Estimated Dissociation Constant Research Articles

Design and construction of a phage-displayed Camelid nanobody library using a simple bioinformatics method

BackgroundRational design of synthetic phage-displayed libraries requires the identification of the most appropriate positions for randomization using defined amino acid sets to recapitulate the natural occurrence. The present study uses position-specific scoring matrixes (PSSMs) for identifying and randomizing Camelidae nanobody (VHH) CDR3. The functionality of a synthetic VHH repertoire designed by this method was tested for discovering new VHH binders to recombinant coagulation factor VII (rfVII). MethodsBased on PSSM analysis, the CDR3 of cAbBCII10 VHH framework was identified, and a set of amino acids for the substitution of each PSSM-CDR3 position was defined. Using the Rosetta design SwiftLib tool, the final repertoire was back-translated to a degenerate nucleotide sequence. A synthetic phage-displayed library was constructed based on this repertoire and screened for anti-rfVII binders. ResultsA synthetic phage-displayed VHH library with 1 × 108 variants was constructed. Three VHH binders to rfVII were isolated from this library with estimated dissociation constants (KD) of 1 × 10−8 M, 5.8 × 10−8 M and 2.6 × 10-7 M. ConclusionPSSM analysis is a simple and efficient way to design synthetic phage-displayed libraries.

Dissociation Constant of Di‐ and Tri‐Basic Solvents Based on Piperazine and Its Derivatives for Post‐Combustion CO2 Capture

AbstractAmine solvents remain popular in the industries for post combustion carbon dioxide (CO2) capture. Dissociation constant of basic amine solvents plays significant role to influence the CO2 absorption. It is thus important to study the dissociation of new amine solvents for assessing their viability for CO2 capture. In the present work, potentiometric titration method is employed to determine the dissociation constants of dibasic (e. g., Piperazine (PZ), 1‐methyl piperazine (1‐MPZ) and tribasic amines (1‐(2‐aminoethyl‐piperazine) (AEPZ) which could be promising for formulating blended solvent system towards enhancing the rate of CO2 absorption. The estimated dissociation constants for the PZ and 1‐MPZ while match well with reported results, the dissociation constants for AEPZ has been determined newly here within the temperature range 298—318 K. In addition, the structural geometric parameters and Natural Bond Order (NBO) calculations of the amine solvents are performed based on Density Functional Theory (DFT). The NBO calculations predict charge distribution on the nitrogen and other elements, and envisage its effect on the pKa values of the amines. Further, potential energy surface (PES) scanning calculations are performed for AEPZ to explain it's ring inversion which is responsible for obtaining a single pKa value in experimental studies.

In vitro and in vivo characterization of human serum albumin-based PEGylated nanoparticles for BDNF and NT3 codelivery

The utilization of neurotrophins in medicine shows significant potential for addressing neurodegenerative conditions, such as age-related macular degeneration (AMD). However, the therapeutic use of neurotrophins has been restricted due to their short half-life. Here, we aimed to synthesize PEGylated nanoparticles based on electrostatic-driven interactions between human serum albumin (HSA), a carrier for adsorption; neurotrophin-3 (NT3); and brain-derived neurotrophic factor (BDNF). Electrophoretic (ELS) and multi-angle dynamic light scattering (MADLS) revealed that the PEGylated HSA-NT3-BDNF nanoparticles ranged from 10 to 430 nm in diameter and exhibited a low polydispersity index (<0.4) and a zeta potential of −8 mV. Based on microscale thermophoresis (MST), the estimated dissociation constant (Kd) from the HSA molecule of BDNF was 1.6 μM, and the Kd of NT3 was 732 μM. The nanoparticles were nontoxic toward ARPE-19 and L-929 cells in vitro and efficiently delivered BDNF and NT3. Based on the biodistribution of neurotrophins after intravitreal injection into BALB/c mice, both nanoparticles were gradually released in the mouse vitreous body within 28 days. PEGylated HSA-NT3-BDNF nanoparticles stabilize neurotrophins and maintain this characteristic in vivo. Thus, given the simplicity of the system, the nanoparticles may enhance the treatment of a variety of neurological disorders in the future.

Biosensing with Surface-Charge-Modulated Graphene Field-Effect Transistors beyond Nonlinear Electrolytic Screening.

In field-effect transistor (FET) biosensors, charge screening in electrolyte solutions limits the sensitivity, thereby restricting the applicability of FET sensors. This is particularly pronounced in graphene FET (GFET) biosensors, where the bare graphene surface possesses a strongly negative charge, which impedes the high sensitivity of GFETs owing to nonlinear electrolytic screening at the interfaces between graphene and liquid. In this study, we counteracted the negative surface charge of graphene by decorating positively charged compounds and demonstrated the sensing of C-reactive protein (CRP) with surface-charge-modulated GFETs (SCM-GFETs). We integrated multiple SCM-GFETs with anti-CRP antibodies and nonfunctionalized GFETs into a chip and measured differentials to eliminate background changes to improve measurement reliability. The FET response corresponded to the fluorescence images, which visualized the specific adsorption of CRP. The estimated dissociation constant was consistent with previously reported values; this supports the conclusion that the results are attributed to specific adsorption. Conversely, the signal in GFETs without decoration was obscured by noise because of nonlinear electrolytic screening, further emphasizing the significance of surface-charge modulation. The limit of detection of the system was determined to be 2.9 nM. This value has the potential to be improved through further optimization of the surface charges to align with specific applications. Our devices effectively circumvent nonlinear electrolytic screening, opening the door for further advancements in GFET biosensor technology.

Finding a perfect match of ion-exchanger and plasticizer for ion-selective sensors

Application of neutral ionophore based ion-selective sensors requires presence of ion-exchanger in the receptor phase, silently assuming that it is not only soluble but also dissociates to ions in the applied plasticizer. Although for typically applied ion-selective membrane constituents (plasticizers - ion-exchanger pairs) dissociation of ion-exchangers to ions is proven by theoretical (or close to) performance of resulting sensors, search for alternative plasticizers or ion-exchangers requires a method allowing estimation of the match of properties of involved compounds. In this work we propose a simple optical approach allowing estimation of ion-exchanger interactions with plasticizer. The results were confirmed by conductivity studies of model plasticizers solutions. The estimated dissociation constants of model ion-exchangers in plasticizers used are in excellent agreement with the results of optical studies. It was shown that solubility coupled with poor dissociation to ions of ion-exchanger affects performance of the resulting ion-selective membrane. Rational choice of properties of ion-exchanger and plasticizer allows finding a perfect match of the two, that results in improvements in performance of sensors (e.g. detection limits). As model sensors potassium and sodium ion-selective electrodes with poly(vinyl chloride) (PVC) based membranes, plasticized with classical plasticizer bis(2-ethylhexyl sebacate) or biodegradable alternative acetyl tributyl citrate, were prepared and studied using selected ion-exchangers.

Superoxide dismutase A (SodA) is a c-di-GMP effector protein governing oxidative stress tolerance in Stenotrophomonas maltophilia

C-di-GMP is a bacterial second messenger implicated in the regulation of many key functions including antibiotic tolerance and biofilm formation. Our understanding of how c-di-GMP exerts its action via receptors to modulate different biological functions is still limited. Here we used a c-di-GMP affinity pull-down assay coupled to LC-MS/MS to identify c-di-GMP-binding proteins in the opportunistic pathogen Stenotrophomonas maltophilia. This analysis identified Smlt3238 (SodA), a protein of the superoxide dismutase family, as a c-di-GMP-binding protein. Microscale thermophoresis showed that purified SodA protein bound c-di-GMP with an estimated dissociation constant (Kd) value of 141.5 μM. Using various in vivo and in vitro experiments, we demonstrated that c-di-GMP modulates the enzyme activity of SodA directly. Circular dichroism experiments revealed that SodA protein gradually altered its basic structure with increasing levels of c-di-GMP. Phenotypic experiments conducted in the presence of a range of intracellular c-di-GMP levels showed that SodA function is modulated by c-di-GMP. The findings thus identify a novel c-di-GMP binding protein that governs oxidative stress tolerance in S. maltophilia.

Screening of EWI-2-Derived Peptides for Targeting Tetraspanin CD81 and Their Effect on Cancer Cell Migration.

CD81, a transmembrane protein belonging to the tetraspanin family, has recently been suggested as a therapeutic target for cancers. Here, we screened peptides that bind to the tetraspanin CD81 protein, and evaluated their inhibitory activity in cancer cell migration. To screen for CD81-binding peptides (CD81-BP), a peptide array membrane was prepared from the amino acid sequence of the EWI-2 protein, a major partner of CD81, before binding to fluorescently labeled CD81. As a result, four candidate CD81-BPs were identified and characterized. In particular, the CFMKRLRK peptide (called P152 in this study) was found to be the best candidate that preferentially binds to the extracellular loop of CD81, with an estimated dissociation constant of 0.91 µM. Since CD81 was reported to promote cancer cell migration, an initial step in metastasis, the Boyden chamber assay, was next performed to assess the effect of CD81-BP candidates on the migration of MDA-MB-231 human breast cancer cells. Interestingly, our result indicated that P152 could suppress MDA-MB-231 cell migration at the level comparable to that of an anti-human CD81 antibody (5A6). Thus, we propose these CD81-BPs with the anti-migration property against cancer cells for the development of novel therapeutic strategies.

Construction of the lactate-sensing fibremats by confining sensor fluorescent protein of lactate inside nanofibers of the poly(HPMA/DAMA)/ADH-nylon 6 core-shell fibremat.

The development of a new materials platform capable of sustaining the functionality of proteinous sensor molecules over an extended period without being affected by biological contaminants in living systems, such as proteases, is highly demanded. In this study, our primary focus was on fabricating new core-shell fibremats using unique polymer materials, capable of functionalizing encapsulated sensor proteins while resisting the effects of proteases. The core-fibre parts of core-shell fibremats were made using a newly developed post-crosslinkable water-soluble copolymer, poly(2-hydroxypropyl methacrylamide)-co-poly(diacetone methacrylamide), and the bifunctional crosslinking agent, adipic dihydrazide, while the shell layer of the nanofibers was made of nylon 6. Upon encapsulating the lactate-sensor protein eLACCO1.1 at the core-fibre part, the fibremat exhibited a distinct concentration-dependent fluorescence response, with a dynamic range of fluorescence alteration exceeding 1000% over the lactate concentration range of 0 to 100 mM. The estimated dissociation constant from the titration data was comparable to that estimated in a buffer solution. The response remained stable even after 5 cycles and in the presence of proteases. These results indicates that our core-shell fibremat platform could serve as effective immobilizing substrates for various sensor proteins, facilitating continuous and quantitative monitoring of various low-molecular-weight metabolites and catabolites in a variety of biological samples.

Kinetics and interaction studies of anti-tetraspanin antibodies and ICAM-1 with extracellular vesicle subpopulations using continuous flow quartz crystal microbalance biosensor

Continuous flow quartz crystal microbalance (QCM) was utilized to study binding kinetics between EV subpopulations (exomere- and exosome-sized EVs) and four affinity ligands: monoclonal antibodies against tetraspanins (anti-CD9, anti-CD63, and anti-CD81) and recombinant intercellular adhesion molecule-1 (ICAM-1) or CD54 protein). High purity CD9+, CD63+, and CD81+ EV subpopulations of <50 nm exomeres and 50–80 nm exosomes were isolated and fractionated using our recently developed on-line coupled immunoaffinity chromatography – asymmetric flow field-flow fractionation system. Adaptive Interaction Distribution Algorithm (AIDA), specifically designed for the analysis of complex biological interactions, was used with a four-step procedure for reliable estimation of the degree of heterogeneity in rate constant distributions. Interactions between exomere-sized EVs and anti-tetraspanin antibodies demonstrated two interaction sites with comparable binding kinetics and estimated dissociation constants Kd ranging from nM to fM. Exomeres exhibited slightly higher affinity compared to exosomes. The highest affinity with anti-tetraspanin antibodies was achieved with CD63+ EVs. The interaction of EV subpopulations with ICAM-1 involved in cell internalization of EVs was also investigated. EV – ICAM-1 interaction was also of high affinity (nM to pM range) with overall lower affinity compared to the interactions of anti-tetraspanin antibodies and EVs. Our findings proved that QCM is a valuable label-free tool for kinetic studies with limited sample concentration, and that advanced algorithms, such as AIDA, are crucial for proper determination of kinetic heterogeneity. To the best of our knowledge, this is the first kinetic study on the interaction between plasma-derived EV subpopulations and anti-tetraspanin antibodies and ICAM-1.

BioMIPs: molecularly imprinted silk fibroin nanoparticles to recognize the iron regulating hormone hepcidin

The possibility to prepare molecularly imprinted nanoparticles from silk fibroin was recently demonstrated starting from methacrylated silk fibroin and choosing a protein as template. Here, we attempted the imprinting of fibroin-based molecularly imprinted polymers (MIPs), called bioMIPs, using as a template hepcidin that is a iron-metabolism regulator-peptide, possessing a hairpin structure. A homogeneous population (PDI < 0.2) of bioMIPs with size ~50 nm was produced. The bioMIPs were selective for the template; the estimated dissociation constant for hepcidin was KD = 3.6 ± 0.5 10−7 M and the average number of binding sites per bioMIP was equal to 2. The bioMIPs used in a competitive assay for hepcidin in serum showed a detection range of 1.01 10−7– 6.82 10−7 M and a limit of detection of 3.29 10−8 M.Graphical abstract

Selection of a Nuclease-Resistant RNA Aptamer Targeting CD19.

Simple SummaryHaematological malignancies show a constantly growing incidence, accounting for 6.5% of new cancer cases worldwide. Among them, B-cell neoplasms often show resistance to conventional chemotherapy that is also associated with numerous adverse effects. Therefore, in order for the treatment outcome to be improved, the development of new safe and effective targeted therapeutic approaches represents a main challenge. In this regard, nucleic acid aptamers are very attractive molecules. Indeed, they show high affinity and specificity for their target, increased tumour penetration, and low toxicity. Recently, CD19 has emerged as a key surface marker of malignant B cells, suitable for the development of new compounds for malignant B-cell targeting. Here, we isolated an RNA aptamer targeting the human CD19 antigen on malignant B cells that was able to rapidly internalise into target cells. Therefore, it represents a useful carrier for secondary reagents and a promising tool for the development of new safe and effective targeted therapies for B-cell malignancy treatment.The transmembrane glycoprotein cluster of differentiation 19 (CD19) is a B cell–specific surface marker, expressed on the majority of neoplastic B cells, and has recently emerged as a very attractive biomarker and therapeutic target for B-cell malignancies. The development of safe and effective ligands for CD19 has become an important need for the development of targeted conventional and immunotherapies. In this regard, aptamers represent a very interesting class of molecules. Additionally referred to as ‘chemical antibodies’, they show many advantages as therapeutics, including low toxicity and immunogenicity. Here, we isolated a nuclease-resistant RNA aptamer binding to the human CD19 glycoprotein. In order to develop an aptamer also useful as a carrier for secondary reagents, we adopted a cell-based SELEX (Systematic Evolution of Ligands by EXponential Enrichment) protocol adapted to isolate aptamers able to internalise upon binding to their cell surface target. We describe a 2′-fluoro pyrimidine modified aptamer, named B85.T2, which specifically binds to CD19 and shows an exquisite stability in human serum. The aptamer showed an estimated dissociation constant (KD) of 49.9 ± 13 nM on purified human recombinant CD19 (rhCD19) glycoprotein, a good binding activity on human B-cell chronic lymphocytic leukaemia cells expressing CD19, and also an effective and rapid cell internalisation, thus representing a promising molecule for CD19 targeting, as well as for the development of new B-cell malignancy-targeted therapies.

Withanolide Derivative 2,3-Dihydro-3β-methoxy Withaferin-A Modulates the Circadian Clock via Interaction with RAR-Related Orphan Receptor α (RORa).

Withanolide derivatives have anticancer, anti-inflammatory, and other functions and are components of Indian traditional Ayurvedic medicine. Here, we found that 2,3-dihydro-3β-methoxy withaferin-A (3βmWi-A), a derivative of withaferin-A (Wi-A) belonging to a class of withanolides that are abundant in Ashwagandha (Withania somnifera), lengthened the period of the circadian clock. This compound dose-dependently elongated circadian rhythms in Sarcoma 180 cancer cells and in normal fibroblasts including NIH3T3 and spontaneously immortalized mouse embryonic fibroblasts (MEF). Furthermore, 3βmWi-A dose-dependently upregulated the mRNA expression and promoter activities of Bmal1 after dexamethasone stimulation and of the nuclear orphan receptors, Rora and Nr1d1, that comprise the stabilization loop for Bmal1 oscillatory expression. We showed that 3βmWi-A functions as an inverse agonist for RORa with an IC50 of 11.3 μM and that 3βmWi-A directly, but weakly, interacts with RORa (estimated dissociation constant [Kd], 5.9 μM). We propose that 3βmWi-A is a novel modulator of circadian rhythms.

A general model to optimise CuII labelling efficiency of double-histidine motifs for pulse dipolar EPR applications.

Electron paramagnetic resonance (EPR) distance measurements are making increasingly important contributions to studies of biomolecules underpinning health and disease by providing highly accurate and precise geometric constraints. Combining double-histidine (dH) motifs with CuII spin labels shows promise for further increasing the precision of distance measurements, and for investigating subtle conformational changes. However, non-covalent coordination-based spin labelling is vulnerable to low binding affinity. Dissociation constants of dH motifs for CuII-nitrilotriacetic acid were previously investigated via relaxation induced dipolar modulation enhancement (RIDME), and demonstrated the feasibility of exploiting the dH motif for EPR applications at sub-μM protein concentrations. Herein, the feasibility of using modulation depth quantitation in CuII-CuII RIDME to simultaneously estimate a pair of non-identical independent KD values in such a tetra-histidine model protein is addressed. Furthermore, we develop a general speciation model to optimise CuII labelling efficiency, depending upon pairs of identical or disparate KD values and total CuII label concentration. We find the dissociation constant estimates are in excellent agreement with previously determined values, and empirical modulation depths support the proposed model.

AChE Inhibition Study for Alzheimer's Disease Treatment: An In-silico Study

Alzheimer's disease (AD) is the most common form of dementia associated with plaques and tangles in the brain. Several acetylcholinesterase inhibitors have been clinically used to delay or halt the progression of the disease. Solanadine (Snd) and gamma solamargine (Gsm) have been shown to inhibit acetylcholinesterase (AChE). The current study attempts to describe the molecular interactions between human brain AChE and inhibitors Snd and Gsm. The free energy of binding and estimated dissociation constant (Ki) for the 'Snd-AChE Catalytic Anionic site (CAS)-interaction' were determined to be –9.51 kcal/mol and 107.54 nM, respectively and the free energy of binding and estimated Ki for the 'Gsm -AChE CAS-interaction' were determined to be – 8.64 kcal/mol and 463.88 nM, respectively. Hydrophobic interactions, polar interactions, and hydrogen bonding play an important role in the correct positioning of Snd within the 'catalytic site' of AChE to permit docking, while hydrophobic interactions and polar interactions play a significant role in the correct positioning of Gsm within the 'catalytic site' of AChE to permit docking. It is hoped that the information provided in this study will help in the design of AChE-inhibitors as anti-Alzheimer agents.

Snare Priming Inhibition via Phosphatidic Acid Induced Sec18 Conformational Changes and Competitive Small Molecule Binding to Sec18

Membrane fusion is mediated by SNARE proteins, and is vital for a multitude of cellular transport phenomena. Post fusion, cis-SNAREs are recycled for continued fusion, in an ATP dependent process called priming. The unique job of priming all cellular SNAREs is the responsibility of AAA+ ATPase NSF or Sec18. In a joint computational and experimental effort, we show that Phosphatidic Acid (PA) inhibits the priming of cis-SNARE complexes by inducing conformational changes in Sec18 protomer. We further studied priming inhibition via a new small molecule inhibitor of Sec18 called IPA, which inhibits fusion, priming, and competitively inhibits Sec18 binding to PA. We identify potential PA binding sites to Sec18 using computational flooding of short tailed PA, as well as using an HMMM PA rich membrane to capture unbiased spontaneous membrane insertion of Sec18 monomers. In addition, to further sample potential binding sites of both PA as well as IPA on Sec18 we employed ensemble molecular docking using AutoDock Vina, with consecutive molecular dynamics simulations performed for the top poses using NAMD for characterization of ligand stability. Finally, we performed Random Accelerated Molecular dynamics simulations on the most stable resultant poses to estimate dissociation constants, which were compared to results from liposome binding experiments providing insight into the mechanism of priming regulation via Sec18.

Assembly–disassembly is coupled to the ATPase cycle of tobacco Rubisco activase

The carbon-fixing activity of enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is regulated by Rubisco activase (Rca), a ring-forming ATPase that catalyzes inhibitor release. For higher plant Rca, the catalytic roles played by different oligomeric species have remained obscure. Here, we utilized fluorescence-correlation spectroscopy to estimate dissociation constants for the dimer-tetramer, tetramer-hexamer, hexamer-12-mer, and higher-order assembly equilibria of tobacco Rca. A comparison of oligomer composition with ATPase activity provided evidence that assemblies larger than hexamers are hydrolytically inactive. Therefore, supramolecular aggregates may serve as storage forms at low-energy charge. We observed that the tetramer accumulates only when both substrate and product nucleotides are bound. During rapid ATP turnover, about one in six active sites was occupied by ADP, and ∼36% of Rca was tetrameric. The steady-state catalytic rate reached a maximum between 0.5 and 2.5 μm Rca. In this range, significant amounts of dimers, tetramers, and hexamers coexisted, although none could fully account for the observed activity profile. Therefore, we propose that dynamic assembly-disassembly partakes in the ATPase cycle. According to this model, the association of dimers with tetramers generates a hexamer that forms a closed ring at high ATP and magnesium levels. Upon hydrolysis and product release, the toroid breaks open and dissociates into a dimer and tetramer, which may be coupled to Rubisco remodeling. Although a variant bearing the R294V substitution assembled in much the same way, highly stabilized states could be generated by binding of a transition-state analog. A tight-binding pre-hydrolysis state appears to become more accessible in thermally labile Rcas.

Presence of a Haloarchaeal Halorhodopsin-Like Cl- Pump in Marine Bacteria.

Light-driven ion-pumping rhodopsins are widely distributed among bacteria, archaea, and eukaryotes in the euphotic zone of the aquatic environment. H+-pumping rhodopsin (proteorhodopsin: PR), Na+-pumping rhodopsin (NaR), and Cl−-pumping rhodopsin (ClR) have been found in marine bacteria, which suggests that these genes evolved independently in the ocean. Putative microbial rhodopsin genes were identified in the genome sequences of marine Cytophagia. In the present study, one of these genes was heterologously expressed in Escherichia coli cells and the rhodopsin protein named Rubricoccus marinus halorhodopsin (RmHR) was identified as a light-driven inward Cl− pump. Spectroscopic assays showed that the estimated dissociation constant (Kd,int.) of this rhodopsin was similar to that of haloarchaeal halorhodopsin (HR), while the Cl−-transporting photoreaction mechanism of this rhodopsin was similar to that of HR, but different to that of the already-known marine bacterial ClR. This amino acid sequence similarity also suggested that this rhodopsin is similar to haloarchaeal HR and cyanobacterial HRs (e.g., SyHR and MrHR). Additionally, a phylogenetic analysis revealed that retinal biosynthesis pathway genes (blh and crtY) belong to a phylogenetic lineage of haloarchaea, indicating that these marine Cytophagia acquired rhodopsin-related genes from haloarchaea by lateral gene transfer. Based on these results, we concluded that inward Cl−-pumping rhodopsin is present in genera of the class Cytophagia and may have the same evolutionary origins as haloarchaeal HR.

Lipophilicity Influences Drug Binding to α1-Acid Glycoprotein F1/S Variants But Not to the A Variant.

ObjectiveHuman α1-acid glycoprotein has genetic variants, the F1, S, and A variants, which can be separated isoelectrophoretically. These variants show differences in their affinity of binding to several drugs. In this study, we investigated the factors determining drug binding to these α1-acid glycoprotein genetic variants using disopyramide, warfarin, and tamsulosin as marker compounds.MethodsBinding of the marker drugs to human α1-acid glycoprotein was determined by ultra-filtration in the presence or absence of various other drugs. For screening of the α1-acid glycoprotein variants to which the marker drugs became bound, the effects of various other drugs on their binding were studied. The binding data were analyzed using a competitive inhibition model and the relationship between the estimated dissociation constants and physicochemical properties, such as log P, was also analyzed.ResultsThe binding of tamsulosin was significantly decreased by aprindine, carvedilol, erythromycin, thioridazine, and warfarin, but not by disopyramide. The dissociation constants of drugs bound to F1/S variants were significantly correlated with their lipophilicity, but those for the A variant were not.ConclusionsWe were able to develop a simple screening method for determining individual α1-acid glycoprotein variants to which drugs would bind. The binding of drugs to F1/S variants may be determined mainly by drug lipophilicity.Electronic supplementary materialThe online version of this article (doi:10.1007/s40268-017-0193-9) contains supplementary material, which is available to authorized users.

Ghrelin binding to serum albumin and its biological impact

Ghrelin is an octanoylated peptide hormone that plays a key role in the regulation of the body weight and glucose homeostasis. In plasma, ghrelin circulates bound to larger proteins whose identities are partially established. Here, we used size exclusion chromatography, mass spectrometry and isothermal titration microcalorimetry to show that ghrelin interacts with serum albumin. Furthermore, we found that such interaction displays an estimated dissociation constant (KD) in the micromolar range and involves albumin fatty-acid binding sites as well as the octanoyl moiety of ghrelin. Notably, albumin-ghrelin interaction reduces the spontaneous deacylation of the hormone. Both in vitro experiments—assessing ghrelin ability to inhibit calcium channels—and in vivo studies—evaluating ghrelin orexigenic effects—indicate that the binding to albumin affects the bioactivity of the hormone. In conclusion, our results suggest that ghrelin binds to serum albumin and that this interaction impacts on the biological activity of the hormone.

Characterization of muscarinic receptor binding by the novel radioligand, [3H]imidafenacin, in the bladder and other tissues of rats

The present study aimed to directly characterize specific binding sites of tritium ([3H])-labeled imidafenacin, a new radioligand for labeling muscarinic receptors, in the bladder and other peripheral or central nervous tissues of rats.Muscarinic receptors in rat tissues were measured by radioligand binding assay using [3H]imidafenacin.Specific [3H]imidafenacin binding in rat tissues was saturable, reversible, and of high affinity. Estimated dissociation constants (Kd values) were significantly lower in submaxillary gland and prostate and higher in heart than in bladder, indicating lower Kd values in M1 and M3 subtype- than M2 subtype-dominating tissues. Unlabeled imidafenacin and clinically used antimuscarinic agents competed with [3H]imidafenacin for binding sites in bladder and other tissues in a concentration-dependent manner, which indicated pharmacological specificity of [3H]imidafenacin binding sites. Pretreatment with N-(2-chloroethyl)-4-piperidinyl diphenylacetate (4-DAMP mustard), an irreversible inactivating agent of M3 subtype, significantly decreased the number of [3H]imidafenacin binding sites in bladder, submaxillary gland, and colon, but not in heart. [3H]imidafenacin labeled muscarinic receptors in M1 and M3 subtype-dominating tissues with higher affinity than [N-methyl-3H]scopolamine methyl chloride (NMS).[3H]imidafenacin is a useful radioligand to label muscarinic receptors in M1- and M3-dominating tissues with high affinity.