Competitive Binding Experiments Research Articles

Synthesis, crystallographic studies, molecular modeling and in vitro biological studies of silver(I) complexes with aminoadamantane ligands

Silver(I) complexes with amantadine (atd) and memantine (mtn) were synthesized and characterized. Elemental, thermogravimetric and mass spectrometric analyses indicated a 1:2 metal/ligand ratio, with the molecular composition AgC20H34N2·NO3 for Ag–atd and AgC24H42N2·NO3·H2O for Ag–mtn. The crystal structures of the silver(I) complexes were determined by single crystal X-ray diffractometric studies and show the coordination of amantadine and memantine to the Ag(I) ion by the nitrogen atom of the NH2 group. The spectral analysis by infrared and 1H, 13C and {15N,1H} nuclear magnetic resonance (NMR) spectroscopies confirmed the coordination sites of the ligands to the silver ions. Computational studies revealed modes of vibration and bond lengths similar to those found experimentally. The in vitro antibacterial activity assays showed that amantadine is not active over the tested strains while memantine showed a low activity against Staphylococcus aureus and Pseudomonas aeruginosa. On the other hand, the complexes had a pronounced antibacterial activity over the same strains with minimum inhibitory concentration (MIC) values in the micromolar range. Biophysical assays based on fluorescence spectroscopy indicated that the silver(I) complexes interact weakly with bovine serum albumin, while agarose gel electrophoresis and competitive binding experiments revealed that the compounds interact with DNA by non-covalent interactions.

Assessment of Chemotherapy-Induced Organ Damage with Ga-68 Labeled Duramycin.

Evaluation of [68Ga]NODAGA-duramycin as a positron emission tomography (PET) tracer of cell death for whole-body detection of chemotherapy-induced organ toxicity. Tracer specificity of Ga-68 labeled NODAGA-duramycin was determined in vitro using competitive binding experiments. Organ uptake was analyzed in untreated and doxorubicin, busulfan, and cisplatin-treated mice 2h after intravenous injection of [68Ga]NODAGA-duramycin. In vivo data were validated by immunohistology and blood parameters. In vitro experiments confirmed specific binding of [68Ga]NODAGA-duramycin. Organ toxicities were detected successfully using [68Ga]NODAGA-duramycin PET/X-ray computed tomography (CT) and confirmed by immunohistochemistry and blood parameter analysis. Organ toxicities in livers and kidneys showed similar trends in PET/CT and immunohistology. Busulfan and cisplatin-related organ toxicities in heart, liver, and lungs were detected earlier by PET/CT than by blood parameters and immunohistology. [68Ga]NODAGA-duramycin PET/CT was successfully applied to non-invasively detect chemotherapy-induced organ toxicity with high sensitivity in mice. It, therefore, represents a promising alternative to standard toxicological analyses with a high translational potential.

High-Affinity Binding of Chemokine Analogs that Display Ligand Bias at the HIV-1 Coreceptor CCR5

The chemokine receptor CCR5 is a drug target to prevent transmission of HIV/AIDS. We studied four analogs of the native chemokine regulated, on activation, normal T-cell-expressed, and secreted (RANTES) (CCL5) that have anti-HIV potencies of around 25 pM, which is more than four orders of magnitude higher than that of RANTES itself. It has been hypothesized that the ultrahigh potency of the analogs is due to their ability to bind populations of receptors not accessible to native chemokines. To test this hypothesis, we developed a homogeneous dual-color fluorescence cross-correlation spectroscopy assay for saturation- and competition-binding experiments. The fluorescence cross-correlation spectroscopy assay has the advantage that it does not rely on competition with radioactively labeled native chemokines used in conventional assays. We prepared site-specifically labeled fluorescent analogs using native chemical ligation of synthetic peptides, followed by bioorthogonal fluorescent labeling. We engineered a mammalian cell expression construct to provide fluorescently labeled CCR5, which was purified using a tandem immunoaffinity and size-exclusion chromatography approach to obtain monomeric fluorescent CCR5 in detergent solution. We found subnanomolar binding affinities for the two analogs 5P12-RANTES and 5P14-RANTES and about 20-fold reduced affinities for PSC-RANTES and 6P4-RANTES. Using homologous and heterologous competition experiments with unlabeled chemokine analogs, we conclude that the analogs all bind at the same binding site, whereas the native chemokines (RANTES and MIP-1α) fail to displace bound fluorescent analogs even at tens of micromolar concentrations. Our results can be rationalized with de novo structural models of the N-terminal tails of the synthetic chemokines that adopt a different binding mode as compared to the parent compound.

Interaction between an (−)-epigallocatechin-3-gallate-copper complex and bovine serum albumin: Fluorescence, circular dichroism, HPLC, and docking studies

The interaction of copper complexed with (−)-epigallocatechin-3-gallate (EGCG) and bovine serum albumin (BSA) was investigated using fluorescence, circular dichroism (CD) spectroscopy, HPLC and protein-ligand docking. The fluorescence quenching efficiency of BSA by EGCG was enhanced after EGCG was complexed with copper, and the EGCG-Cu complex exhibited a higher apparent binding affinity (8.88 × 105 M−1) to BSA compared with EGCG alone (5.17 × 105 M−1). The CD experiment showed that both the EGCG-BSA and [EGCG-Cu]-BSA interactions resulted in the unfolding of the secondary structure of the protein. Results of competitive binding experiments confirmed that the location of EGCG and EGCG-Cu complex binding in BSA was site I. Furthermore, molecular modeling was used to identify the amino acid residue in site I and site II that play key roles in this binding interaction. The data suggest that most of the residues involved in the [EGCG-Cu]-BSA reaction belong to the subdomains IIa (site I) of BSA.

Probing the interaction of a coumarin-di(2-picolyl)amine hybrid drug-like molecular entity with human serum albumin: Multiple spectroscopic and molecular modeling techniques.

HSA is an important plasma protein responsible for transport of drug molecules. Coumarin derivatives play critical role as anticancer, antidiabetic and antiparkinson agents. In our lab we have synthesized coumarin-based pharmacophore, di(2-picolyl)amine-3(bromoacetyl) coumarin (ligand-L) endowed with anticancer activity. Anticancer agents binding mode of HSA provides valuable pharmacological information and is a structural guidance in synthesizing new drugs with greater efficacy. Thus, binding mechanism of ligand-L with HSA was explored using spectroscopic and molecular docking techniques. UV-Vis spectroscopy demonstrates hyperchromism in the absorbance spectra of HSA on addition of ligand-L suggesting interaction of ligand-L with HSA. Fluorescence spectroscopy indicates quenching in the fluorescence of HSA in the presence of ligand-L confirming the complex formation and this binding follows static mechanism. Steady state fluorescence spectroscopy revealed high binding affinity between ligand-L and HSA with a 1:1 stoichiometry. Thermodynamic parameters obtained by ITC suggest that the interaction between ligand-L and HSA is mainly driven by van der Waals forces and hydrogen bonds, and the negative value of ΔG is an indication of spontaneous binding process. Competitive binding and molecular docking experiments showed that the binding site of ligand-L mainly resides in sub-domain IIA of HSA. CD experiments revealed no significant conformational changes in the secondary structure of HSA on binding of ligand-L. We also found that esterase-like activity of HSA was not affected by ligand-L. In conclusion, this study demonstrates binding mechanism of ligand-L with HSA, and the binding did not induce conformational changes in HSA. This study is likely to provide better understanding of transport and delivery of ligand-L via HSA. Overall, it will provide insights into pharmacokinetic properties of ligand-L and designing new ligand-L based derivatives with greater efficacy.

A Simple and Label-Free Detection of As3+ using 3-nitro-L-tyrosine as an As3+-chelating Ligand.

A simple and rapid As3+ detection method using 3-nitro-L-tyrosine (N-Tyr) is reported. We discovered the specific property of N-Tyr, which specifically chelates As3+. The reaction between As3+ and N-Tyr induces a prompt color change to vivid yellow, concomitantly increasing the absorbance at 430 nm. The selectivity for As3+ is confirmed by competitive binding experiments with various metal ions (Hg2+, Pb2+, Cd2+, Cr3+, Mg2+, Ni2+, Cu2+, Fe2+, Ca2+, Zn2+, and Mn2+). Also, the N-Tyr binding site, binding affinity, and As3+/N-Tyr reaction stoichiometry are investigated. The specific reaction is utilized to design a sensor that enables the quantitative detection of As3+ in the 0.1–100 μM range with good linearity (R2 = 0.995). Furthermore, the method’s applicability for the analysis of real samples, e.g., tap and river water, is successfully confirmed, with good recoveries (94.32–109.15%) using As3+-spiked real water samples. We believe that our discovering and its application for As3+ analysis can be effectively utilized in environmental analyses such as those conducted in water management facilities, with simplicity, rapidity, and ease.

Tissue Inhibitor of Metalloproteinase-1 Promotes Polymorphonuclear Neutrophil (PMN) Pericellular Proteolysis by Anchoring Matrix Metalloproteinase-8 and -9 to PMN Surfaces.

Matrix metalloproteinase (MMP)-8 and -9 released by degranulating polymorphonuclear cells (PMNs) promote pericellular proteolysis by binding to PMN surfaces in a catalytically active tissue inhibitor of metalloproteinases (TIMP)-resistant forms. The PMN receptor(s) to which MMP-8 and MMP-9 bind(s) is not known. Competitive binding experiments showed that Mmp-8 and Mmp-9 share binding sites on murine PMN surfaces. A novel form of TIMP-1 (an inhibitor of soluble MMPs) is rapidly expressed on PMN surfaces when human PMNs are activated. Membrane-bound TIMP-1 is the PMN receptor for pro- and active MMP-8 and -9 as shown by the following: 1) TIMP-1 is strikingly colocalized with MMP-8 and -9 on activated human PMN surfaces and in PMN extracellular traps; 2) minimal immunoreactive and active Mmp-8 or Mmp-9 are detected on the surface of activated Timp-1-/- murine PMNs; and 3) binding of exogenous Timp-1 (but not Timp-2) to Timp-1-/- murine PMNs reconstitutes the binding of exogenous pro-Mmp-8 and pro-Mmp-9 to the surface of Timp-1-/- PMNs. Unlike full-length pro-Mmp-8 and pro-Mmp-9, mutant pro-Mmp proteins lacking the COOH-terminal hemopexin domain fail to bind to Mmp-8-/-x Mmp-9-/- murine PMNs. Soluble hemopexin inhibits the binding of pro-Mmp-8 and pro-Mmp-9 to Mmp-8-/-x Mmp-9-/- murine PMNs. Thus, the COOH-terminal hemopexin domains of pro-Mmp-8 and pro-Mmp-9 are required for their binding to membrane-bound Timp-1 on murine PMNs. Exposing nonhuman primates to cigarette smoke upregulates colocalized expression of TIMP-1 with MMP-8 and MMP-9 on peripheral blood PMN surfaces. By anchoring MMP-8 and MMP-9 to PMN surfaces, membrane-bound TIMP-1 plays a counterintuitive role in promoting PMN pericellular proteolysis occurring in chronic obstructive pulmonary disease and other diseases.

The chemistry and pharmacology of putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) 5F-PY-PICA, 5F-PY-PINACA, and their analogs.

5F-PY-PICA and 5F-PY-PINACA are pyrrolidinyl 1-(5-fluoropentyl)ind (az)ole-3-carboxamides identified in 2015 as putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS). 5F-PY-PICA, 5F-PY-PINACA, and analogs featuring variation of the 1-alkyl substituent or contraction, expansion, or scission of the pyrrolidine ring were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS). In competitive binding experiments against HEK293 cells expressing human cannabinoid receptor type 1 (hCB1 ) or type 2 (hCB2 ), all analogs showed minimal affinity for CB1 (pKi <5), although several demonstrated moderate CB2 binding (pKi 5.45-6.99). In fluorescence-based membrane potential assays using AtT20-hCB1 or -hCB2 cells, none of the compounds (at 10μM) produced an effect >50% of the classical cannabinoid agonist CP55,940 (at 1μM) at hCB1 , although several showed slightly higher relative efficacy at hCB2 . Expansion of the pyrrolidine ring of 5F-PY-PICA to an azepane (8) conferred the greatest hCB2 affinity (pKi 6.99) and activity (pEC50 7.54, Emax 72%) within the series. Unlike other SCRA NPS evaluated in vivo using radio biotelemetry, 5F-PY-PICA and 5F-PY-PINACA did not produce cannabimimetic effects (hypothermia, bradycardia) in mice at doses up to 10mg/kg.

Non-covalent interaction of BODIPY-benzimidazole conjugate with bovine serum albumin–A photophysical and molecular docking study

Non-covalent interactions of different dyes/drugs with various biological agents, which lead to large modulations in photophysical properties, have attracted attention of many scientists over the decades due to their versatile applicability in different fields. Herein, we report the spectroscopic investigation of non-covalent interaction of a synthesized BODIPY-benzimidazole conjugate (BDZ) dye with bovine serum albumin (BSA), which results in distinctive modulations in the photophysical properties of the dye. The structural immobilization, and reduced propensity of forming the weakly emissive intramolecular charge transfer (ICT) state of the dye, inside the confined BSA cavity, results into a significant increase in fluorescence intensity, which is also supported by the increase in the excited-state lifetime and fluorescence anisotropy decay time of the dye. Molecular docking calculations and competitive binding experiments reveal that the BDZ dye binds selectively to site IB with a reasonably high binding constant Kb ˜ 1.38 × 104 M−1. The BDZ-BSA complex shows a notable decrease in fluorescence intensity in the presence of guanidinium hydrochloride, indicating the selective binding of the dye with the native form of the BSA protein as compared to its denatured form. Formation of BDZ-BSA complex, which emits in the biologically favorable red region, makes the studied system a probable choice to monitor the denatured form of the BSA protein, biological sensing study, and to understand the transport mechanism of similar kind of drugs and other metabolites with serum albumin protein.

New transition metal complexes of 9,10‐phenanthrenequinone p‐toluyl hydrazone Schiff base: Synthesis, spectroscopy, DNA and HSA interactions, antimicrobial, DFT and docking studies

The new complexes of Cu (II) and Ni (II) of a tridentate Schiff base ligand derived from 9,10‐phenanthrenequinone and p‐toluic hydrazide have been synthesized and characterized by elemental analysis, electrical conductometry, FT‐IR, Mass, NMR and UV–Vis. The DFT calculations were carried out at B3LYP/6‐31G*(d) level for the determination of the optimized structure of the ligand and its complexes. The as‐synthesized compounds were screened for their antimicrobial activity. Also, their binding behavior with fish salmon‐DNA (FS‐DNA) and human serum albumin (HSA) were studied by different kinds of spectroscopic and molecular modeling techniques. The fluorescence data at different temperatures were applied in order to estimate the thermodynamics parameters of interactions of ligand and its complexes with DNA and HSA. The results showed that the as‐made compounds could bind to FS‐DNA and HSA via the groove binding as the major binding mode. According to molecular docking calculation and competitive binding experiments, these compounds bind to the minor groove of DNA and hydrophobic residues located in the subdomain IB of HSA. In addition, the molecular docking results kept in good consistence with experimental data.

Liquiritinapioside – A mineralocorticoid-like substance from liquorice

Surface plasmon resonance (SPR) analysis of the main components of liquorice was performed and a novel strong mineralocorticoid receptor (MR) agonist, namely liquiritinapioside (LA), whose the binding constant was 1.093 × 10−5 M, was reported. As a supplement, LA has been further demonstrated to have a strong MR binding capacity through competitive binding experiments (the enrichment factor of LA was 10.22%). This study also validated the activity of LA on H9c2 cells. The expression of collagen I and the results of Masson staining were used to determine the ability of this substance to cause H9c2 cell fibrosis. Moreover, western blotting was used to verify the mechanism of compound-induced myocardial fibrosis. Overall, the attained results showed that LA could induce the activation of the TGF-β1/p38 MAPK signalling pathway through the MR to induce H9c2 cell fibrosis.

Assessment on the binding affinity between ritonavir with model transport protein: a combined multi-spectroscopic approaches with computer simulation

The binding affinity between ritonavir (RTV) and model transport protein, BSA was assessed through multi-spectroscopic approaches and computer simulation. The findings revealed RTV statically quenched the fluorescence of BSA and formed the 1:1 RTV-BSA complex with the binding constant (Kb) of 1.06 × 103 ∼ 5.08 × 103 M−1 under the studied temperatures (298 ∼ 310 K). During the interaction of RTV with BSA, the hydrogen bonds and van der Waals forces acted as predominant function while the hydrophobicity played an assistant function. Molecular modeling further verified the result obtained from the competitive binding experiments, RTV preferentially fit into in the sub-domain IIIA of BSA. The perturbation in the secondary structures of BSA upon acting with RTV was observed from IR results, whereas synchronous and 3D fluorescence spectral findings unraveled the slight change in the hydrophobicity surrounding Tyr and Trp residues.Communicated by Ramaswamy H. Sarma

Aromatic Bromination Abolishes the Psychomotor Features and Pro-social Responses of MDMA ("Ecstasy") in Rats and Preserves Affinity for the Serotonin Transporter (SERT).

The entactogen MDMA (3,4-methylenedioxy-methamphetamine, “Ecstasy”) exerts its psychotropic effects acting primarily as a substrate of the serotonin transporter (SERT) to induce a non-exocytotic release of serotonin. Nevertheless, the roles of specific positions of the aromatic ring of MDMA associated with the modulation of typical entactogenic effects, using analogs derived from the MDMA template, are still not fully understood. Among many possibilities, aromatic halogenation of the phenylalkylamine moiety may favor distribution to the brain due to increased lipophilicity, and sometimes renders psychotropic substances of high affinity for their molecular targets and high potency in humans. In the present work, a new MDMA analog brominated at C(2) of the aromatic ring (2-Br-4,5-MDMA) has been synthesized and pharmacologically characterized in vitro and in vivo. First, binding competition experiments against the SERT-blocker citalopram were carried out in human platelets and compared with MDMA. Besides, its effects on platelet aggregation were performed in platelet enriched human plasma using collagen as aggregation inductor. Second, as platelets are considered an appropriate peripheral model for estimating central serotonin availability, the functional effects of 2-Br-4,5-MDMA and MDMA on ATP release during human platelet aggregation were evaluated. The results obtained showed that 2-Br-4,5-MDMA exhibits higher affinity for SERT than MDMA and fully abolishes both platelet aggregation and ATP release, resembling the pharmacological profile of citalopram. Subsequent in vivo evaluation in rats at three dose levels showed that 2-Br-4,5-MDMA lacks all key MDMA-like behavioral responses in rats, including hyperlocomotion, enhanced active avoidance conditioning responses and increased social interaction. Taken together, the results obtained are consistent with the notion that 2-Br-4,5-MDMA should not be expected to be an MDMA-like substrate of SERT, indicating that aromatic bromination at C(2) modulates the pharmacodynamic properties of the substrate MDMA, yielding a citalopram-like compound.

Complex formation between the Escherichia coli [NiFe]-hydrogenase nickel maturation factors.

The biosynthesis of the dinuclear metal cluster at the active sites of the [NiFe]-hydrogenase enzymes is a multi-step process executed by a suite of accessory proteins. Nickel insertion during maturation of Escherichia coli [NiFe]-hydrogenase 3 is achieved by the metallochaperones HypA, SlyD and the GTPase HypB, but how these proteins cooperate to ensure nickel delivery is not known. In this study, the complexes formed between the individual purified proteins were examined by using several methods. Size exclusion chromatography (SEC) indicated that SlyD and HypB interact primarily in a 1:1 complex. The affinity of HypB-SlyD was measured by using surface plasmon resonance, which revealed a KD of 24 ± 10nM in the absence of nucleotide and an interaction several fold tighter in the presence of GDP. A ternary complex between all three proteins was not detected, and instead SlyD blocked the interaction of HypA with HypB in competitive binding experiments. Furthermore, cross-linking experiments suggest a weak interaction between HypA and SlyD, which is not detectable by SEC. Electrochemical analysis confirmed each of the pairwise interactions and that the relative affinities of these complexes are on the order of HypB-SlyD > HypB-HypA > HypA-SlyD. These results indicate a hierarchy of interactions, as opposed to a single multiprotein complex, and provide insight into the nickel delivery process during hydrogenase enzyme maturation.

Fluorinated Bambusurils as Highly Effective and Selective Transmembrane Cl−/HCO3− Antiporters

Summary The exchange of chloride and bicarbonate across lipid bilayers is an important biological process. Synthetic molecules can act as mobile carriers for these anions, although most show little selectivity. Here we report on three bambus[6]uril macrocycles functionalized with fluorinated benzyl groups, which are able to exchange Cl− and HCO3− efficiently. Remarkably, rates for Cl−/NO3− exchange are two orders of magnitude lower. The higher rates of Cl−/HCO3− transport can be explained by the ability of the bambusurils to complex Cl− and HCO3− simultaneously, facilitating their exchange at the bilayer interface. Furthermore, the exceptionally high affinity and selectivity of these systems for NO3− appear to contribute to the poor Cl−/NO3− exchange. This work not only demonstrates the importance of anion binding characteristics on anion transport but also the potential relevance of bambusurils for anion transport applications considering the high rate observed for Cl−/HCO3− exchange.

Reinterpreting anomalous competitive binding experiments within G protein-coupled receptor homodimers using a dimer receptor model

An increasing number of G protein-coupled receptors (GPCRs) have been reported to be expressed in the plasma membrane as dimers. Since most ligand binding data are currently fitted by classical equations developed only for monomeric receptors, the interpretation of data could be misleading in the presence of GPCR dimers. On the other hand, the equations developed from dimer receptor models assuming the existence of two orthosteric binding sites within the dimeric molecule offer the possibility to directly calculate macroscopic equilibrium dissociation constants for the two sites, an index of cooperativity (DC) that reflects the molecular communication within the dimer and, importantly, a constant of radioligand-competitor allosteric interaction (KDAB) in competitive assays. Here, we provide a practical way to fit competitive binding data that allows the interpretation of apparently anomalous results, such as competition curves that could be either bell-shaped, monophasic or biphasic depending on the assay conditions. The consideration of a radioligand-competitor allosteric interaction allows fitting these curve patterns both under simulation conditions and in real radioligand binding experiments, obtaining competitor affinity parameters closer to the actual values. Our approach is the first that, assuming the formation of receptor homodimers, is able to explain several experimental results previously considered erroneous due to their impossibility to be fitted. We also deduce the radioligand concentration responsible for the conversion of biphasic to monophasic or to bell-shaped curves in competitive radioligand binding assays. In conclusion, bell-shaped curves in competitive binding experiments constitute evidence for GPCR homodimerization.

Insights into product release dynamics through structural analyses of thymidylate kinase

Several studies on enzyme catalysis have pointed out that the product release event could be a rate limiting step. In this study, we have compared the release event of two products, Adenosine di-phosphate (ADP) and Thymidine di-phosphate (TDP) from the active-site of human and Thermus thermophilus thymidine mono-phosphate kinase (TMPK), referred to as hTMPK and ttTMPK, respectively. TMPK catalyses the conversion of Thymidine mono-phosphate (TMP) to TDP using ATP as phosphoryl donor in the presence of Mg2+ ion. Most of the earlier studies on this enzyme have focused on understanding substrate binding and catalysis, but the critical product release event remains elusive. Competitive binding experiments of the substrates and the products using ttTMPK apo crystals have indicated that the substrate (TMP) can replace the bound product (TDP), even in the presence of an ADP molecule. Further, the existing random accelerated molecular dynamics (RAMD) simulation program was modified to study the release of both the products simultaneously from the active site. The RAMD simulations on product-bound structures of both ttTMPK and hTMPK, revealed that while several exit patterns of the products are permissible, the sequential exit mode is the most preferred pattern for both ttTMPK and hTMPK enzymes. Additionally, the product release from the hTMPK was found to be faster and more directional as compared to ttTMPK. Structural investigation revealed that the critical changes in the residue composition in the LID-region of ttTMPK and hTMPK have an effect on the product release and can be attributed to the observed differences during product release event. Understanding of these dissimilarities is of considerable utility in designing potent inhibitors or prodrugs that can distinguish between eukaryotic and prokaryotic homologues of thymidylate kinase.

Glycosaminoglycans are differentially involved in bacterial binding to healthy and cystic fibrosis lung cells

BackgroundGlycosaminoglycans (GAGs) are essential in many infections, including recurrent bacterial respiratory infections, the main cause of mortality in cystic fibrosis (CF) patients. MethodsUsing a cellular model of healthy and CF lung epithelium, a comparative transcriptomic study of GAG encoding genes was performed using qRT-PCR, and their differential involvement in the adhesion of bacterial pathogens analyzed by enzymatic degradation and binding competition experiments. ResultsVarious alterations in gene expression in CF cells were found which affect GAG structures and seem to influence bacterial adherence to lung epithelium cells. Heparan sulfate appears to be the most important GAG species involved in bacterial binding. ConclusionsAdherence to lung epithelial cells of some of the main pathogens involved in CF is dependent on GAGs, and the expression of these polysaccharides is altered in CF cells, suggesting it could play an essential role in the development of infectious pathology.

Receptor-Ligand Interaction Measured by Inductively Coupled Plasma Mass Spectrometry and Selenium Labeling.

In the search for an alternative strategy to the radioactivity measurement conventionally performed to probe receptor-ligand interactions in pharmacological assays, we demonstrated that selenium labeling of the studied ligand combined with elemental mass spectrometry was as efficient and robust as the reference method but devoid of its environmental and health hazards. The proof-of-concept was illustrated on two GPCR receptors, vasopressin (V1A) and cholecystokinin B (CCK-B), involving peptides as endogenous ligands. We proposed several methodologies to produce selenium-labeled ligands according to peptide sequences along with binding affinity constraints. A selection of selenopeptides that kept high affinities toward the targeted receptor were engaged in saturation and competitive binding experiments with subsequent sensitive RP-LC-ICP-MS measurements. Experimental values of affinity constant ( Ki) were perfectly correlated to literature data, illustrating the general great potency of replacing radioactive iodine by selenium for ligand labeling to further undergo unaffected pharmacology experiments efficiently monitored by elemental mass spectrometry.

Dual redox labeling of DNA as a tool for electrochemical detection of p53 protein-DNA interactions

We present a novel dual redox labeling approach enabling a facile relative evaluation of protein-DNA interactions based on immunoprecipitation at magnetic beads (MBIP) with subsequent electrochemical detection. DNA probes labeled with two different electroactive markers, benzofurazane and nitrobenzene, which yield reduction peaks at distinct potentials, were synthesized using primer extension (PEX) reaction. We show that using the labeled DNA probes, specific and non-specific binding of the p53 protein can be distinguished in a simple competition binding experiment, as a strong preference of the p53 protein was observed towards DNA probes bearing a specific p53 binding site (p53CON), which is in agreement with known binding properties of the p53 protein. The p53 binding to the individual DNA probes can be modulated by specific monoclonal antibodies used for the immunoprecipitation. This approach can potentially be applied, after selection of appropriate DNA probes and monoclonal antibodies, for investigations of DNA-binding properties of other proteins and thus represents a versatile tool for studies of any DNA-binding proteins.