Accurate measurement of avidin and streptavidin in crude biofluids with a new, optimized biotin-fluorescein conjugate

A new biotin-fluorescein conjugate with an ethylene diamine spacer was found to be the first fluorescent biotin derivative which truly mimicked d-biotin in terms of high affinity, fast association, and non-cooperative binding to avidin and streptavidin tetramers. These exceptional properties were attributed to the small size/length of the new ligand since all larger/longer biotin derivatives are known for their mutual steric hindrance and anti-cooperative binding in 4:1 complexes with avidin and streptavidin tetramers. Specific binding of the new biotin-fluorescein conjugate towards avidin and streptavidin was accompanied by 84-88% quenching of ligand fluorescence. In the accompanying study this effect was used for rapid estimation of avidin and streptavidin in a new 'single tube assay'. In the present study the strong quenching effect was utilized to accurately monitor stoichiometric titration of biotin-binding sites in samples with >/=200 pM avidin or streptavidin. The concentration was calculated from the consumption of fluorescent ligand up to the distinct breakpoint in the fluorescence titration profile which was marked by the abrupt appearance of strongly fluorescent ligands which were in excess. Due to this protocol the assay was not perturbed by background fluorescence or coloration in the unknown samples. The new fluorescence titration assay is particularly suited for quick checks on short notice because getting started only means to thaw an aliquot of a standardized stock solution of fluorescent ligand. No calibration is required for the individual assay and the ligand stock solution needs to be restandardized once per week (or once per year) when stored at -25 degrees C (or at -70 degrees C, respectively).     

Application of an allosteric model to describe the interactions among retinol binding protein 4, transthyretin, and small molecule retinol binding protein 4 ligands

Retinol binding protein 4 (RBP4) is a serum protein that serves as the major transport protein for retinol (vitamin A). Recent reports suggest that elevated levels of RBP4 are associated with insulin resistance and that insulin sensitivity may be improved by reducing serum RBP4 levels. This can be accomplished by administration of small molecules, such as fenretinide, that compete with retinol for binding to RBP4 and disrupt the protein-protein interaction between RBP4 and transthyretin (TTR), another serum protein that protects RBP4 from renal clearance. We developed a fluorescence resonance energy transfer (FRET) assay that measures the interaction between RBP4 and TTR and can be used to determine the binding affinities of RBP4 ligands. We present an allosteric model that describes the pharmacology of interaction among RBP4, TTR, retinol, and fenretinide, and we show data that support the model. We show that retinol increases the affinity of RBP4 for TTR by a factor of 4 and determine the affinity constants of fenretinide and retinyl acetate. The assay may be useful for characterizing small molecule ligands that bind to RBP4 and disrupt its interaction with TTR. In addition, such a model could be used to describe other protein-protein interactions that are modulated by small molecules.     

Surface plasmon resonance assay of inhibition by pharmaceuticals for thyroxine hormone binging to transport proteins

We developed a surface plasmon resonance (SPR) assay to estimate the competitive inhibition by pharmaceuticals for thyroxine (T4) binding to thyroid hormone transport proteins, transthyretin (TTR) and thyroxine binding globulin (TBG). In this SPR assay, the competitive inhibition of pharmaceuticals for introducing T4 into immobilized TTR or TBG on the sensor chip can be estimated using a running buffer containing pharmaceuticals. The SPR assay showed reproducible immobilization of TTR and TBG, and the kinetic binding parameters of T4 to TTR or TBG were estimated. The equilibrium dissociation constants of TTR or TBG measured by SPR did not clearly differ from data reported for other binding assays. To estimate the competitive inhibition of tetraiodothyroacetic acid, diclofenac, genistein, ibuprofen, carbamazepine, and furosemide, reported to be competitive or noncompetitive pharmaceuticals for T4 binding to TTR or TBG, their 50% inhibition concentrations (IC₅₀) (or 80% inhibition concentration, IC₈₀) were calculated from the change of T4 responses in sensorgrams obtained with various concentrations of the pharmaceuticals. Our SPR method should be a useful tool for predicting the potential of thyroid toxicity of pharmaceuticals by evaluating the competitive inhibition of T4 binding to thyroid hormone binding proteins, TTR and TBG.     

Transthyretin chemical chaperoning by flavonoids: Structure–activity insights towards the design of potent amyloidosis inhibitors

BackgroundMany polyphenols have been proposed as broad-spectrum inhibitors of amyloid formation. To investigate structure–activity relationships relevant for the interaction of flavonoids with transthyretin (TTR), the protein associated with familial amyloid polyneuropathy (FAP), we compared the effects of major tea catechins and their larger polymers theaflavins, side-by-side, on TTR amyloid formation process.MethodsInteraction of flavonoids with TTR and effect on TTR stability were assessed through binding assays and isoelectric focusing in polyacrylamide gel. TTR aggregation was studied, in vitro, by dynamic light scattering (DLS), transmission electron microscopy (TEM) and in cell culture, through cytotoxicity assays.ResultsTested flavonoids bound to TTR and stabilized the TTR tetramer, with different potencies. The flavonoids also inhibited in vitro formation of TTR small oligomeric species and in cell culture inhibited pathways involving caspase-3 activation and ER stress that are induced by TTR oligomers. In all assays performed the galloyl esters presented higher potency to inhibit aggregation than the non-gallated flavonoids tested.ConclusionsOur results highlight the presence of gallate ester moiety as key structural feature of flavonoids in chemical chaperoning of TTR aggregation. Upon binding to the native tetramer, gallated flavonoids redirect the TTR amyloidogenic pathway into unstructured nontoxic aggregation assemblies more efficiently than their non-gallated forms.General significanceOur findings suggest that galloyl moieties greatly enhance flavonoid anti-amyloid chaperone activity and this should be taken into consideration in therapeutic candidate drug discovery.     

Semi-quantitative models for identifying potent and selective transthyretin amyloidogenesis inhibitors

Rate-limiting dissociation of the tetrameric protein transthyretin (TTR), followed by monomer misfolding and misassembly, appears to cause degenerative diseases in humans known as the transthyretin amyloidoses, based on human genetic, biochemical and pharmacologic evidence. Small molecules that bind to the generally unoccupied thyroxine binding pockets in the native TTR tetramer kinetically stabilize the tetramer, slowing subunit dissociation proportional to the extent that the molecules stabilize the native state over the dissociative transition state—thereby inhibiting amyloidogenesis. Herein, we use previously reported structure-activity relationship data to develop two semi-quantitative algorithms for identifying the structures of potent and selective transthyretin kinetic stabilizers/amyloidogenesis inhibitors. The viability of these prediction algorithms, in particular the more robust in silico docking model, is perhaps best validated by the clinical success of tafamidis, the first-in-class drug approved in Europe, Japan, South America, and elsewhere for treating transthyretin aggregation-associated familial amyloid polyneuropathy. Tafamidis is also being evaluated in a fully-enrolled placebo-controlled clinical trial for its efficacy against TTR cardiomyopathy. These prediction algorithms will be useful for identifying second generation TTR kinetic stabilizers, should these be needed to ameliorate the central nervous system or ophthalmologic pathology caused by TTR aggregation in organs not accessed by oral tafamidis administration.     

Quantitation of aflatoxin B1 and aflatoxin B1 antibody by an enzyme-linked immunosorbent microassay.

A specific microtest plate enzyme immunoassay has been developed for the rapid quantitation of aflatoxin B1 at levels as low as 25 pg per assay. Multiple-site injection of rabbits with an aflatoxin B1 carboxymethyloxime-bovine serum albumin conjugate was used for the production of hyperimmune sera. Dilutions of the purified antibody were air dried onto microplates previously treated with bovine serum albumin and glutaraldehyde and then incubated with an aflatoxin B1 carboxymethyloxime-horseradish peroxidase conjugate. The amount of enzyme bound to antibody was determined by monitoring the change in absorbance at 414 nm after the addition of a substrate solution consisting of hydrogen peroxide and 2,2'-azino-di-3-ethyl-benzthiazoline-6-sulfonate. Antibody titers determined in this manner closely correlated with those determined by radioimmunoassay. Competition assays as performed by incubation of different aflatoxin analogs with the peroxidase conjugate showed that aflatoxins B1 and B2 and aflatoxicol caused the most inhibition of conjugate binding to antibody. Aflatoxins G1 and G2 inhibited the conjugate binding to a lesser degree, whereas aflatoxins M1 and B2a had no effect of the assay.     

Structural basis for the stabilization of amyloidogenic immunoglobulin light chains by hydantoins

Misfolding and aggregation of immunoglobulin light chains (LCs) leads to the degeneration of post-mitotic tissue in the disease immunoglobulin LC amyloidosis (AL). We previously reported the discovery of small molecule kinetic stabilizers of the native dimeric structure of full-length LCs, which slow or stop the LC aggregation cascade at the outset. A predominant structural category of kinetic stabilizers emerging from the high-throughput screen are coumarins substituted at the 7-position, which bind at the interface between the two variable domains of the light chain dimer. Here, we report the binding mode of another, more polar, LC kinetic stabilizer chemotype, 3,5-substituted hydantoins. Computational docking, solution nuclear magnetic resonance experiments, and x-ray crystallography show that the aromatic substructure emerging from the hydantoin 3-position occupies the same LC binding site as the coumarin ring. Notably, the hydantoin ring extends beyond the binding site mapped out by the coumarin hits. The hydantoin ring makes hydrogen bonds with both LC monomers simultaneously. The alkyl substructure at the hydantoin 5-position partially occupies a novel binding pocket proximal to the pocket occupied by the coumarin substructure. Overall, the hydantoin structural data suggest that a larger area of the LC variable-domain–variable-domain dimer interface is amenable to small molecule binding than previously demonstrated, which should facilitate development of more potent full-length LC kinetic stabilizers.     

Determination of bradykinin in rat urine by coupled-column high pressure liquid chromatography with precolumn derivatization with a water-soluble fluorogenic reagent

The green fluorescent protein (GFP) and its mutants have been extensively used to study various cellular processes and, more recently, as labels in binding assays. We have employed a mutant of GFP, an enhanced GFP (EGFP), in the development of homogeneous assays for biotin and cortisol. To demonstrate the feasibility of using EGFP as a label with different kinds of binders in the development of homogeneous assays, we employed the biotin-avidin and an antigen-antibody as the binding pairs. Biotin and cortisol were chemically conjugated to EGFP. A quenching of fluorescence intensity of EGFP was observed upon binding of avidin to the EGFP-biotin conjugate. The percentage fluorescence quenching observed decreased as the concentration of free biotin in the sample increased due to the fewer binding sites on avidin available for binding to the EGFP-biotin conjugate. A dose-response curve for biotin was generated by relating percentage fluorescence quenched with free biotin in the sample. To determine whether EGFP can undergo a similar type of homogeneous change when used as a label for antigen-antibody type of binding, cortisol was selected as a model analyte. In the presence of an anti-cortisol antibody the fluorescence signal of the EGFP-cortisol conjugate was quenched. A dose-response curve for cortisol was generated by relating the quenching in the fluorescence signal with varying amounts of free cortisol in the sample. This is the first time that GFP or one of its mutants has been employed as a label in homogeneous assays, thus enhancing the versatility of employing GFP or its mutants in a number of bioanalytical applications, such as clinical analysis and high-throughput screening systems.     

A simple and sensitive enzyme-mediated assay of biotin.

Free biotin was quantitated by a competition by coating biotin-bovine serum albumin conjugate on a polystyrene microplate for binding to avidin-beta-galactosidase conjugate. The enzyme conjugate remaining on the plate surface as a result of the competition was detected by reaction with one of the following fluorogenic substrates, resorufin beta-D-galactoside and fluorescein di-beta-D-galactoside, in a fluorescence plate reader. Free biotin as little as 0.1 nmol can be routinely detected.     

Retention of misfolded mutant transthyretin by the chaperone BiP/GRP78 mitigates amyloidogenesis

Carriers of the D18G transthyretin (TTR) mutation display an unusual central nervous system (CNS) phenotype with late onset of disease. D18G TTR is monomeric and highly prone to misfold and aggregate even at physiological conditions. Extremely low levels of mutant protein circulate both in human serum and cerebrospinal fluid, indicating impaired secretion of D18G TTR. Recent data show efficient selective ER-associated degradation (ERAD) of D18G TTR. One essential component of the ER-assisted folding machinery is the molecular chaperone BiP. Co-expression of BiP and D18G TTR, or BiP and wild-type (wt) TTR, or mutants A25T TTR and L55P TTR in Escherichia coli showed that only D18G TTR was significantly captured by BiP. Negligible capture of wt TTR and L55P TTR was seen and a sixfold smaller amount of A25T TTR bound to BiP compared to D18G TTR. These data correlate very well with thermodynamic and kinetic stability of the TTR variants, indicating that folding efficiency is inversely correlated to BiP capture. The complexes between BiP and D18G TTR were stable and could be isolated through affinity chromatography. Analytical ultracentrifugation and size-exclusion chromatography revealed that D18G TTR and BiP formed a mixture of 1:1 complexes and large soluble oligomers. The stoichiometry of captured D18G TTR versus BiP increased with increasing size of the oligomers. This indicates that BiP either worked as a molecular shepherd collecting the aggregation-prone mutant into stable oligomers or that BiP could bind to oligomers formed from misfolded mutant protein. Sequence analysis of bound TTR peptides to BiP revealed a bound sequence corresponding to residues 88-103 of TTR, comprising beta-strand F in the folded TTR monomer constituting part of the hydrogen bonding tetramer interface in native TTR. The F-strand has also been suggested as a possible elongation region of amyloid fibrils, implicating how substoichiomeric amounts of BiP could sequester prefibrillar amyloidogenic oligomers through binding to this part of TTR. BiP binding to D18G TTR was abolished by addition of ATP. The released D18G TTR completely misfolded into amyloid aggregates as shown by ThT fluorescence and Congo red binding.     

Transthyretin binding to A-Beta peptide--impact on A-Beta fibrillogenesis and toxicity

It has been suggested that transthyretin (TTR) is involved in preventing A-Beta fibrillization in Alzheimer’s disease (AD). Here, we characterized the TTR/A-Beta interaction by competition binding assays. TTR binds to different A-Beta peptide species: soluble (Kd, 28 nM), oligomers and fibrils; diverse TTR variants bind differentially to A-Beta. Transmission electron microscopy (TEM) analysis demonstrated that TTR is capable of interfering with A-Beta fibrillization by both inhibiting and disrupting fibril formation. Co-incubation of the two molecules resulted in the abolishment of A-Beta toxicity. Our results confirmed TTR as an A-Beta ligand and indicated the inhibition/disruption of A-Beta fibrils as a possible mechanism underlying the protective role of TTR in AD.     

The pathway by which the tetrameric protein transthyretin dissociates

The homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to its constituent monomers in order to enable partial denaturation that allows the process of amyloidogenesis associated with human pathology to ensue. The TTR quaternary structure contains two distinct dimer interfaces, one of which creates the two binding sites for the natural ligand thyroxine. Tetramer dissociation could proceed through three distinct pathways; scission into dimers along either of the two unique quaternary interfaces followed by dimer dissociation represents two possibilities. Alternatively, the tetramer could lose monomers sequentially. To elucidate the TTR dissociation pathway, we employed two different TTR constructs, each featuring covalent attachment of proximal subunits. We demonstrate that tethering the A and B subunits of TTR with a disulfide bond (as well as the symmetrically disposed C and D subunits) allows urea-mediated dissociation of the resulting (TTR-S-S-TTR)(2) construct, affording (TTR-S-S-TTR)(1) retaining a stable 16-stranded beta-sheet structure that is equivalent to the dimer not possessing a thyroid binding site. In contrast, linking the A and C subunits employing a peptide tether (TTR-L-TTR)(2) affords a kinetically stable quaternary structure that does not dissociate or denature in urea. Both tethered constructs and wild-type TTR exhibit analogous stability based on guanidine hydrochloride denaturation curves. The latter denaturant can denature the tetramer, unlike urea, which can only denature monomeric TTR; hence urea requires dissociation to monomers to function. Under native conditions, the (TTR-S-S-TTR)(2) construct is able to dissociate and incorporate subunits from labeled WT TTR homotetramers at a rate equivalent to that exhibited by WT TTR. In contrast, the (TTR-L-TTR)(2) construct is unable to exchange any subunits, even after 180 h. All of the data presented herein and elsewhere demonstrate that the pathway of TTR tetramer dissociation occurs by scission of the tetramer along the crystallographic C(2) axis affording AB and CD dimers that rapidly dissociate into monomers. Determination of the mechanism of dissociation provides an explanation for why small molecules that bind at the AB/CD dimer-dimer interface impose kinetic stabilization upon TTR and disease-associated variants thereof.     

In vivo method to monitor changes in HER2 expression using near-infrared fluorescence imaging

Human epidermal growth factor receptor type 2 (HER2) is a well-known biomarker that is overexpressed in many breast carcinomas. HER2 expression level is an important factor to optimize the therapeutic strategy and monitor the treatment. We used albumin binding domain-fused HER2-specific Affibody molecules, labeled with Alexa Fluor750 dye, to characterize HER2 expression in vivo. Near-infrared optical imaging studies were carried out using mice with subcutaneous HER2-positive tumors. Animals were divided into groups of five: no treatment and 12 hours and 1 week after treatment of the tumors with the Hsp90 inhibitor 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG). The compartmental ligands-receptor model, describing binding kinetics, was used to evaluate HER2 expression from the time sequence of the fluorescence images after the intravenous probe injection. The normalized rate of accumulation of the specific fluorescent biomarkers, estimated from this time sequence, linearly correlates with the conventional ex vivo enzyme-linked immunosorbent assay (ELISA) readings for the same tumor. Such correspondence makes properly arranged fluorescence imaging an excellent candidate for estimating HER2 overexpression in tumors, complementing ELISA and other ex vivo assays. Application of this method to the fluorescence data from HER2-positive xenografts reveals that the 17-DMAG treatment results in downregulation of HER2. Application of the AngioSense 750 probe confirmed the antiangiogenic effect of 17-DMAG found with Affibody-Alexa Fluor 750 conjugate.     

Post‐translational modifications of transthyretin affect the triiodonine‐binding potential

Transthyretin (TTR) is a visceral protein, which facilitates the transport of thyroid hormones in blood and cerebrospinal fluid. The homotetrameric structure of TTR enables the simultaneous binding of two thyroid hormones per molecule. Each TTR subunit provides a single cysteine residue (Cys₁₀), which is frequently affected by oxidative post‐translational modifications. As Cys₁₀ is part of the thyroid hormone‐binding channel within the TTR molecule, PTM of Cys₁₀ may influence the binding of thyroid hormones. Therefore, we analysed the effects of Cys₁₀ modification with sulphonic acid, cysteine, cysteinylglycine and glutathione on binding of triiodothyronine (T3) by molecular modelling. Furthermore, we determined the PTM pattern of TTR in serum of patients with thyroid disease by immunoprecipitation and mass spectrometry to evaluate this association in vivo. The in silico assays demonstrated that oxidative PTM of TTR resulted in substantial reorganization of the intramolecular interactions and also affected the binding of T3 in a chemotype‐ and site‐specific manner with S‐glutathionylation as the most potent modulator of T3 binding. These findings were supported by the in vivo results, which indicated thyroid function‐specific patterns of TTR with a substantial decrease in S‐sulphonated, S‐cysteinylglycinated and S‐glutathionylated TTR in hypothyroid patients. In conclusion, this study provides evidence that oxidative modifications of Cys₁₀ seem to affect binding of T3 to TTR probably because of the introduction of a sterical hindrance and induction of conformational changes. As oxidative modifications can be dynamically regulated, this may represent a sensitive mechanism to adjust thyroid hormone availability.     

A universal homogeneous assay for high-throughput determination of binding kinetics

There is an increasing demand for assay technologies that enable accurate, cost-effective, and high-throughput measurements of drug–target association and dissociation rates. Here we introduce a universal homogeneous kinetic probe competition assay (kPCA) that meets these requirements. The time-resolved fluorescence energy transfer (TR–FRET) procedure combines the versatility of radioligand binding assays with the advantages of homogeneous nonradioactive techniques while approaching the time resolution of surface plasmon resonance (SPR) and related biosensors. We show application of kPCA for three important target classes: enzymes, protein–protein interactions, and G protein-coupled receptors (GPCRs). This method is capable of supporting early stages of drug discovery with large amounts of kinetic information.     

Solid phase enzyme-linked competitive binding assay for riboflavin

A new solid-phase enzyme-linked assay for riboflavin (vitamin B2) is described. The assay is based on the competition between analyte vitamin molecules and a glucose-6-phosphate dehydrogenase-3-carboxymethylriboflavin conjugate for a limited number of riboflavin-binding protein sites immobilized on Sepharose particles. Significant improvements in conjugate catalytic activity and thus detectability are achieved by optimizing the reaction conditions used to covalently link 3-carboxymethylriboflavin to the enzyme. Optimization experiments include studying the effects of reaction pH and organic solvent composition. Final assay detection limits and the sensitivity of the dose-response curves are dependent on the ratio of conjugate to binding protein sites utilized in an equilibrium assay protocol. Selectivity of the method correlates well with that predicted based on the known association constants of riboflavin-binding protein with flavin analogs. The assay is shown to offer adequate detection limits and selectivity for direct measurement of riboflavin in urine, infant formula, and vitamin capsules.     

Kinetic stabilization of the native state by protein engineering: implications for inhibition of transthyretin amyloidogenesis

The amyloidogenic homotetrameric protein transthyretin (TTR) must undergo rate-limiting dissociation to partially denatured monomers in order to aggregate. TTR contains two distinct quaternary interfaces, one of which defines the binding sites for thyroxine and small-molecule amyloidogenesis inhibitors. Kinetic stabilization of the tetramer can be accomplished either by the binding of amyloidogenesis inhibitors selectively to the native state over the dissociative transition state or by the introduction of trans-suppressor subunits (T119M) into heterotetramers to destabilize the dissociative transition state. In each case, increasing the dissociation activation barrier prevents tetramer dissociation. Herein, we demonstrate that tethering two subunits whose quaternary interface defines the thyroxine binding site also dramatically increases the barrier for tetramer dissociation, apparently by destabilization of the dissociative transition state. The tethered construct (TTR-L-TTR)2 is structurally and functionally equivalent to wild-type TTR. Urea is unable to denature (TTR-L-TTR)2, yet it is able to maintain the denatured state once denaturation is achieved by GdnHCl treatment, suggesting that (TTR-L-TTR)2 is kinetically rather than thermodynamically stabilized, consistent with the identical wild-type TTR and (TTR-L-TTR)2 GdnHCl denaturation curves. Studies focused on a construct containing a single TTR-L-TTR chain and two normal monomer subunits establish that alteration of only one quaternary structural interface is sufficient to impose kinetic stabilization on the entire quaternary structure.     

Kinetic stabilization of an oligomeric protein under physiological conditions demonstrated by a lack of subunit exchange: implications for transthyretin amyloidosis

Kinetic stabilization of transthyretin (TTR) is established to prevent human neurodegeneration. Therefore, small molecule-mediated kinetic stabilization of the native state is an attractive strategy to prevent the misfolding and misassembly associated with TTR amyloid disease. Since the physiological microenvironment resulting in human TTR amyloidogenesis remains unclear, the conservative approach is to identify inhibitors that function under a variety of conditions. Small molecule kinetic stabilization of TTR has been established by concentration-dependent inhibition of acid-mediated amyloidogenesis and urea-induced tetramer dissociation. Since denaturing conditions reduce the binding affinity of inhibitors making it difficult to predict inhibitor efficacy under physiological conditions, we introduce a method for quantifying kinetic stabilization under physiological conditions. The rate of subunit exchange between wild-type TTR homotetramers and wild-type TTR homotetramers tagged with an N-terminal acidic flag tag is dictated by the rate of tetramer dissociation to its monomeric subunits prior to reassembly, rendering this method ideally suited for assessing the kinetic stabilization of TTR imparted by small molecule binding and evaluating small molecule binding constants. Addition of amyloidogenesis inhibitors to this exchange reaction slows tetramer dissociation in a concentration-dependent manner, stopping dissociation at concentrations where at least one inhibitor is bound to each tetramer in solution. Subunit exchange enables the rate of tetramer dissociation and the kinetic stabilization imparted by small molecule binding to be evaluated under physiological conditions in which the TTR concentration is not reduced by aggregation or irreversible dissociation.