK6 in turmeric genome, and its intron-exon composition showed highly conserved nature of these signaling kinases across different species. Quantitative RT-PCR showed high expression of ClMPK6 in rhizome tissues of mature turmeric plants. Analysis of temporal expression revealed significant induction of ClMPK6 transcript in response to defense signaling molecules and pathogen attack at the early stages. Ectopic overexpression of ClMPK6 in Arabidopsis plants resulted in enhanced resistance to Botrytis cinerea and constitutively high expression of defense responsive genes like PDF1.2, PAD3, AOS, ACS2, ACS6, etc. Our results suggest that ClMPK6 substantiate the characteristics of AtMPK6 orthologs in defense against necrotrophic infection in plants.

An Automated Strategy for Binding-Pose Selection and Docking Assessment in Structure-Based Drug Design.

Molecular docking is a widely used technique in drug design to predict the binding pose of a candidate compound in a defined therapeutic target. Numerous docking protocols are available, each characterized by different search methods and scoring functions, thus providing variable predictive capability on a same ligand-protein system. To validate a docking protocol, it is necessary to determine a priori the ability to reproduce the experimental binding pose (i.e., by determining the docking accuracy (DA)) in order to select the most appropriate docking procedure and thus estimate the rate of success in docking novel compounds. As common docking programs use generally different root-mean-square deviation (RMSD) formulas, scoring functions, and format results, it is both difficult and time-consuming to consistently determine and compare their predictive capabilities in order to identify the best protocol to use for the target of interest and to extrapolate the binding poses (i.e., best-docked (BD), best-cluster (BC), and best-fit (BF) poses) when applying a given docking program over thousands/millions of molecules during virtual screening. To reduce this difficulty, two new procedures called Clusterizer and DockAccessor have been developed and implemented for use with some common and "free-for-academics" programs such as AutoDock4, AutoDock4(Zn), AutoDock Vina, DOCK, MpSDockZn, PLANTS, and Surflex-Dock to automatically extrapolate BD, BC, and BF poses as well as to perform consistent cluster and DA analyses. Clusterizer and DockAccessor (code available over the Internet) represent two novel tools to collect computationally determined poses and detect the most predictive docking approach. Herein an application to human lysine deacetylase (hKDAC) inhibitors is illustrated.