Maximal Binding Affinity Research Articles

Anti-lung Cancer Activity of Synthesized Substituted 1,4-Benzothiazines: An Insight from Molecular Docking and Experimental Studies.

Thiazine, a 6-membered distinctive heterocyclic motif with sulfur and nitrogen atoms, is one of the heterocyclic compounds that functions as a core scaffold in a number of medicinally significant molecules. Small thiazine-based compounds may operate simultaneously on numerous therapeutic targets and by employing a variety of methods to halt the development, proliferation, and vasculature of cancer cells. We have, herein, reported a series of substituted 1,4 benzothiazines as potential anticancer agents for the treatment of lung cancer. In order to synthesize 2,3-disubstituted-1,4 benzothiazines in good yield, a facile green approach for the oxidative cycloaddition of 2-amino benzenethiol and 1,3-dicarbonyls employing a catalytic amount of ceric ammonium nitrate has been devised. All the molecules have been characterized by spectral analysis and tested for anticancer activity against the A-549 lung cancer cell line using various functional assays. Further in silico screening of compound 3c against six crucial inflammatory molecular targets, such as Il1-α (PDB ID: 5UC6), Il1- β (PDB ID: 6Y8I), Il6 (PDB ID: 1P9M), vimentin (PDB ID: 3TRT), COX-2 (PDB ID: 5KIR), Il8 (PDB ID: 5D14), and TNF-α (PDB ID: 2AZ5), was done using AutoDock tool. Among the synthesized compounds, propyl 3-methyl-3,4-dihydro-2H-benzo[b][1,4]thiazine-2- carboxylate (3c) was found to be most active based on cell viability assays using A-549 lung cancer cell line and was found to effectively downregulate various pro-inflammatory genes, like Il1-α, Il1-β, Il6, vimentin, COX-2, Il8, and TNF-α in vitro. The ability of the molecule to effectively suppress the proliferation and migration of lung cancer cells in vitro has been further demonstrated by the colony formation unit assay and wound healing assay. Molecular docking analysis showed the maximal binding affinity (- 7.54 kcal/mol) to be exhibited by compound 3c against IL8. A green unconventional route for the synthesis of 2,3-disubstituted-1,4 benzothiazines has been developed. All the molecules were screened for their activity against lung cancer and the data suggested that the presence of an additional unbranched alkyl group attached to the thiazine ring increased their activity. Also, in vitro and in silico modeling confirmed the anti-cancer efficiency of compound 3c, encouraging the exploration of such small molecules against cancer.

Utilization of Computational Tools for the Discovery of Schiff Base-based 1, 3, 4-thiadiazole Scaffold as SGLT2 Inhibitors

Background:: High or abnormal blood sugar levels are the hallmark of diabetes mellitus (DM), a metabolic disorder that will be one of the major causes of mortality in 2021. SGLT2 inhibitors have recently shown beneficial effects in the treatment of diabetes by reducing hyperglycemia and glucosuria. Objective:: Molecular docking and ADMET studies of Schiff base- based 1, 3, 4-thiadiazole scaffold as SGLT2 inhibitors. Methods:: Chem draw Ultra 16.0 software was used to draw the structures of newly designed molecules of Schiff base-based 1, 3, 4-thiadiazole, which were then translated into 3D structures. For the molecular docking study, AutoDock Vina 1.5.6 software was employed. Lazar in silico and Swiss ADME predictors were used to calculate in silico ADMET characteristics. Results:: We have designed 111 novel Schiff base-based 1, 3, 4-thiadiazole derivatives as SGLT2 inhibitors. A total of 10 compounds from the thiadiazole series were found to have higher binding affinity to the SGLT2 protein than dapagliflozin. SSS 56 had the best docking scores and binding affinities, with -10.4 Kcal/mol, respectively. In silico ADMET parameters demonstrated that the best binding compounds were found to be non-carcinogenic with LogP = 2.53-4.02. Conclusion:: Novel Schiff base-based 1, 3, 4-thiadiazole were designed and binding affinity was assessed against SGLT2 protein, which resulted in a new lead molecule with a maximal binding affinity and estimated to be noncarcinogenic with an optimal partition coefficient (iLogP = 2.53- 4.02).

Systematic exploration of the interactions between anionic polyacrylamide and manganese peroxidase based on docking and molecular dynamics simulations

The biodegradation of anionic polyacrylamide (HPAM) mediated by manganese peroxidase (Mn P) has been experimentally discovered and reported by researchers, but the interaction mechanism of Mn P with HPAM at molecular level is still unclear. Here, the molecular mechanisms of HPAM structure models with Mn P obtained from Phanerochaete chrysosporium were investigated by docking and molecular dynamics (MD) simulations. The results indicate that both the number of H-bonds formed and key residues in MnP-HPAM-5 are the largest, leading to its maximal binding affinity. HPAM models contact with an active site of Mn P by H-bonds and hydrophobic interactions to maintain their stability. The binding of HPAM-5 results in the significant change and unfolding of enzyme structure, and the thicker hydration shell produced by a large amount of water molecule near the enzyme surface and the binding region makes HPAM-5 difficult to access the active site of enzyme. The enzyme structure is the most stable when it binds to HPAM-4, and the higher water molecules may form H-bonds with its binding region. These facilitate mass transfer, beneficial to its binding. This study provides the theoretical reference for designing the enzyme with higher activity and stability to enhance the degradation performance of HPAM.

Polycyclic Aromatic Bioactive Compounds from Eclipta Alba and Its Anticancer Potential against Breast Cancer Target Proteins: An Antibreast Cancer Intervention through In Silico and In Vitro Validations

The present study is to identify polycyclic aromatic bioactive phytocompounds from Eclipta alba against breast cancer target protein through in silico approach. Among 52 phytocompounds ecliptalbine, wedelolactone, ursolic acid and beta amyrin they have exhibited strong binding affinity against all three screened breast cancer target proteins such as matrix metallo protein (PDB ID 1RM8), estrogen receptor (PDB ID 3ERT) and progesterone receptor (PDB ID 4OAR). In drug likeliness 11 phytocompounds showed zero violations against all five drug likeliness rule, whereas all three anticancer drugs showed three minimal violations against the drug likeliness rule. Docking score of all screened compounds lies in the range of −4.3 Kcal/mol to −9.4 Kcal/mol in that ecliptalbine and ursolic acid show maximal binding affinity with all screened target proteins of breast cancer. Phytocompound ecliptalbine, wedelolactone and ursolic acid has shown excellent simulation trajectories with two screened target proteins in MDS analysis. Further compound ecliptalbine shown good phracokinetic and DFT scores which affirm that reason behind the good binding affinity with breast cancer target proteins. MMGBSA analysis also affirms the excellent binding affinity of ecliptalbine with breast cancer target proteins (–51.42 and −72.74 Kcal/mol) than the standard drug score. Finally ethanolic leaf extract of Eclipta alba showed excellent In vitro antioxidant (DPPH-IC50 83.40 µg/ml and ABTS–IC50 48.80 µg/ml) and cytotoxic potential (IC50 97.20 µg/ml) against triple-negative breast cancer cells (MDA-MB-231). Further these drug candidates can be validated through in vitro and preclinical studies to discover novel drug for breast cancer therapeutics.

Ab-CoV: a curated database for binding affinity and neutralization profiles of coronavirus-related antibodies.

We have developed a database, Ab-CoV, which contains manually curated experimental interaction profiles of 1780 coronavirus-related neutralizing antibodies. It contains more than 3200 datapoints on half maximal inhibitory concentration (IC50), half maximal effective concentration (EC50) and binding affinity (KD). Each data with experimentally known three-dimensional structures are complemented with predicted change in stability and affinity of all possible point mutations of interface residues. Ab-CoV also includes information on epitopes and paratopes, structural features of viral proteins, sequentially similar therapeutic antibodies and Collier de Perles plots. It has the feasibility for structure visualization and options to search, display and download the data. Ab-CoV database is freely available at https://web.iitm.ac.in/bioinfo2/ab-cov/home. Supplementary data are available at Bioinformatics online.

Regulation of melanocortin-5 receptor pharmacology by two isoforms of MRAP2 in ricefield eel (Monopterus albus)

The melanocortin-5 receptor (MC5R) has been implicated in the regulation of exocrine gland secretion, immune regulation, and muscle fatty acid oxidation in mammals. Melanocortin-2 receptor accessory protein 2 (MRAP2) can modulate trafficking, ligand binding, and signaling of melanocortin receptors. To explore potential interaction between ricefield eel (Monopterus albus) MC5R and MRAP2s (maMC5R, maMRAP2X1, and maMRAP2X2), herein we studied the pharmacological characteristics of maMC5R and its modulation by maMRAP2s expressed in the human embryonic kidney cells. Three agonists, α-melanocyte-stimulating hormone (α-MSH), ACTH (1–24), and [Nle4, D-Phe7]-α-MSH, could bind to maMC5R and induce intracellular cAMP production dose-dependently. Compared with human MC5R (hMC5R), maMC5R displayed decreased maximal binding but higher binding affinity to α-MSH or ACTH (1–24). When stimulated with α-MSH or ACTH (1–24), maMC5R showed significantly lower EC50 and maximal response than hMC5R. Two maMRAP2s had no effect on cell surface expression of maMC5R, whereas they significantly increased maximal binding. Only maMRAP2X2 significantly decreased the binding affinity of ACTH (1–24). Both maMRAP2X1 and maMRAP2X2 significantly reduced maMC5R efficacy but did not affect ligand sensitivity. The availability of maMC5R pharmacological characteristics and modulation by maMRAP2s will assist the investigation of its roles in regulating diverse physiological processes in ricefield eel.

Effects of side chain length of 10-methyl-aplog-1, a simplified analog of debromoaplysiatoxin, on PKC binding, anti-proliferative, and pro-inflammatory activities.

10-Methyl-aplog-1 (1), a simplified analog of debromoaplysiatoxin, exhibits a high binding affinity for protein kinase C (PKC) isozymes and potent antiproliferative activity against several cancer cells with few adverse effects. A recent study has suggested that its phenol group in the side chain is involved in hydrogen bonding and CH/π interactions with the binding cleft-forming loops in the PKCδ-C1B domain. To clarify the effects of the side chain length on these interactions, four analogs of 1 with various lengths of side chains (2-5) were prepared. The maximal PKC binding affinity and antiproliferative activity were observed in 1. Remarkably, the introduction of a bromine atom into the phenol group of 2 increased not only these activities but also proinflammatory activity. These results indicated that 1 has the optimal side chain length as an anticancer seed. This conclusion was supported by docking simulations of 1-5 to the PKCδ-C1B domain.

In-Vitro and In-Silico Anti-inflammatory Activity of Lupeol Isolated from Crateva adansonii and Its Hidden Molecular Mechanism

The aim of the present study is to reveal the possible mechanism of anti-inflammatory activity of Crateva adansonii leaf extract to claim folkoric use of the plant in inflammation disease conditions. Isolation of chloroform leaf extract fraction (CEF) containing anti-inflammatory marker compound of the plant lupeol is done through column chromatography procedure, which is further confirmed by HPLC analysis with standard lupeol compound. The isolated marker compound lupeol from CEF shows significant in vitro anti-inflammatory effect compared to methanolic and chloroform leaf extracts (ME & CE) of the plant Crateva adansonii and reference standard indomethacin. Further in silico screening of lupeol and indomethacin against five crucial inflammatory molecular targets such as COX-2 (PDB ID: 4COX), MPO (PDB ID: 3ZS0), IL1β (PDBID: 1T4Q), IL6 (PDBID: 19PM) and TNFα (PDBID: 2AZ5) was done using autodock tool. Maximal binding affinity (− 11.6, − 9.0, − 9.9, − 7.5, − 9.0 kcal/mol) was exhibited by lupeol against all five targets of inflammation COX-2, MPO, TNFα, IL1β & IL6 respectively. In vitro and in silico modeling confirms the anti-inflammatory efficiency of lupeol and also unveils the hidden molecular mechanisms of folkoric use of Crateva adansonii plant in inflammatory disease conditions.

Platform for Screening Abiotic/Biotic Interactions Using Indicator Displacement Assays.

This paper describes novel adaptations of optically sectioned planar format assays to screen compounds for their affinities to materials surfaces. The novel platform, which we name optically sectioned indicator displacement assays (O-IDA), makes use of displaceable dyes in a format adaptable to high-throughput multiwell plate technologies. We describe two approaches: the first being where the dye exhibits fluorescence in both the surface bound and unbound state and the second, where fluorescence is lost upon displacement of the dye from the surface. Half maximal inhibitory concentration (IC50), binding affinity (Ki), and binding free energy (ΔGads) values can be extracted from the raw data. Representative biomolecules were tested for interactions with silica in an aqueous environment and ZnO(0001)-Zn and (10-10) facets in a nonaqueous environment. We provide the first experimental values for both the binding of small molecules to silica and the facet-dependent ZnO binding affinity of key amino acids associated with ZnO-specific oligopeptides. The specific data will be invaluable to those studying interactions at interfaces both experimentally and computationally. O-IDA provides a general framework for the high-throughput screening of molecule binding to materials surfaces, which has important applications in drug delivery, (bio-) catalysis, biosensing, and biomaterial engineering.

Inheritance of a Phenotypically Neutral Epimutation Evokes Gene Silencing in Later Generations.

SummarySmall RNAs trigger the formation of epialleles that are silenced across generations. Consequently, RNA-directed epimutagenesis is associated with persistent gene repression. Here, we demonstrate that small interfering RNA-induced epimutations in fission yeast are still inherited even when the silenced gene is reactivated, and descendants can reinstate the silencing phenotype that only occurred in their ancestors. This process is mediated by the deposition of a phenotypically neutral molecular mark composed of tri-methylated histone H3 lysine 9 (H3K9me3). Its stable propagation is coupled to RNAi and requires maximal binding affinity of the Clr4/Suvar39 chromodomain to H3K9me3. In wild-type cells, this mark has no visible impact on transcription but causes gene silencing if RNA polymerase-associated factor 1 complex (Paf1C) activity is impaired. In sum, our results reveal a distinct form of epigenetic memory in which cells acquire heritable, transcriptionally active epialleles that confer gene silencing upon modulation of Paf1C.

Development and Validation of Molecular Overlays Derived from Three-Dimensional Hydrophobic Similarity with PharmScreen.

Molecular alignment is a standard procedure for three-dimensional (3D) similarity measurements and pharmacophore elucidation. This process is influenced by several factors, such as the physicochemical descriptors utilized to account for the molecular determinants of biological activity and the reference templates. Relying on the hypothesis that the maximal achievable binding affinity for a drug-like molecule is largely due to desolvation, we explore a novel strategy for 3D molecular overlays that exploits the partitioning of molecular hydrophobicity into atomic contributions in conjunction with information about the distribution of hydrogen-bond (HB) donor/acceptor groups. A brief description of the method, as implemented in the software package PharmScreen, including the derivation of the fractional hydrophobic contributions within the quantum mechanical version of the Miertus-Scrocco-Tomasi (MST) continuum model, and the procedure utilized for the optimal superposition between molecules, is presented. The computational procedure is calibrated by using a data set of 402 molecules pertaining to 14 distinct targets taken from the literature and validated against the AstraZeneca test, which comprises 121 experimentally derived sets of molecular overlays. The results point out the suitability of the MST-based hydrophobic parameters for generating molecular overlays, as correct predictions were obtained for 94%, 79%, and 54% of the molecules classified into easy, moderate, and hard sets, respectively. Moreover, the results point out that this accuracy is attained at a much lower degree of identity between the templates used by hydrophobic/HB fields and electrostatic/steric ones. These findings support the usefulness of the hydrophobic/HB descriptors to generate complementary overlays that may be valuable to rationalize structure-activity relationships and for virtual screening campaigns.

Quantitative Prediction of Multivalent Ligand-Receptor Binding Affinities for Influenza, Cholera, and Anthrax Inhibition.

Multivalency achieves strong, yet reversible binding by the simultaneous formation of multiple weak bonds. It is a key interaction principle in biology and promising for the synthesis of high-affinity inhibitors of pathogens. We present a molecular model for the binding affinity of synthetic multivalent ligands onto multivalent receptors consisting of n receptor units arranged on a regular polygon. Ligands consist of a geometrically matching rigid polygonal core to which monovalent ligand units are attached via flexible linker polymers, closely mimicking existing experimental designs. The calculated binding affinities quantitatively agree with experimental studies for cholera toxin ( n = 5) and anthrax receptor ( n = 7) and allow to predict optimal core size and optimal linker length. Maximal binding affinity is achieved for a core that matches the receptor size and for linkers that have an equilibrium end-to-end distance that is slightly longer than the geometric separation between ligand core and receptor sites. Linkers that are longer than optimal are greatly preferable compared to shorter linkers. The angular steric restriction between ligand unit and linker polymer is shown to be a key parameter. We construct an enhancement diagram that quantifies the multivalent binding affinity compared to monovalent ligands. We conclude that multivalent ligands against influenza viral hemagglutinin ( n = 3), cholera toxin ( n = 5), and anthrax receptor ( n = 7) can outperform monovalent ligands only for a monovalent ligand affinity that exceeds a core-size dependent threshold value. Thus, multivalent drug design needs to balance core size, linker length, as well as monovalent ligand unit affinity.

Heterochromatin assembly by interrupted Sir3 bridges across neighboring nucleosomes.

Heterochromatin is a conserved feature of eukaryotic chromosomes with central roles in regulation of gene expression and maintenance of genome stability. Heterochromatin formation involves spreading of chromatin-modifying factors away from initiation points over large DNA domains by poorly understood mechanisms. In Saccharomyces cerevisiae, heterochromatin formation requires the SIR complex, which contains subunits with histone-modifying, histone-binding, and self-association activities. Here, we analyze binding of the Sir proteins to reconstituted mono-, di-, tri-, and tetra-nucleosomal chromatin templates and show that key Sir-Sir interactions bridge only sites on different nucleosomes but not sites on the same nucleosome, and are therefore 'interrupted' with respect to sites on the same nucleosome. We observe maximal binding affinity and cooperativity to unmodified di-nucleosomes and propose that nucleosome pairs bearing unmodified histone H4-lysine16 and H3-lysine79 form the fundamental units of Sir chromatin binding and that cooperative binding requiring two appropriately modified nucleosomes mediates selective Sir recruitment and spreading.

Radioligand binding assay for accurate determination of nuclear retinoid X receptors: A case of triorganotin endocrine disrupting ligands.

Nuclear 9-cis retinoic acid receptors (retinoid X receptors, RXR) are promiscuous dimerization partners for a number of nuclear receptors. In the present study, we established a novel in vitro method for quantitative determination of the nuclear retinoid X receptors in rat liver. One type of high affinity and limited capacity RXR specific binding sites with the Ka value ranging from 1.011 to 1.727×10(9)l/mol and the Bmax value ranging from 0.346 to 0.567pmol/mg, was demonstrated. Maximal 9-cis retinoic acid (9cRA) specific binding to nuclear retinoid X receptors was achieved at 20°C, and the optimal incubation time for the 9cRA-RXR complex formation was 120min. From a number of endocrine disruptors, tributyltins and triphenyltins are known as RXR ligands. Our data confirmed the property of tributyltin chloride or triphenyltin chloride to bind to a high affinity and limited capacity RXR binding sites. Described optimal conditions for ligand binding to RXR molecules enabled us to calculate maximal binding capacity (Bmax) and affinity (Ka) values. This study provides an original RXR radioligand binding assay that can be employed for investigation of novel RXR ligands that comprise both drugs and endocrine disruptors.

The Mps1 kinase modulates the recruitment and activity of Cnn1(CENP-T) at Saccharomyces cerevisiae kinetochores.

Kinetochores are conserved protein complexes that bind the replicated chromosomes to the mitotic spindle and then direct their segregation. To better comprehend Saccharomyces cerevisiae kinetochore function, we dissected the phospho-regulated dynamic interaction between conserved kinetochore protein Cnn1(CENP-T), the centromere region, and the Ndc80 complex through the cell cycle. Cnn1 localizes to kinetochores at basal levels from G1 through metaphase but accumulates abruptly at anaphase onset. How Cnn1 is recruited and which activities regulate its dynamic localization are unclear. We show that Cnn1 harbors two kinetochore-localization activities: a C-terminal histone-fold domain (HFD) that associates with the centromere region and a N-terminal Spc24/Spc25 interaction sequence that mediates linkage to the microtubule-binding Ndc80 complex. We demonstrate that the established Ndc80 binding site in the N terminus of Cnn1, Cnn1(60-84), should be extended with flanking residues, Cnn1(25-91), to allow near maximal binding affinity to Ndc80. Cnn1 localization was proposed to depend on Mps1 kinase activity at Cnn1-S74, based on in vitro experiments demonstrating the Cnn1-Ndc80 complex interaction. We demonstrate that from G1 through metaphase, Cnn1 localizes via both its HFD and N-terminal Spc24/Spc25 interaction sequence, and deletion or mutation of either region results in anomalous Cnn1 kinetochore levels. At anaphase onset (when Mps1 activity decreases) Cnn1 becomes enriched mainly via the N-terminal Spc24/Spc25 interaction sequence. In sum, we provide the first in vivo evidence of Cnn1 preanaphase linkages with the kinetochore and enrichment of the linkages during anaphase.

Hepatic glucocorticoid and α1- and β2-adrenergic receptors in calves change during neonatal maturation and are related to energy regulation

Catecholamines and glucocorticoids are involved in fetal maturation of organ systems to prepare the fetus for extrauterine life. Calves, especially when born preterm, depend on function of the adrenergic system and the glucocorticoid axis to adapt energy metabolism for the neonatal period. We tested the hypothesis that hepatic glucocorticoid and α1- and β2-adrenergic receptors in neonatal calves are involved in adaptation of energy metabolism around birth and that respective binding capacities depend on stage of maturation during the neonatal period. Calves (n=7 per group) were delivered by section preterm (PT, 9d before term) or were born at term (full-term, FT; spontaneous vaginal delivery), or spontaneously born and fed colostrum for 4d (FTC). Blood samples were taken immediately after birth and before and 2h after feeding at 24h after birth (PT, FT) or on d 4 of life (FTC) to determine metabolic and endocrine changes. After slaughter at 26h after birth (PT, FT) or on d 4 of life (FTC), liver tissue was obtained to measure hepatic binding capacity of glucocorticoid and α1- and β2-adrenergic receptors. Maximal binding capacity and binding affinity were calculated by saturation binding assays using [3H]-prazosin and [3H]-CGP-12177 for determination of α1- and β2-adrenergic receptors, respectively, and [3H]-dexamethasone for determination of glucocorticoid receptor in liver. Additional liver samples were taken to measure mRNA abundance of glucocorticoid and α1- and β2-adrenergic receptors, of key enzymes and factors related to hepatic lipid metabolism, and of insulin-like growth factor 1 (IGF1). Plasma concentrations of β-hydroxybutyrate and leptin changed with time, and leptin concentrations were affected by stage of maturation. The binding capacities for hepatic glucocorticoid and β2-adrenergic receptors as well as gene expression of IGF1 were greater in FTC than in FT and PT, and binding affinity for β2-adrenergic receptor was lowest in PT. The binding capacity of hepatic α1-adrenergic receptor was greatest in FTC and greater in FT than in PT. The binding capacities of glucocorticoid and α1-adrenergic receptors were mainly related to variables of glucose and lipid metabolism. In conclusion, our results indicate dependence of hepatic glucocorticoid and adrenergic receptors on stage of maturation in neonatal calves and emphasize the association of α1-adrenergic receptor and glucocorticoid receptor with neonatal glucose and lipid metabolism.

Effects of colostrum versus formula feeding on hepatic glucocorticoid and α1- and β2-adrenergic receptors in neonatal calves and their effect on glucose and lipid metabolism

Neonatal energy metabolism in calves has to adapt to extrauterine life and depends on colostrum feeding. The adrenergic and glucocorticoid systems are involved in postnatal maturation of pathways related to energy metabolism and calves show elevated plasma concentrations of cortisol and catecholamines during perinatal life. We tested the hypothesis that hepatic glucocorticoid receptors (GR) and α1- and β2-adrenergic receptors (AR) in neonatal calves are involved in adaptation of postnatal energy metabolism and that respective binding capacities depend on colostrum feeding. Calves were fed colostrum (CF; n=7) or a milk-based formula (FF; n=7) with similar nutrient content up to d 4 of life. Blood samples were taken daily before feeding and 2h after feeding on d 4 of life to measure metabolites and hormones related to energy metabolism in blood plasma. Liver tissue was obtained 2h after feeding on d 4 to measure hepatic fat content and binding capacity of AR and GR. Maximal binding capacity and binding affinity were calculated by saturation binding assays using [3H]-prazosin and [3H]-CGP-12177 for determination of α1- and β2-AR and [3H]-dexamethasone for determination of GR in liver. Additional liver samples were taken to measure mRNA abundance of AR and GR, and of key enzymes related to hepatic glucose and lipid metabolism. Plasma concentrations of albumin, triacylglycerides, insulin-like growth factor I, leptin, and thyroid hormones changed until d 4 and all these variables except leptin and thyroid hormones responded to feed intake on d 4. Diet effects were determined for albumin, insulin-like growth factor I, leptin, and thyroid hormones. Binding capacity for GR was greater and for α1-AR tended to be greater in CF than in FF calves. Binding affinities were in the same range for each receptor type. Gene expression of α1-AR (ADRA1) tended to be lower in CF than FF calves. Binding capacity of GR was related to parameters of glucose and lipid metabolism, whereas β2-AR binding capacity was negatively associated with glucose metabolism. In conclusion, our results indicate a dependence of GR and α1-AR on milk feeding immediately after birth and point to an involvement of hepatic GR and AR in postnatal adaptation of glucose and lipid metabolism in calves.

Examination of the Polypeptide Substrate Specificity for Escherichia coli ClpA

Enzyme-catalyzed protein unfolding is essential for a large array of biological functions, including microtubule severing, membrane fusion, morphogenesis and trafficking of endosomes, protein disaggregation, and ATP-dependent proteolysis. These enzymes are all members of the ATPases associated with various cellular activity (AAA+) superfamily of proteins. Escherichia coli ClpA is a hexameric ring ATPase responsible for enzyme-catalyzed protein unfolding and translocation of a polypeptide chain into the central cavity of the tetradecameric E. coli ClpP serine protease for proteolytic degradation. Further, ClpA also uses its protein unfolding activity to catalyze protein remodeling reactions in the absence of ClpP. ClpA recognizes and binds a variety of protein tags displayed on proteins targeted for degradation. In addition, ClpA binds unstructured or poorly structured proteins containing no specific tag sequence. Despite this, a quantitative description of the relative binding affinities for these different substrates is not available. Here we show that ClpA binds to the 11-amino acid SsrA tag with an affinity of 200 ± 30 nM. However, when the SsrA sequence is incorporated at the carboxy terminus of a 30-50-amino acid substrate exhibiting little secondary structure, the affinity constant decreases to 3-5 nM. These results indicate that additional contacts beyond the SsrA sequence are required for maximal binding affinity. Moreover, ClpA binds to various lengths of the intrinsically unstructured protein, α-casein, with an affinity of ∼30 nM. Thus, ClpA does exhibit modest specificity for SsrA when incorporated into an unstructured protein. Moreover, incorporating these results with the known structural information suggests that SsrA makes direct contact with the domain 2 loop in the axial channel and additional substrate length is required for additional contacts within domain 1.

Druggability Assessment of Allosteric Proteins by Dynamics Simulations in the Presence of Probe Molecules

Druggability assessment of a target protein has emerged in recent years as an important concept in hit-to-lead optimization. A reliable and physically relevant measure of druggability would allow informed decisions on the risk of investing in a particular target. Here, we define “druggability” as a quantitative estimate of binding sites and affinities for a potential drug acting on a specific protein target. In the present study, we describe a new methodology that successfully predicts the druggability and maximal binding affinity for a series of challenging targets, including those that function through allosteric mechanisms. Two distinguishing features of the methodology are (i) simulation of the binding dynamics of a diversity of probe molecules selected on the basis of an analysis of approved drugs and (ii) identification of druggable sites and estimation of corresponding binding affinities on the basis of an evaluation of the geometry and energetics of bound probe clusters. The use of the methodology for a variety of targets such as murine double mutant-2, protein tyrosine phosphatase 1B (PTP1B), lymphocyte function-associated antigen 1, vertebrate kinesin-5 (Eg5), and p38 mitogen-activated protein kinase provides examples for which the method correctly captures the location and binding affinities of known drugs. It also provides insights into novel druggable sites and the target’s structural changes that would accommodate, if not promote and stabilize, drug binding. Notably, the ability to identify high affinity spots even in challenging cases such as PTP1B or Eg5 shows promise as a rational tool for assessing the druggability of protein targets and identifying allosteric or novel sites for drug binding.

Transcriptional repressor CopR acts by inhibiting RNA polymerase binding

CopR is a transcriptional repressor encoded by the broad-host-range streptococcal plasmid pIP501, which also replicates in Bacillus subtilis. It acts in concert with the antisense RNA, RNAIII, to control pIP501 replication. CopR represses transcription of the essential repR mRNA about 10- to 20-fold. In previous work, DNA binding and dimerization constants were determined and the motifs responsible localized. The C terminus of CopR was shown to be required for stability. Furthermore, SELEX of the copR operator revealed that in vivo evolution was for maximal binding affinity. Here, we elucidate the repression mechanism of CopR. Competition assays showed that CopR-operator complexes are 18-fold less stable than RNA polymerase (RNAP)-pII complexes. DNase I footprinting revealed that the binding sites for CopR and RNAP overlap. Gel-shift assays demonstrated that CopR and B. subtilis RNAP cannot bind simultaneously, but compete for binding at promoter pII. Due to its higher intracellular concentration CopR inhibits RNAP binding. Additionally, KMnO(4) footprinting experiments indicated that prevention of open complex formation at pII does not further contribute to the repression effect of CopR.