Transfer of ANS-Like Drugs from Micellar Drug Delivery Systems to Albumin Is Highly Favorable and Protected from Competition with Surfactant by "Reserved" Binding Sites.

Micellar drug delivery systems (MDDS) for the intravenous administration of poorly soluble drugs have great advantages over alternative formulations in terms of the safety of their excipients, storage stability, and straightforward production. A classic example is mixed micelles of glycocholate (GC) and lecithin, both endogenous substances in human blood. What limits the use of MDDS is the complexity of the transitions after injection. In particular, as the MDDS disintegrate partially or completely after injection, the drug has to be transferred safely to endogenous carriers in the blood, such as human serum albumin (HSA). If this transfer is compromised, the drug might precipitate─a process that needs to be excluded under all circumstances. The key question of this paper is whether the high local concentration of GC at the moment and site of MDDS dissolution might transiently saturate HSA binding sites and, hence, endanger quick drug transfer. To address this question, we have used a new approach, which is time-resolved fluorescence spectroscopy of the single tryptophan in HSA, Trp-214, to characterize the competitive binding of GC and the drug substitute anilinonaphthalenesulfonate (ANS) to HSA. Time-resolved fluorescence of Trp-214 showed important advantages over established methods for tackling this problem. ANS has been the standard "model drug" to study albumin binding for decades, given its structural similarity to the class of naphthalene-containing acidic drugs and the fact that it is displaced from HSA by numerous drugs (which presumably bind to the same sites). Our complex global fit uses the critical approximation that the average lifetimes behave similarly to a single lifetime, but the resulting errors are found to be moderate and the results provide a convincing explanation of the, at first glance, counterintuitive behavior. Accordingly, and largely in line with the literature, we observed two types of sites binding ANS at HSA: 3 type A, rather peripheral, and 2 type B, likely more central sites. The latter quench Trp-214 by Förster Resonance Energy Transfer (FRET) with a rate constant of ≈0.4 ns-1 per ANS. Adding millimolar concentrations of GC displaces ANS from the A sites but not from B sites. At incomplete ANS saturation, this causes a GC-induced translocation of ANS from A to the more FRET-active B sites. This leads to the apparent paradox that the partial displacement of ANS from HSA increases its quenching effect on Trp-214. The most important conclusion is that (ANS-like) drugs cannot be displaced from the type-B sites, and consequently, drug transfer to these sites is not impaired by competitive binding of GC in the vicinity of a dissolving micelle. The second conclusion is that for unbound GC above the CMC (9 mM), ANS equilibrates between HSA and GC micelles but with a strong preference for free sites on HSA. That means that even persisting micelles would lose their cargo readily once exposed to HSA. For all MDDS sharing this property, targeted drug delivery approaches involving them as the nanocarrier would be pointless.

Assessment of oxygen kinetic parameters for closely related ammonia-oxidizing bacteria.

The reaction kinetics of lithotrophic ammonia-oxidizing bacteria (AOB) are strongly dependent on dissolved oxygen (DO) as their metabolism is an aerobic process. In this study, we estimate the kinetic parameters, including the oxygen affinity constant (Km[O2]) and the maximum oxygen consumption rate (Vmax[O2]), of different AOB species, by fitting the data to the Michaelis-Menten equation using nonlinear regression analysis. An example for three different species of Nitrosomonas bacteria (N. europaea, N. eutropha, and N. mobilis) in monoculture is given, finding a Km[O2] of 0.25 ± 0.05 mg l-1, 0.47 ± 0.09 mg l-1, and 0.28 ± 0.08 mg l-1, and a Vmax[O2] of 0.07 ± 0.04 pg h-1cell-1, 0.25 ± 0.06 pg h-1cell-1, and 0.02 ± 0.001 pg h-1cell-1 for N. europaea, N. eutropha, and N. mobilis, respectively. This study shows that of the analyzed AOB, N. europaea has the highest affinity towards oxygen and N. eutropha the lowest affinity towards oxygen, indicating that the former can convert ammonia even under low DO conditions. These results improve the understanding of the ecophysiology of AOB in the environment. The accuracy of mathematically modelled ammonia oxidation can be improved, allowing the implementation of better management practices to restore the nitrogen cycle in natural and engineered water systems.

Experimental kS estimation: A comparison of methods for Corynebacterium glutamicum from lab to microfluidic scale.

Knowledge about the specific affinity of whole cells toward a substrate, commonly referred to as kS , is a crucial parameter for characterizing growth within bioreactors. State-of-the-art methodologies measure either uptake or consumption rates at different initial substrate concentrations. Alternatively, cell dry weight or respiratory data like online oxygen and carbon dioxide transfer rates can be used to estimate kS . In this work, a recently developed substrate-limited microfluidic single-cell cultivation (sl-MSCC) method is applied for the estimation of kS values under defined environmental conditions. This method is benchmarked with two alternative microtiter plate methods, namely high-frequency biomass measurement (HFB) and substrate-limited respiratory activity monitoring (sl-RA). As a model system, the substrate affinity kS of Corynebacterium glutamicum ATCC 13032 regarding glucose was investigated assuming a Monod-type growth response. A kS of <70.7 mg/L (with 95% probability) with HFB, 8.55 ± 1.38 mg/L with sl-RA, and 2.66 ± 0.99 mg/L with sl-MSCC was obtained. Whereas HFB and sl-RA are suitable for a fast initial kS estimation, sl-MSCC allows an affinity estimation by determining tD at concentrations less or equal to the kS value. Thus, sl-MSCC lays the foundation for strain-specific kS estimations under defined environmental conditions with additional insights into cell-to-cell heterogeneity.

An Overlooked Hepcidin-Cadmium Connection.

Hepcidin (DTHFPICIFCCGCCHRSKCGMCCKT), an iron-regulatory hormone, is a 25-amino-acid peptide with four intramolecular disulfide bonds circulating in blood. Its hormonal activity is indirect and consists of marking ferroportin-1 (an iron exporter) for degradation. Hepcidin biosynthesis involves the N-terminally extended precursors prepro-hepcidin and pro-hepcidin, processed by peptidases to the final 25-peptide form. A sequence-specific formation of disulfide bonds and export of the oxidized peptide to the bloodstream follows. In this study we considered the fact that prior to export, reduced hepcidin may function as an octathiol ligand bearing some resemblance to the N-terminal part of the α-domain of metallothioneins. Consequently, we studied its ability to bind Zn(II) and Cd(II) ions using the original peptide and a model for prohepcidin extended N-terminally with a stretch of five arginine residues (5R-hepcidin). We found that both form equivalent mononuclear complexes with two Zn(II) or Cd(II) ions saturating all eight Cys residues. The average affinity at pH 7.4, determined from pH-metric spectroscopic titrations, is 1010.1 M-1 for Zn(II) ions; Cd(II) ions bind with affinities of 1015.2 M-1 and 1014.1 M-1. Using mass spectrometry and 5R-hepcidin we demonstrated that hepcidin can compete for Cd(II) ions with metallothionein-2, a cellular cadmium target. This study enabled us to conclude that hepcidin binds Zn(II) and Cd(II) sufficiently strongly to participate in zinc physiology and cadmium toxicity under intracellular conditions.

The action of aripiprazole and brexpiprazole at the receptor level in singultus.

The hiccup (Latin singultus) is an involuntary periodic contraction of the diaphragm followed by glottic closure, which can be a rare side effect of aripiprazole. In contrast to the structurally closely related aripiprazole, brexpiprazole was not associated with this particular adverse drug reaction. Having two very similar drugs that differ in their ability to induce hiccups represents a unique opportunity to gain insight into the receptors involved in the pathophysiology of the symptom and differences in clinical effects between aripiprazole and brexpiprazole. The overlap between maneuvers used to terminate paroxysmal supraventricular tachycardia and those employed to terminate bouts of hiccups suggests that activation of efferent vagal fibers can be therapeutic in both instances. Recent work seems to support a pivotal role for serotonin receptors in such vagal activation. It is unlikely that a unique receptor-drug interaction could explain the different effects of the examined drugs on hiccup. The different effect is most likely the consequence of several smaller effects at more than one receptor. Brexpiprazole is a highly affine (potent) α2C antagonist and, therefore, also an indirect 5-HT1A agonist. In contrast, aripiprazole is a partial 5-HT1A agonist (weak antagonist) and an HT3 antagonist. Activation of 5-HT1A receptors enhances vagal activity while HT3 blockade reduces it. Vagus nerve activation is therapeutic for hiccups. A definitive answer continues to be elusive.

The glycosylation of anti-rhIFN-α2b recombinant antibodies influences the antigen-neutralizing activity.

OBJECTIVES: The influence of glycosylation on the antigen-neutralizing ability of two potential biotherapeutic anti-human IFN-α2b antibodies composed by murine and humanized single-chain Fv fused to human Fcγ1 (chimeric and humanized scFv-Fc, respectively) was studied. RESULTS: Chimeric antibodies produced in CHO-K1 and HEK293 mammalian cells showed no differences in the antigen-antibody affinity but demonstrated differences in the in vitro neutralization of IFN-α2b activity. On the other hand, the humanized antibodies produced in the same cell types showed differences in both the antigen-antibody affinity and the antigen-neutralizing ability. These differences are due to the scFv domain, as evidenced by its expression in CHO-K1 and HEK293 cells. In order to determine if the Fc glycosylation influences the antigen binding ability, both parameters were analyzed on chimeric and humanized deglycosylated scFv-Fc. Surprisingly, no differences in the antigen-antibody affinity were observed, but differences in the antigen-neutralizing ability of both chimeric and humanized antibodies, and their respectively deglycosylated glycoforms were found. CONCLUSIONS: Fc glycosylation influences the antigen neutralization ability of two anti-rhIFN-α2b recombinant antibodies. Although affinity is the widely accepted parameter to analyze antibody antigen binding, it does not appear to be sufficient to describe the behavior of recombinant antibodies in vitro. This work contributes with a high impact knowledge to develop therapeutic recombinant antibodies where glycosylation and producer cell lines must be taken into account for their influence on the antigen binding capacity and not only for their impact on the effector properties as it has been historically considered for antibodies.

Label-free evaluation of small-molecule-protein interaction using magnetic capture and electrochemical detection.

The evaluation of interaction between small molecules and protein is an important step in the discovery of new drugs and to study complex biological systems. In this work, an alternative method was presented to evaluate small-molecule-protein interaction by using ligand capture by protein-coated magnetic particles (MPs) and disposable electrochemical cells. The interaction study was conducted using [10]-gingerol from ginger rhizome and a transmembrane protein αVß3 integrin. Initially, the electrochemical behavior of the natural compound [10]-gingerol was evaluated with the disposable carbon-based electrodes and presented an irreversible oxidation process controlled by diffusion. The analytical curve for [10]-gingerol was obtained in the range of 1.0 to 20.0 µmol L-1, with limit of detection of 0.26 µmol L-1. Then MPs coated with αVß3 integrin were incubated with standard solutions and extracts of ginger rhizome for [10]-gingerol capture and separation. The bioconjugate obtained was dropped to the disposable electrochemical cells, keeping a permanent magnet behind the working electrode, and the binding process was evaluated by the electrochemical detection of [10]-gingerol. The assay method proposed was also employed to calculate the [10]-gingerol-αVß3 integrin association constant, which was calculated as 4.3 × 107 M-1. The method proposed proved to be a good label-free alternative to ligand-protein interaction studies. Graphical abstract ᅟ.

Reusable chemiluminescent fiber optic aptasensor for the determination of 17ß-estradiol in water samples.

A reusable fiber optic chemiluminescent aptasensor (FOCA) is reported for the rapid and sensitive on-site detection of 17ß-estradiol (E2), an endocrine-disrupting compound frequently found in water samples. The E2-ovalbumin conjugate (E2-OVA) was covalently immobilized onto the optical fiber as a biorecognition element as well as a transducer. The affinity constant of the E2/aptamer complex was determined to be 1.35 × 106 M-1 using the FOCA. An indirect competitive assay was then developed for E2 detection. A certain concentration of HRP-E2 aptamers pre-reacted with samples containing E2 in various concentrations. Part of HRP-E2 aptamers specially bound to the sensor surface after introduction of the mixture. This catalyzed the chemiluminescece reaction of a chemiluminescent system composed of luminol and H2O2. A higher concentration of E2 led to less HRP-E2 aptamer bound to the biosensor surface, thus resulting in less chemiluminescence. Highly sensitive detection of E2 was achieved under optimal conditions, and the limit of detection is 48 ng ·L-1 (0.18 nM). The whole analytical process, including measurement and regeneration, can be performed in <15 min. The robustness of the biosensor allows its application to multiple assays with little activity loss. The selectivity, recovery, and accuracy of the sensor was demonstrated by evaluating its response to potentially interfering endocrine disruptors in spiked water samples. Graphical abstract Schematic diagram of the fiber optic chemiluminescent aptasensor system (A), detection mechanism of 17ß-estradiol (B), and its application for detection of 17ß-estradiol with rapidity and sensitivity (C and D).

Kinetic analysis of the inhibition of the drug efflux protein AcrB using surface plasmon resonance.

Multidrug efflux protein complexes such as AcrAB-TolC from Escherichia coli are paramount in multidrug resistance in Gram-negative bacteria and are also implicated in other processes such as virulence and biofilm formation. Hence efflux pump inhibition, as a means to reverse antimicrobial resistance in clinically relevant pathogens, has gained increased momentum over the past two decades. Significant advances in the structural and functional analysis of AcrB have informed the selection of efflux pump inhibitors (EPIs). However, an accurate method to determine the kinetics of efflux pump inhibition was lacking. In this study we standardised and optimised surface plasmon resonance (SPR) to probe the binding kinetics of substrates and inhibitors to AcrB. The SPR method was also combined with a fluorescence drug binding method by which affinity of two fluorescent AcrB substrates were determined using the same conditions and controls as for SPR. Comparison of the results from the fluorescent assay to those of the SPR assay showed excellent correlation and provided validation for the methods and conditions used for SPR. The kinetic parameters of substrate (doxorubicin, novobiocin and minocycline) binding to AcrB were subsequently determined. Lastly, the kinetics of inhibition of AcrB were probed for two established inhibitors (phenylalanine arginyl ß-naphthylamide and 1-1-naphthylmethyl-piperazine) and three novel EPIs: 4-isobutoxy-2-naphthamide (A2), 4-isopentyloxy-2-naphthamide (A3) and 4-benzyloxy-2-naphthamide (A9) have also been probed. The kinetic data obtained could be correlated with inhibitor efficacy and mechanism of action. This study is the first step in the quantitative analysis of the kinetics of inhibition of the clinically important RND-class of multidrug efflux pumps and will allow the design of improved and more potent inhibitors of drug efflux pumps. This article is part of a Special Issue entitled: Beyond the Structure-Function Horizon of Membrane Proteins edited by Ute Hellmich, Rupak Doshi and Benjamin McIlwain.

Enolase from Trypanosoma cruzi is inhibited by its interaction with metallocarboxypeptidase-1 and a putative acireductone dioxygenase.

Purification of enolase (ENO) from the cytosol of Trypanosoma cruzi indicated that it may interact with at least five other proteins. Two of them were identified as metallocarboxypeptidase-1 (TcMCP-1) and a putative acireductone dioxygenase (ARDp). Subcellular localization studies confirmed the presence of ARDp in the cytosol, as is the case for ENO and TcMCP-1. Analysis of the ARDp sequence showed that this protein has two domains, an N-terminal ARD and a C-terminal TRP14 (thioredoxin-related protein) domain. The interactions between ENO, TcMCP-1 and ARDp were confirmed for the natural proteins from the trypanosome (using size-exclusion chromatography and co-immunoprecipitation from a cytosolic fraction) and recombinant forms (using ELISA ligand-binding assay and ENO activity assays). The ELISA ligand-binding assays permitted to verify the optimal physicochemical conditions for the interactions (representative for the physiological conditions) and to determine the affinity constants (Kd): ENO/ARDp: 9.54 ±â€¯0.82 nM, ARDp/ENO 10.05 ±â€¯1.11 nM, and ENO/TcMCP-1: 5.66 ±â€¯0.61 nM. The data also show that the interaction between TcMCP-1 and ARDp is mediated by ENO acting as a "bridge". Furthermore, considerable inhibition of the ENO activity, up to 85%, is observed when the enzyme interacts with TcMCP-1 and ARDp simultaneously. All these data confirm that the interaction between ENO, TcMCP-1 and ARDp, occurring in T. cruzi's cytosol, modulates the ENO activity and suggest a possible physiological mechanism for regulation of the ENO activity by the protein-protein interaction.

Revisiting the streptavidin-biotin binding by using an aptamer and displacement isothermal calorimetry titration.

The association constant of a well-known streptavidin-biotin binding has only been inferred from separately measured kinetic parameters. In a single experiment, we obtained Ka 1 × 10(12) M(-1) by using a streptavidin-binding aptamer and ligand-displacement isothermal titration calorimetry. This study explores the challenges of determining thermodynamic parameters and the derived equilibrium binding affinity of tight ligand-receptor binding.

A kinetic model of GPCRs: analysis of G protein activity, occupancy, coupling and receptor-state affinity constants.

CONTEXT: G protein-coupled receptors are vital macromolecules for a wide variety of physiological processes. Upon agonist binding, these receptors accelerate the exchange of GDP for GTP in G proteins coupled to them. The activated G protein interacts with effector proteins to implement downstream biological functions. OBJECTIVE: We present a kinetic, quaternary complex model, based on a system of coupled linear first-order differential equations, which accounts for the binding attributes of the ligand, receptor, G protein and two types of guanine nucleotide (GDP and GTP) as well as for GTPase activity. METHODS: We solved the model numerically to predict the extents of G protein activation, receptor occupancy by ligand and receptor coupling that result from varying the ligand concentration, presence of GDP and/or GTP, the ratio of G protein to receptor and the equilibrium constants governing receptor pre-coupling and constitutive activity. We also simulated responses downstream from G protein activation using a transducer function. RESULTS: Our model shows that agonist-induced G protein activation can occur with either a net decrease or increase in total receptor-G protein coupling. In addition, we demonstrate that affinity constants of the ligand for both the active and inactive states of the receptor can be derived to a close approximation from analysis of simulated responses downstream from receptor activation. DISCUSSION AND CONCLUSION: The latter result validates our prior methods for estimating the active state affinity constants of ligands, and our results on receptor coupling have relevance to studies investigating receptor-G protein interactions using fluorescence techniques.

Fast, metadynamics-based method for prediction of the stereochemistry-dependent relative free energies of ligand-receptor interactions.

The computational approach applicable for the molecular dynamics (MD)-based techniques is proposed to predict the ligand-protein binding affinities dependent on the ligand stereochemistry. All possible stereoconfigurations are expressed in terms of one set of force-field parameters [stereoconfiguration-independent potential (SIP)], which allows for calculating all relative free energies by only single simulation. SIP can be used for studying diverse, stereoconfiguration-dependent phenomena by means of various computational techniques of enhanced sampling. The method has been successfully tested on the ß2-adrenergic receptor (ß2-AR) binding the four fenoterol stereoisomers by both metadynamics simulations and replica-exchange MD. Both the methods gave very similar results, fully confirming the presence of stereoselective effects in the fenoterol-ß2-AR interactions. However, the metadynamics-based approach offered much better efficiency of sampling which allows for significant reduction of the unphysical region in SIP.

Bile enhances glucose uptake, reduces permeability, and modulates effects of lectins, trypsin inhibitors and saponins on intestinal tissue.

Antinutritional factors (ANFs) can disrupt digestive and other intestinal functions. ANFs in soybean meal (SBM) are implicated in proliferative and inflammatory responses in the intestine of various (functionally) monogastric animals, including Atlantic salmon (Salmo salar L.). The goal of the current study was to investigate the effect of ex vivo exposure of mid and distal intestinal tissue of salmon to soybean saponins (SAP), lectin (LEC) and Kunitz' trypsin inhibitor (KTI), singly and in combination, on epithelial function, as assessed by measuring in vitro glucose uptake pathways along a glucose concentration gradient. As solubilization of SAP in the calcium-containing Ringer's solution was problematic but resolved with the addition of a physiological concentration of bile collected from the gall bladder of salmon, an evaluation of bile effects became an added element. Results indicated that bile increased baseline glucose absorption and possibly transport, and also had a protective effect on the epithelial barrier, at least partially due to taurocholate. Compared to controls, tissues exposed to LEC+bile, KTI+bile and LEC+KTI+bile exhibited increased glucose uptake at the higher glucose concentrations, apparently due to markedly increased tissue permeability. Addition of SAP, however, attenuated the response, possibly by binding bile components. SAP+bile, also in combination with LEC and/or KTI, as well as LEC, KTI and LEC+KTI without bile often reduced transcellular glucose uptake pathways, while maintaining low tissue permeability. SAP+LEC+KTI+bile, LEC and KTI caused the most marked reductions. The distal intestine was more affected, reflecting the restriction of in vivo SBM-induced inflammatory changes to this region.

Comparative evaluation of in vitro efficacy of colesevelam hydrochloride tablets.

CONTEXT: Colesevelam hydrochloride is used as an adjunct to diet and exercise to reduce elevated low-density lipoprotein (LDL) cholesterol in patients with primary hyperlipidemia as well as to improve glycemic control in patients with type 2 diabetes. This is likely to result in submission of abbreviated new drug applications (ANDA). OBJECTIVE: This study was conducted to compare the efficacy of two tablet products of colesevelam hydrochloride based on the in vitro binding of bile acid sodium salts of glycocholic acid (GC), glycochenodeoxycholic acid (GCDA) and taurodeoxycholic acid (TDCA). METHODS: Kinetic binding study was carried out with constant initial bile salt concentrations as a function of time. Equilibrium binding studies were conducted under conditions of constant incubation time and varying initial concentrations of bile acid sodium salts. The unbound concentration of bile salts was determined in the samples of these studies. Langmuir equation was utilized to calculate the binding constants k1 and k2. RESULTS: The amount of the three bile salts bound to both the products reached equilibrium at 3 h. The similarity factor (f2) was 99.5 based on the binding profile of total bile salts to the test and reference colesevelam tablets as a function of time. The 90% confidence interval for the test to reference ratio of k2 values were 96.06-112.07 which is within the acceptance criteria of 80-120%. CONCLUSION: It is concluded from the results that the test and reference tablets of colesevelam hydrochloride showed a similar in vitro binding profile and capacity to bile salts.

Cyclodextrins: Establishing building blocks for AI-driven drug design by determining affinity constants in silico.

Cyclodextrins (CDs) are cyclic carbohydrate polymers that hold significant promise for drug delivery and industrial applications. Their effectiveness depends on their ability to encapsulate target molecules with strong affinity and specificity, but quantifying affinities in these systems accurately is challenging for a variety of reasons. Computational methods represent an exceptional complement to in vitro assays because they can be employed for existing and hypothetical molecules, providing high resolution structures in addition to a mechanistic, dynamic, kinetic, and thermodynamic characterization. Here, we employ potential of mean force (PMF) calculations obtained from guided metadynamics simulations to characterize the 1:1 inclusion complexes between four different modified ßCDs, with different type, number, and location of substitutions, and two sterol molecules (cholesterol and 7-ketocholesterol). Our methods, validated for reproducibility through four independent repeated simulations per system and different post processing techniques, offer new insights into the formation and stability of CD-sterol inclusion complexes. A systematic distinct orientation preference where the sterol tail projects from the CD's larger face and significant impacts of CD substitutions on binding are observed. Notably, sampling only the CD cavity's wide face during simulations yielded comparable binding energies to full-cavity sampling, but in less time and with reduced statistical uncertainty, suggesting a more efficient approach. Bridging computational methods with complex molecular interactions, our research enables predictive CD designs for diverse applications. Moreover, the high reproducibility, sensitivity, and cost-effectiveness of the studied methods pave the way for extensive studies of massive CD-ligand combinations, enabling AI algorithm training and automated molecular design.

Unraveling the molecular dynamics of sugammadex-rocuronium complexation: A blueprint for cyclodextrin drug design.

Sugammadex, marketed as Bridion™, is an approved cyclodextrin (CD) based drug for the reversal of neuromuscular blockade in adults undergoing surgery. Sugammadex forms an inclusion complex with the neuromuscular blocking agent (NMBA) rocuronium, allowing rapid reversal of muscle paralysis. In silico methods have been developed for studying CD inclusion complexes, aimed at accurately predicting their structural, energetic, dynamic, and kinetic properties, as well as binding constants. Here, a computational study aimed at characterizing the sugammadex-rocuronium system from the perspective of docking calculations, free molecular dynamics (MD) simulations, and biased metadynamics simulations with potential of mean force (PMF) calculations is presented. The aim is to provide detailed information about this system, as well as to use it as a model system for validation of the methods. This method predicts results in line with experimental evidence for both the optimal structure and the quantitative value for the binding constant. Interestingly, there is a less profound preference for the orientation than might be assumed based on electrostatic interactions, suggesting that both orientations may exist in solution. These results show that this technology can efficiently analyze CD inclusion complexes and could be used to facilitate the development and optimization of novel applications for CDs.

Designing a Simple Electrochemical Genosensor for the Detection of Urinary PCA3, a Prostate Cancer Biomarker.

This study investigates the feasibility of a simple electrochemical detection of Prostate Cancer Antigen 3 (PCA3) fragments extracted from patients' urine, using a thiolated single-strand DNA probe immobilized on a gold surface without using a redox probe. To enhance the PCA3 recognition process, we conducted a comparative analysis of the hybridization location using two thiolated DNA probes: Probe 1 targets the first 40 bases, while Probe 2 targets the fragment from bases 47 to 86. Hybridization with PCA3 followed, using square wave voltammetry. The limit of detection of the designed genosenors were of the order of (2.2 ng/mL), and (1.6 ng/mL) for Probes 1 and 2, respectively, and the subsequent sensitivities were of the order of (0.09 ± 0.01) µA-1 · µg-1 · mL and (0.10 ± 0.01) µA-1 · µg-1 · mL. Specificity tests were then conducted with the sensor functionalized with Probe 2, as it presents better analytical performances. The electrochemical results indicate that the designed sensor can clearly discriminate a complementary target from a non-complementary one. A further modeling of the calibration curves with the Power Law/Hill model indicates that the dissociation constant increases by one order of magnitude, confirming the ability of the designed sensor to perfectly discriminate complementary targets from non-complementary ones.

Expression of VEGFR2 Ligand Binding Domain in Pichia pink™ 4 Cells and Evaluation of Its Interactions with VEGF-A165 Receptor Binding Domain.

Vascular endothelial growth factor A165 (VEGF-A165) and VEGF receptor 2 (KDR) are important mediators of angiogenesis. We aimed to express the soluble KDR ligand-binding domain (sKDR1-3) and evaluate its interaction with the VEGF-A165 receptor-binding domain (VEGFA165-RBD). sKDR1-3 DNA was designed and subcloned into pPinkα-HC plasmid. The cassette was transfected into the Pichia pink™ 4 genome by homologous recombination. We optimized the expression of sKDR1-3 under the induction of different methanol concentrations. VEGFA165-RBD was expressed in E. coli BL21 harboring pET28a( +)─VEGFA165-RBD vector under induction with IPTG with/without lactose. Interaction and biological activity of sKDR1-3 and VEGFA165-RBD were investigated by ELISA and anti-proliferation tests. sKDR1-3 migrated on SDS-PAGE gel as a 35-180 kDa protein due to glycosylation. The relative expression level of sKDR1-3 under 1% methanol was higher than 0.5% and 4% methanol induction. IPTG and cysteine were suitable for induction and refolding of VEGFA165-RBD. 25 ng sKDR1-3 and 20 ng VEGFA165-RBD showed strong binding. sKDR1-3 bound to VEGFA165-RBD and VEGF-A165 with dissociation constants of 0.148 and 0.2 nM, respectively. 4-10 nM concentrations of sKDR1-3 inhibited the proliferation of HUVE cells induced by 5 nM VEGFA165-RBD. In consideration, sKDR1-3 in the nanomolar concentration range, is a promising anticancer drug to inhibit angiogenesis.

Exploring a potential palonosetron allosteric binding site in the 5-HT(3) receptor.

Palonosetron (Aloxi) is a potent second generation 5-HT(3) receptor antagonist whose mechanism of action is not yet fully understood. Palonosetron acts at the 5-HT(3) receptor binding site but recent computational studies indicated other possible sites of action in the extracellular domain. To test this hypothesis we mutated a series of residues in the 5-HT3A receptor subunit (Tyr(73), Phe(130), Ser(163), and Asp(165)) and in the 5-HT3B receptor subunit (His(73), Phe(130), Glu(170), and Tyr(143)) that were previously predicted by in silico docking studies to interact with palonosetron. Homomeric (5-HT(3)A) and heteromeric (5-HT(3)AB) receptors were then expressed in HEK293 cells to determine the potency of palonosetron using both fluorimetric and radioligand methods to test function and ligand binding, respectively. The data show that the substitutions have little or no effect on palonosetron inhibition of 5-HT-evoked responses or binding. In contrast, substitutions in the orthosteric binding site abolish palonosetron binding. Overall, the data support a binding site for palonosetron at the classic orthosteric binding pocket between two 5-HT3A receptor subunits but not at allosteric sites previously identified by in silico modelling and docking.