Structural Adaptation of the Single-Stranded DNA-Binding Protein C-Terminal to DNA Metabolizing Partners Guides Inhibitor Design.

Single-stranded DNA-binding protein (SSB) is a bacterial interaction hub and an appealing target for antimicrobial therapy. Understanding the structural adaptation of the disordered SSB C-terminus (SSB-Ct) to DNA metabolizing enzymes (e.g., ExoI and RecO) is essential for designing high-affinity SSB mimetic inhibitors. Molecular dynamics simulations revealed the transient interactions of SSB-Ct with two hot spots on ExoI and RecO. The residual flexibility of the peptide-protein complexes allows adaptive molecular recognition. Scanning with non-canonical amino acids revealed that modifications at both termini of SSB-Ct could increase the affinity, supporting the two-hot-spot binding model. Combining unnatural amino acid substitutions on both segments of the peptide resulted in enthalpy-enhanced affinity, accompanied by enthalpy-entropy compensation, as determined by isothermal calorimetry. NMR data and molecular modeling confirmed the reduced flexibility of the improved affinity complexes. Our results highlight that the SSB-Ct mimetics bind to the DNA metabolizing targets through the hot spots, interacting with both of segments of the ligands.

Proteomimetic surface fragments distinguish targets by function.

The fragment-centric design promises a means to develop complex xenobiotic protein surface mimetics, but it is challenging to find locally biomimetic structures. To address this issue, foldameric local surface mimetic (LSM) libraries were constructed. Protein affinity patterns, ligand promiscuity and protein druggability were evaluated using pull-down data for targets with various interaction tendencies and levels of homology. LSM probes based on H14 helices exhibited sufficient binding affinities for the detection of both orthosteric and non-orthosteric spots, and overall binding tendencies correlated with the magnitude of the target interactome. Binding was driven by two proteinogenic side chains and LSM probes could distinguish structurally similar proteins with different functions, indicating limited promiscuity. Binding patterns displayed similar side chain enrichment values to those for native protein-protein interfaces implying locally biomimetic behavior. These analyses suggest that in a fragment-centric approach foldameric LSMs can serve as useful probes and building blocks for undruggable protein interfaces.

Pellitorine, an extract of Tetradium daniellii, is an antagonist of the ion channel TRPV1.

BACKGROUND: Transient Receptor Potential Vanilloid 1 (TRPV1) confers noxious heat and inflammatory pain signals in the peripheral nervous system. Clinical trial of resiniferatoxin from Euphorbia species is successfully aimed at TRPV1 in cancer pain management and heading toward new selective painkiller status that further validates this target for drug discovery efforts. Evodia species, used in traditional medicine for hundreds of years, are a recognised source of different TRPV1 agonists, but no antagonist has yet been reported. HYPOTHESIS/PURPOSE: In a search for painkiller leads, we noted for the first time a TRPV1 antagonist activity in the fresh fruits of Tetradium daniellii (Benn.) T.G. Hartley (syn. Evodia hupehensis Dode). METHODS: Through a combination of extraction and purification methods with functional TRPV1-specific Ca2+ uptake assays (bioactivity-guided fractionation/isolation/purification); we isolated a new painkiller candidate that is a distant structural homologue of capsiate exovanilloids and endovanilloids such as anandamide, but a putative competitive inhibitor of the TRPV1. Four additional inactive compounds (N-isobutyl-4,5-epoxy-2E-decadienamide, geranylpsoralen, 8-(7',8'-epoxygeranyloxy)psoralen, and xanthotoxol) were also co-purified with pellitorine. Their structures were established by extensive 1D- and 2D-NMR spectroscopic analysis. RESULTS: 1H- and 13C NMR determination of the chemical structure revealed it to be pellitorine, (2E,4E)-N-(2-methylpropyl)deca-2,4-dienamide, which can compete structurally with algesics released in inflammation. In contrast to previous isolates from Evodia species, pellitorine blocked capsaicin-evoked Ca2+ uptake with an IC50 of 154 µg/ml (0.69 mM/l). N-Isobutyl-4,5-epoxy-2E-decadienamide and geranylpsoralen, 8-(7',8'-epoxygeranyloxy)psoralen, and xanthotoxol did not affect the TRPV1. CONCLUSION: This is the first evidence that pellitorine, an aliphatic alkylamide analogue of capsaicin, can serve as an antagonist of the TRPV1 and may inhibit exovanilloid-induced pain.

De Novo Modular Development of a Foldameric Protein-Protein Interaction Inhibitor for Separate Hot Spots: A Dynamic Covalent Assembly Approach.

Protein-protein interactions stabilized by multiple separate hot spots are highly challenging targets for synthetic scaffolds. Surface-mimetic foldamers bearing multiple recognition segments are promising candidate inhibitors. In this work, a modular bottom-up approach is implemented by identifying short foldameric recognition segments that interact with the independent hot spots, and connecting them through dynamic covalent library (DCL) optimization. The independent hot spots of a model target (calmodulin) are mapped with hexameric ß-peptide helices using a pull-down assay. Recognition segment hits are subjected to a target-templated DCL ligation through thiol-disulfide exchange. The most potent derivative displays low nanomolar affinity towards calmodulin and effectively inhibits the calmodulin-TRPV1 interaction. The DCL assembly of the folded segments offers an efficient approach towards the de novo development of a high-affinity inhibitor of protein-protein interactions.

Competitive inhibition of TRPV1-calmodulin interaction by vanilloids.

There is enormous interest toward vanilloid agonists of the pain receptor TRPV1 in analgesic therapy, but the mechanisms of their sensory neuron-blocking effects at high or repeated doses are still a matter of debate. Our results have demonstrated that capsaicin and resiniferatoxin form nanomolar complexes with calmodulin, and competitively inhibit TRPV1-calmodulin interaction. These interactions involve the protein recognition interface of calmodulin, which is responsible for all of the cell-regulatory calmodulin-protein interactions. These results draw attention to a previously unknown vanilloid target, which may contribute to the explanation of the paradoxical pain-modulating behavior of these important pharmacons.

Besides neuro-imaging, the Thy1-YFP mouse could serve for visualizing experimental tumours, inflammation and wound-healing.

The B6.Cg-Tg(Thy1-YFP)16Jrs/J transgenic mouse strain, widely used to study neuronal development and regeneration, expresses the yellow fluorescent protein (YFP) in the peripheral nerves and the central nervous system under the control of regulatory sequences of the Thy1 gene. The Thy1 (CD90) cell surface glycoprotein is present on many cell types besides neurons, and is known to be involved in cell adhesion, migration and signal transduction. We hypothesized that Thy1-activating conditions could probably activate the truncated Thy1 regulatory sequences used in the Thy1-YFP construct, resulting in YFP transgene expression outside the nervous system. We demonstrated that the stroma of subcutaneous tumours induced by the injection of 4T1 or MC26 carcinoma cells into BALB/c(Thy1-YFP) mice, carrying the same construct, indeed expressed the YFP transgene. In the tumour mass, the yellow-green fluorescent stromal cells were clearly distinguishable from 4T1 carcinoma cells stably transfected with red fluorescent protein. Local inflammation induced by subcutaneous injection of complete Freund's adjuvant, as well as the experimental wound-healing milieu, also triggered YFP fluorescence in both the BALB/c(Thy1-YFP) and B6.Cg-Tg(Thy1-YFP)16Jrs/J mice, pointing to eventual overlapping pathways of wound-healing, inflammation and tumour growth.

Dosage uniformity problems which occur due to technological errors in extemporaneously prepared suppositories in hospitals and pharmacies.

The availability of suppositories in Hungary, especially in clinical pharmacy practice, is usually provided by extemporaneous preparations. Due to the known advantages of rectal drug administration, its benefits are frequently utilized in pediatrics. However, errors during the extemporaneous manufacturing process can lead to non-homogenous drug distribution within the dosage units. To determine the root cause of these errors and provide corrective actions, we studied suppository samples prepared with exactly known errors using both cerimetric titration and HPLC technique. Our results show that the most frequent technological error occurs when the pharmacist fails to use the correct displacement factor in the calculations which could lead to a 4.6% increase/decrease in the assay in individual dosage units. The second most important source of error can occur when the molding excess is calculated solely for the suppository base. This can further dilute the final suppository drug concentration causing the assay to be as low as 80%. As a conclusion we emphasize that the application of predetermined displacement factors in calculations for the formulation of suppositories is highly important, which enables the pharmacist to produce a final product containing exactly the determined dose of an active substance despite the different densities of the components.

Simultaneous quantification of paracetamol, acetylsalicylic acid and papaverine with a validated HPLC method.

Combined drug products have the advantages of better patient compliance and possible synergic effects. The simultaneous application of several active ingredients at a time is therefore frequently chosen. However, the quantitative analysis of such medicines can be challenging. The aim of this study is to provide a validated method for the investigation of a multidose packed oral powder that contained acetylsalicylic acid, paracetamol and papaverine-HCl. Reversed-phase high-pressure liquid chromatography was used. The Agilent Zorbax SB-C18 column was found to be the most suitable of the three different stationary phases tested for the separation of the components of this sample. The key parameters in the method development (apart from the nature of the column) were the pH of the aqueous phase (set to 3.4) and the ratio of the organic (acetonitrile) and the aqueous (25 mM phosphate buffer) phases, which was varied from 7:93 (v/v) to 25:75 (v/v) in a linear gradient, preceded by an initial hold. The method was validated: linearity, precision (repeatability and intermediate precision), accuracy, specificity and robustness were all tested, and the results met the ICH guidelines.

Contribution of syndecans to lipoplex-mediated gene delivery.

The long awaited breakthrough of gene therapy significantly depends on the in vivo efficiency of targeted intracellular delivery. Hidden details of cellular uptake present a great hurdle for non-viral gene delivery with liposomes. Growing scientific evidence supports the involvement of polyanionic cell surface carbohydrates in cellular internalization of cationic liposomes. Syndecans, a highly conserved family of transmembrane heparan sulfate proteoglycans serve attachment sites for great variety of cationic ligands including growth factors, cytokines and even parasites. In the present study we quantitatively measured the contribution of various syndecan isoforms to liposome-mediated gene transfer. The obtained data show the superiority of syndecan-4, the ubiquitously expressed isoform of the syndecan family, in cellular uptake of liposomes. Applied mutational analysis demonstrated that gene delivery could be abolished by mutating the glycosaminoglycan attachment site of syndecans, highlighting the importance of polyanionic heparan sulfate side chains in the attachment of cationic liposomes. Blocking sulfation of syndecans also diminished gene delivery, a finding that confirms the essential role of polyanionic charges in binding cationic liposomes. Mutating other parts of the syndecan extracellular domain, including the cell-binding domain, had clearly smaller effect on liposome internalization. Mutational analyses also revealed that superiority of syndecan-4 in liposome-mediated gene delivery is significantly influenced by its cytoplasmic domain that orchestrates signaling pathways leading to macropinocytosis. In summary our study present a mechanistic insight into syndecan-mediated macropinocytic uptake of lipoplexes and highlights syndecan-4 as a superior target for cationic liposomes.

Novel sample preparation method for surfactant containing suppositories: effect of micelle formation on drug recovery.

Rectal drug delivery is currently at the focus of attention. Surfactants promote drug release from the suppository bases and enhance the formulation properties. The aim of our work was to develop a sample preparation method for HPLC analysis for a suppository base containing 95% hard fat, 2.5% Tween 20 and 2.5% Tween 60. A conventional sample preparation method did not provide successful results as the recovery of the drug failed to fulfil the validation criterion 95-105%. This was caused by the non-ionic surfactants in the suppository base incorporating some of the drug, preventing its release. As guidance for the formulation from an analytical aspect, we suggest a well defined surfactant content based on the turbidimetric determination of the CMC (critical micelle formation concentration) in the applied methanol-water solvent. Our CMC data correlate well with the results of previous studies. As regards the sample preparation procedure, a study was performed of the effects of ionic strength and pH on the drug recovery with the avoidance of degradation of the drug during the procedure. Aminophenazone and paracetamol were used as model drugs. The optimum conditions for drug release from the molten suppository base were found to be 100 mM NaCl, 20-40 mM NaOH and a 30 min ultrasonic treatment of the final sample solution. As these conditions could cause the degradation of the drugs in the solution, this was followed by NMR spectroscopy, and the results indicated that degradation did not take place. The determined CMCs were 0.08 mM for Tween 20, 0.06 mM for Tween 60 and 0.04 mM for a combined Tween 20, Tween 60 system.

Functionally important amino acid residues in the transient receptor potential vanilloid 1 (TRPV1) ion channel--an overview of the current mutational data.

This review aims to create an overview of the currently available results of site-directed mutagenesis studies on transient receptor potential vanilloid type 1 (TRPV1) receptor. Systematization of the vast number of data on the functionally important amino acid mutations of TRPV1 may provide a clearer picture of this field, and may promote a better understanding of the relationship between the structure and function of TRPV1. The review summarizes information on 112 unique mutated sites along the TRPV1, exchanged to multiple different residues in many cases. These mutations influence the effect or binding of different agonists, antagonists, and channel blockers, alter the responsiveness to heat, acid, and voltage dependence, affect the channel pore characteristics, and influence the regulation of the receptor function by phosphorylation, glycosylation, calmodulin, PIP2, ATP, and lipid binding. The main goal of this paper is to publish the above mentioned data in a form that facilitates in silico molecular modelling of the receptor by promoting easier establishment of boundary conditions. The better understanding of the structure-function relationship of TRPV1 may promote discovery of new, promising, more effective and safe drugs for treatment of neurogenic inflammation and pain-related diseases and may offer new opportunities for therapeutic interventions.

Divalent heavy metal cations block the TRPV1 Ca(2+) channel.

Transient receptor potential vanilloid 1 (TRPV1) is a non-selective cation channel involved in pain sensation and in a wide range of non-pain-related physiological and pathological conditions. The aim of the present study was to explore the effects of selected heavy metal cations on the function of TRPV1. The cations ranked in the following sequence of pore-blocking activity: Co(2+) [half-maximal inhibitory concentration (IC(50)) = 13 µM] > Cd(2+) (I (50) = 38 µM) > Ni(2+) (IC(50) = 62 µM) > Cu(2+) (IC(50) = 200 µM). Zn(2+) proved to be a weak (IC(50) = 27 µM) and only partial inhibitor of the channel function, whereas Mg(2+), Mn(2+) and La(3+) did not exhibit any substantial effect. Co(2+), the most potent channel blocker, was able not only to compete with Ca(2+) but also to pass with it through the open channel of TRPV1. In response to heat activation or vanilloid treatment, Co(2+) accumulation was verified in TRPV1-transfected cell lines and in the TRPV1+ dorsal root ganglion neurons. The inhibitory effect was also demonstrated in vivo. Co(2+) applied together with vanilloid agonists attenuated the nocifensive eye wipe response in mice. Different rat TRPV1 pore point mutants (Y627W, N628W, D646N and E651W) were created that can validate the binding site of previously used channel blockers in agonist-evoked (45)Ca(2+) influx assays in cells expressing TRPV1. The IC(50) of Co(2+) on these point mutants were determined to be reasonably comparable to those on the wild type, which suggests that divalent cations passing through the TRPV1 channel use the same negatively charged amino acids as Ca(2+).

Human complement receptor 2 (CR2/CD21) as a receptor for DNA: implications for its roles in the immune response and the pathogenesis of systemic lupus erythematosus (SLE).

Human CR2 is a B cell membrane glycoprotein that plays a central role in autoimmunity. Systemic lupus erythematosus (SLE) patients show reduced CR2 levels, and complete deficiency of CR2 and CR1 promotes the development of anti-DNA antibodies in mouse models of SLE. Here we show that multiple forms of DNA, including bacterial, viral and mammalian DNA, bind to human CR2 with moderately high affinity. Surface plasmon resonance studies showed that methylated DNA bound with high affinity with CR2 at a maximal K(D) of 6nM. DNA was bound to the first two domains of CR2 and this binding was blocked by using a specific inhibitory anti-CR2 mAb. DNA immunization in Cr2(-/-) mice revealed a specific defect in immune responses to bacterial DNA. CR2 can act as a receptor for DNA in the absence of complement C3 fixation to this ligand. These results suggest that CR2 plays a role in the recognition of foreign DNA during host-immune responses. This recognition function of CR2 may be a mechanism that influences the development of autoimmunity to DNA in SLE.

Probing the putative active site of YjdL: an unusual proton-coupled oligopeptide transporter from E. coli.

YjdL from E. coli is an unusual proton-coupled oligopeptide transporter (POT). Unlike prototypical POTs, dipeptides are preferred over tripeptides, in particular dipeptides with a positively charged C-terminal residue. To further understand this difference in peptide specificity, the sequences of YjdL and YdgR, a prototypical E. coli POT, were compared in light of the crystal structure of a POT from Shewanella oneidensis. Several residues found in the putative active site were mutated and the activities of the mutated variants were assessed in terms of substrate uptake assays, and changes in specificity in terms of uptake inhibition. Most strikingly, changing the YjdL specific Asp392 to the conserved Ser in YjdL obliterated the preference for a positively charged C-terminal residue. Based on this unique finding and previously published results indicating that the dipeptide N-terminus may interact with Glu388, a preliminary orientation model of a dipeptide in the YjdL cavity is presented. Single site mutations of particularly Ala281 and Trp278 support the presented orientation. A dipeptide bound in the cavity of YjdL appears to be oriented such that the N-terminal side chain protrudes into a sub pocket that opens towards the extracellular space. The C-terminal side chain faces in the opposite direction into a sub pocket that faces the cytoplasm. These data indicated a stabilizing effect on a bulky N-terminal residue by an Ala281Phe variant and on the dipeptide backbone by Trp278. In the presented orientation model, Tyr25 and Tyr58 both appear to be in proximity of the dipeptide backbone while Lys117 appears to be in proximity of the peptide C-terminus. Mutational studies of these conserved residues highlight their functional importance.

Homology modeling and phylogenetic relationships of catalases of an opportunistic pathogen Rhizopus oryzae.

AIMS: A homology modeling methodology was developed and used to obtain the 3D structures for four putative catalases of Rhizopus oryzae in order to assess their functionality. MAIN METHODS: Homology models were built using different modeling strategies using non-protein compounds as steric constraints, a symmetry constraint to force identical chains and an additional loop modeling algorithm. Percent structural overlap values (SO) were calculated for each model-template pair to qualify the homology models. KEY FINDINGS: Comparing the different modeling strategies by the SO values revealed that the quality of the models, i.e. the similarity to the template was greatly increased in the presence of the prosthetic groups, modeling multiple protein chains together, enforcing symmetrical chains and applying additional loop modeling. For the best homology models achieved this way, the SO values express similar evolutionary relationships between the proteins modeled and the templates that were previously established by phylogenetic analysis. In three out of the four catalases of R. oryzae the highest quality models, the active center, i.e. the heme molecule and the surrounding amino acids showed a spatial arrangement identical to that observed experimentally in other catalases. The remaining protein is missing an 11 residue long fragment and has mutated residues within the active center. SIGNIFICANCE: Better homology models can be obtained with templates chosen by phylogenetic relationship, although building an accurate model needs structural constraints too. Calculating the structural overlap between the models and the templates may also help to find the appropriate templates.

The multidrug resistance efflux complex, EmrAB from Escherichia coli forms a dimer in vitro.

Tripartite efflux systems are responsible for the export of toxins across both the inner and outer membranes of gram negative bacteria. Previous work has indicated that EmrAB-TolC from Escherichia coli is such a tripartite system, comprised of EmrB an MFS transporter, EmrA, a membrane fusion protein and TolC, an outer membrane channel. The whole complex is predicted to form a continuous channel allowing direct export from the cytoplasm to the exterior of the cell. Little is known, however, about the interactions between the individual components of this system. Reconstitution of EmrA+EmrB resulted in co-elution of the two proteins from a gel filtration column indicating formation of the EmrAB complex. Electron microscopic single particle analysis of the reconstituted EmrAB complex revealed the presence of particles approximately 240x140A, likely to correspond to two EmrAB dimers in a back-to-back arrangement, suggesting the dimeric EmrAB form is the physiological state contrasting with the trimeric arrangement of the AcrAB-TolC system.

A genomic strategy for cloning, expressing and purifying efflux proteins of the major facilitator superfamily.

A genomic strategy for the overexpression of bacterial multidrug and antibiotic resistance membrane efflux proteins in Escherichia coli is described. Expression is amplified so that the encoded proteins from a range of Gram-positive and Gram-negative bacteria comprise 5% to 35% of E. coli inner membrane protein. Depending upon their topology, proteins are produced with RGS(His)(6)-tag or a Strep-tag at the C terminus. These tags facilitate the purification of the overexpressed proteins in milligram quantities for structural studies. The strategy is illustrated for the bicyclomycin resistance efflux protein, Bcr, of E. coli.

Structure of the Epstein-Barr virus major envelope glycoprotein.

Epstein-Barr virus (EBV) infection of B cells is associated with lymphoma and other human cancers. EBV infection is initiated by the binding of the viral envelope glycoprotein (gp350) to the cell surface receptor CR2. We determined the X-ray structure of the highly glycosylated gp350 and defined the CR2 binding site on gp350. Polyglycans shield all but one surface of the gp350 polypeptide, and we demonstrate that this glycan-free surface is the receptor-binding site. Deglycosylated gp350 bound CR2 similarly to the glycosylated form, suggesting that glycosylation is not important for receptor binding. Structure-guided mutagenesis of the glycan-free surface disrupted receptor binding as well as binding by a gp350 monoclonal antibody, a known inhibitor of virus-receptor interactions. These results provide structural information for developing drugs and vaccines to prevent infection by EBV and related viruses.

Microbial drug efflux proteins of the major facilitator superfamily.

Drug efflux proteins are widespread amongst microorganisms, including pathogens. They can contribute to both natural insensitivity to antibiotics and to emerging antibiotic resistance and so are potential targets for the development of new antibacterial drugs. The design of such drugs would be greatly facilitated by knowledge of the structures of these transport proteins, which are poorly understood, because of the difficulties of obtaining crystals of quality. We describe a structural genomics approach for the amplified expression, purification and characterisation of prokaryotic drug efflux proteins of the 'Major Facilitator Superfamily' (MFS) of transport proteins from Helicobacter pylori, Staphylococcus aureus, Escherichia coli, Enterococcus faecalis, Bacillus subtilis, Brucella melitensis, Campylobacter jejuni, Neisseria meningitides and Streptomyces coelicolor. The H. pylori putative drug resistance protein, HP1092, and the S. aureus QacA proteins are used as detailed examples. This strategy is an important step towards reproducible production of transport proteins for the screening of drug binding and for optimisation of crystallisation conditions to enable subsequent structure determination.