Biophysical Insights into the Binding Interactions of Inhibitors (ICA-1S/1T) Targeting Protein Kinase C-ι

In vitro and in vivo activities of a novel benzothiopyranone candidate NTB-3119M against Mycobacterium tuberculosis.

Apolipoprotein M (apoM) is a plasma protein associated mainly with HDL. ApoM is suggested to be important for the formation of prebeta-HDL, but its mechanism of action is unknown. Homology modeling has suggested apoM to be a lipocalin. Lipocalins share a structurally conserved beta-barrel, which in many lipocalins bind hydrophobic ligands. The aim of this study was to test the ability of apoM to bind different hydrophobic substances. ApoM was produced both in Escherichia coli and in HEK 293 cells. Characterization of both variants with electrophoretic and immunological methods suggested apoM from E. coli to be correctly folded. Intrinsic tryptophan fluorescence of both apoM variants revealed that retinol, all-trans-retinoic acid, and 9-cis-retinoic acid bound (dissociation constant = 2-3 microM), whereas other tested substances (e.g., cholesterol, vitamin K, and arachidonic acid) did not. The intrinsic fluorescence of two apoM mutants carrying single tryptophans was quenched by retinol and retinoic acid to the same extent as wild-type apoM, indicating that the environment of both tryptophans was affected by the binding. In conclusion, the binding of retinol and retinoic acid supports the hypothesis that apoM is a lipocalin. The physiological relevance of this binding has yet to be elucidated.

Scoring functions are often applied in combination with molecular docking methods to predict ligand binding poses and ligand binding affinities or to identify active compounds through virtual screening. An objective benchmark for assessing the performance of current scoring functions is expected to provide practical guidance for the users to make smart choices among available methods. It can also elucidate the common weakness in current methods for future improvements. The primary goal of our comparative assessment of scoring functions (CASF) project is to provide a high-standard, publicly accessible benchmark of this type. Our latest study, i.e., CASF-2013, evaluated 20 popular scoring functions on an updated set of protein-ligand complexes. This data set was selected out of 8302 protein-ligand complexes recorded in the PDBbind database (version 2013) through a fairly complicated process. Sample selection was made by considering the quality of complex structures as well as binding data. Finally, qualified complexes were clustered by 90% similarity in protein sequences. Three representative complexes were chosen from each cluster to control sample redundancy. The final outcome, namely, the PDBbind core set (version 2013), consists of 195 protein-ligand complexes in 65 clusters with binding constants spanning nearly 10 orders of magnitude. In this data set, 82% of the ligand molecules are "druglike" and 78% of the protein molecules are validated or potential drug targets. Correlation between binding constants and several key properties of ligands are discussed. Methods and results of the scoring function evaluation will be described in a companion work in this issue (doi: 10.1021/ci500081m ).

Tumors of the head and neck represent a molecularly diverse set of human cancers, but relatively few proteins have actually been shown to drive the disease at the molecular level. To identify new targets for individualized diagnosis or therapeutic intervention, we performed a kinase centric chemical proteomics screen and quantified 146 kinases across 34 head and neck squamous cell carcinoma (HNSCC) cell lines using intensity-based label-free mass spectrometry. Statistical analysis of the profiles revealed significant intercell line differences for 42 kinases (p < 0.05), and loss of function experiments using siRNA in high and low expressing cell lines identified kinases including EGFR, NEK9, LYN, JAK1, WEE1, and EPHA2 involved in cell survival and proliferation. EGFR inhibition by the small molecule inhibitors lapatinib, gefitinib, and erlotinib as well as siRNA led to strong reduction of viability in high but not low expressing lines, confirming EGFR as a drug target in 10-20% of HNSCC cell lines. Similarly, high, but not low EPHA2-expressing cells showed strongly reduced viability concomitant with down-regulation of AKT and ERK signaling following EPHA2 siRNA treatment or EPHA1-Fc ligand exposure, suggesting that EPHA2 is a novel drug target in HNSCC. This notion is underscored by immunohistochemical analyses showing that high EPHA2 expression is detected in a subset of HNSCC tissues and is associated with poor prognosis. Given that the approved pan-SRC family kinase inhibitor dasatinib is also a very potent inhibitor of EPHA2, our findings may lead to new therapeutic options for HNSCC patients. Importantly, the strategy employed in this study is generic and therefore also of more general utility for the identification of novel drug targets and molecular pathway markers in tumors. This may ultimately lead to a more rational approach to individualized cancer diagnosis and therapy.

A specific tertiary hydrogen bond that is present between the side-chain hydroxyl group of Tyr30 and the side-chain N delta 1 atom of His17 in the small, monomeric, single-domain protein, barstar, has been perturbed by site-directed mutagenesis of the sole histidine residue (His17) to a glutamine residue. The effect of the perturbation has been studied in the resultant mutant protein, H17Q, by equilibrium unfolding methods. Both guanidine hydrochloride (GdnHCl)-induced denaturation and thermal denaturation studies have been performed, with unfolding monitored by UV absorption, intrinsic tryptophan fluorescence, near-UV and far-UV circular dichroism (CD), and size exclusion chromatography. While wild-type (wt) barstar shows a two-state unfolding transition when denatured by either GdnHCl or heat, the mutant protein H17Q undergoes unfolding through a transition that involves at least one equilibrium intermediate I, which is populated at intermediate concentrations of denaturant or at intermediate temperatures. In the case of GdnHCl-induced denaturation, the midpoint of the fluorescence-monitored denaturation curve is 1.4 +/- 0.1 M, that of the near-UV CD-monitored denaturation curve is 1.6 +/- 0.1 M, and that of the far-UV CD-monitored denaturation curve is 1.8 +/- 0.1 M. The accumulation of I is also evident in gel filtration experiments which indicate that I forms slowly from the fully-folded form, F, and that once formed, I rapidly equilibrates with the unfolded form, U. The gel filtration data for H17Q suggest that in 1.5 M GdnHCl, there is no F present and that I is the predominant form. I does not appear to possess hydrated hydrophobic surfaces, which is reflected in its inability to bind 8-anilino-1-naphthalenesulfonic acid (ANS). At least one equilibrium-unfolding intermediate is also observed upon thermal denaturation. The midpoints of the thermal denaturation curves of H17Q are 63.0 +/- 0.5 degrees C when monitored by absorbance at 287 nm or by intrinsic fluorescence at 332 nm; 65.0 +/- 0.5 degrees C when monitored by mean residue ellipticity at 275 nm; and 68.3 +/- 0.5 degrees C when monitored by mean residue ellipticity at 220 nm. In contrast, all four optical probes yield the same midpoint, 71.5 +/- 0.5 degrees C, for the wt protein. The results indicate that perturbation of the tertiary hydrogen bond leads to the accumulation of at least one intermediate (I) in both thermal denaturation studies and GdnHCl-induced denaturation studies. The intermediate(s) I are characterized by a greater disruption of tertiary structure than of secondary structure.

Experimental determination of the thermodynamic parameters affecting the adsorption behaviour and dispersion effectiveness of PCE superplasticizers

Major depression has been interpreted as an inflammatory disease characterized by cell-mediated immune activation, which is generally triggered by various stresses. Microglia has been thought to be the cellular link between inflammation and depression-like behavioural alterations. The expression of cathepsin C (Cat C), a lysosomal proteinase, is predominantly induced in microglia in neuroinflammation. However, little is known about the role of Cat C in pathophysiology of depression. In the present study, Cat C transgenic mice and wild type mice were subjected to an intraperitoneal injection of LPS (0.5mg/kg) and 6-week unpredictable chronic mild stress (UCMS) exposure to establish acute and chronic stress-induced depression model. We examined and compared the behavioural and proinflammatory cytokine alterations in serum and depression-targeted brain areas of Cat C differentially expressed mice in stress, as well as indoleamine 2,3-dioxygenase (IDO) and 5-hydroxytryptamine (5HT) levels in brain. The results showed that Cat C overexpression (Cat C OE) promoted peripheral and central inflammatory response with significantly increased TNFα, IL-1β and IL-6 in serum, hippocampus and prefrontal cortex, and resultant upregulation of IDO and downregulation of 5HT expression in brain, and thereby aggravated depression-like behaviours accessed by open field test, forced swim test and tail suspension test. In contrast, Cat C knockdown (Cat C KD) partially prevented inflammation, which may help alleviate the symptoms of depression in mice. To the best of our knowledge, we are the first to demonstrate that Cat C aggravates neuroinflammation involved in disturbances of behaviour and neurochemistry in acute and chronic stress-induced murine model of depression.

Double-mutant cycles: a powerful tool for analyzing protein structure and function

The development of a new knowledge-based scoring function (DrugScore) and its power to recognize binding modes close to experiment, to predict binding affinities, and to identify ‘hot spots’ in binding pockets is presented. Structural information is extracted from crystallographically determined protein-ligand complexes using ReLiBase and converted into distance-dependent pair-preferences and solvent-accessible surface (SAS) dependent singlet preferences of protein and ligand atoms. The sum of the pair preferences and the singlet preferences is calculated using the 3D structure of protein-ligand complexes either taken directly from the X-raystructure or generated by the docking tool FlexX. DrugScore discriminates efficiently between well-docked ligand binding modes (root-mean-squaredeviation <2.0 A with respect to a crystallographically determined reference complex) and computer-generated ones largely deviating from the native structure. For two test sets (91 and 68 protein-ligand complexes, taken from the PDB) the calculated score recognizes poses deviating <2 A from the crystal structure on rank 1 in three quarters of all possible cases. Compared to the scoring function in FlexX, this is a substantial improvement. For five test sets ofcrystallographically determined protein-ligand complexes as well as for two sets of ligand geometries generated by FlexX, the calculated score is correlated with experimentally determined binding affinities. For a set of 16 crystallographically determined serine protease inhibitor complexes, a R2 value of 0.86 and a standard deviation of 0.95 log units is achievedas best result; for a set of 64 thrombin and trypsin inhibitors docked into their target proteins, aR2 value of 0.48 and a standard deviation of 0.7 log units is calculated. DrugScore performs better than other state-of-the-art scoring functions. To assess DrugScore's capability to reproduce the geometry of directional interactions correctly, ‘hotspots’ are identified and visualized in terms of isocontour surfaces inside the binding pocket. A dataset of 159 X-ray protein-ligand complexes is used to reproduce and highlight the actually observed ligand atom positions. In 74% of all cases, the actually observed atom type corresponds to an atom type predicted by the most favorable score at the nearest grid point. The prediction rate increases to 85% ifat least an atom type of the same class of interaction is suggested. DrugScore is fast to compute and includes implicitly solvation and entropy contributions. Small deviations in the 3D structureare tolerated and, since only contacts to non-hydrogenatoms are regarded, it does not require any assumptions on protonation states.

l-Arginine metabolism by the arginase and nitric oxide (NO) synthase (NOS) families of enzymes is important in NO production, and imbalances between these pathways contribute to airway hyperresponsiveness (AHR) in asthma. To investigate the role of arginase isozymes (ARG1 and ARG2) in AHR, we determined the protein expression of ARG1, ARG2, the NOS isozymes, and other proteins involved in l-arginine metabolism in lung tissues from asthma patients and in acute (3-wk) and chronic (12-wk) murine models of ovalbumin-induced airway inflammation. Expression of ARG1 was increased in human asthma, whereas ARG2, NOS isoforms, and the other l-arginine-related proteins (i.e., cationic amino acid transporters 1 and 2, agmatinase, and ornithine decarboxylase) were unchanged. In the acute murine model of allergic airway inflammation, augmentation of ARG1 expression was similarly the most dramatic change in protein expression. However, ARG2, NOS1, NOS2, and agmatinase were also increased, whereas NOS3 expression was decreased. Arginase inhibition in vivo with nebulized S-(2-boronoethyl)-l-cysteine attenuated the methacholine responsiveness of the central airways in mice from the acute model. Further investigations in the chronic murine model revealed an expression profile that more closely paralleled the human asthma samples: only ARG1 expression was significantly increased. Interestingly, in the chronic mouse model, which generates a remodeling phenotype, arginase inhibition attenuated methacholine responsiveness of the central and peripheral airways. The similarity in arginase expression between human asthma and the chronic model and the attenuation of AHR after in vivo treatment with an arginase inhibitor suggest the potential for therapeutic modification of arginase activity in asthma.

The change in heat capacity, DeltaC(p), on protein unfolding has been usually determined by calorimetry. A noncalorimetric method which employs the Gibbs-Helmholtz relationship to determine DeltaC(p) has seen some use. Generally, in this method the free energy change on unfolding of the protein is determined at a variety of temperatures and the temperature at which DeltaG is zero, T(m), and change in enthalpy at T(m) are determined by thermal denaturation and DeltaC(p) is then calculated using the Gibbs-Helmholtz equation. We show here that an abbreviated method with stability determinations at just two temperatures gives values of DeltaC(p) consistent with values from free energy change on unfolding determination at a much wider range of temperatures. Further, even the free energy change on unfolding from a single solvent denaturation at the proper temperature, when coupled with the melting temperature, T(m), and the van't Hoff enthalpy, DeltaH(vH), from a thermal denaturation, gives a reasonable estimate of DeltaC(p), albeit with greater uncertainty than solvent denaturations at two temperatures. We also find that nonlinear regression of the Gibbs-Helmholtz equation as a function of stability and temperature while simultaneously fitting DeltaC(p), T(m), and DeltaH(vH) gives values for the last two parameters that are in excellent agreement with experimental values.

The interactions of polyacrylic acid (PAA), polymethacrylic acid (PMA), polyacrylamide (PAAm) and polymethacrylamide (PMAm) with some disperse dyes such as azobenzene, p-hydroxyazobenzene and p-aminoazobenzene were studied by an equilibrium dialysis method at 5°, 15° and 25°C.It was found that PMA displays a strong binding affinity toward these disperse dyes and on the contrary PMAm does a weak one. In the cases of PAA and PAAm no similar phenomena were observed. An addition of urea to PMA solution promoted a marked decrease in the binding ability.The results are interpreted in terms of the interaction of these dyes with partially hydrophobic tightly coiled chains of PMA, which are stable in aqueous solution and not in aqueous urea solution.The binding isotherm between the dyes and PMA was exhibited by the Langmuir one, so the thermodynamic parameters, free energy change, enthalpy change and unitary entropy change, can be calculated from the Klotz's equation. The free energy change and the enthalpy change obtained in this work are all negative and the unitary entropy change is large positive. The favorable binding process, therefore, involves two main contributions, an entropy gain and an exothermic interaction. Comparing the effect of the entropy term with that of the enthalpy one, the former is larger than the latter in the course of the binding process. In particular azobenzene which is the least polar compound has larger positive entropy change.From the above data it was deduced that the large positive unitary entropy change may be attributed to the hydrophobic interaction between the dyes and PMA.The viscosity of PMA, PAA, PAAm and PMAm in aqueous solution and aqueous urea solution was measured and discussed in some detal.

We developed a new computational algorithm for the accurate identification of ligand binding envelopes rather than surface binding sites. We performed a large scale classification of the identified envelopes according to their shape and physicochemical properties. The predicting algorithm, called PocketFinder, uses a transformation of the Lennard-Jones potential calculated from a three-dimensional protein structure and does not require any knowledge about a potential ligand molecule. We validated this algorithm using two systematically collected data sets of ligand binding pockets from complexed (bound) and uncomplexed (apo) structures from the Protein Data Bank, 5616 and 11,510, respectively. As many as 96.8% of experimental binding sites were predicted at better than 50% overlap level. Furthermore 95.0% of the asserted sites from the apo receptors were predicted at the same level. We demonstrate that conformational differences between the apo and bound pockets do not dramatically affect the prediction results. The algorithm can be used to predict ligand binding pockets of uncharacterized protein structures, suggest new allosteric pockets, evaluate feasibility of protein-protein interaction inhibition, and prioritize molecular targets. Finally the data base of the known and predicted binding pockets for the human proteome structures, the human pocketome, was collected and classified. The pocketome can be used for rapid evaluation of possible binding partners of a given chemical compound.

Iso-1-cytochromes c having lysine 32 replaced by leucine, glutamine, tyrosine, and tryptophan were prepared from strains of bakers' yeast, Saccharomyces cerevisiae, and chemically blocked at cysteine 107 with methyl methanethiolsulfonate to prevent dimerization. These modified ferricytochromes c were guanidine denatured, and the unfolding thermodynamics were determined by circular dichroism and fluorescence measurements. Thermal unfolding was also monitored by absorbance measurements. The guanidine denaturation midpoints for the altered proteins are smaller than the wild type, while the orders of stability from unfolding free energy changes are: Lys-32 (wild type) approximately Leu-32 approximately Gln-32 (circular dichroism), greater than Gln-32 (fluorescence) greater than Tyr-32 approximately Trp-32. Midpoints and differences in free energy changes for thermal unfolding parallel the fluorescence free energy changes for guanidine-induced unfolding. Thus, the blocked Leu-32 and Lys-32 proteins are equally stable with respect to both chemical and thermal denaturation. The reported data indicate that single replacements may significantly modify protein stability, and that substitution for an evolutionarily retained residue in normal cytochrome c structures does not always destabilize the protein. In addition, in vitro thermal stabilities approximately correlate with in vivo specific activities.

The isolated GTPase-activating-protein-related domain of neurofibromin-1 has a low conformational stability in solution