Determination Of Protein Binding Research Articles

Kinetic Analysis of Arf GAP1 Indicates a Regulatory Role for Coatomer

Arf GAPs are a family of enzymes that catalyze the hydrolysis of GTP bound to Arf. Arf GAP1 is one member of the family that has a critical role in membrane traffic at the Golgi apparatus. Two distinct models for the regulation of Arf GAP1 in membrane traffic have been proposed. In one model, Arf GAP1 functions in a ternary complex with coat proteins and is inhibited by cargo proteins. In another model, Arf GAP1 is recruited to a membrane surface that has defects created by the increased membrane curvature that accompanies transport vesicle formation. Here we have used kinetic and mutational analysis to test predictions of models of regulation of Arf GAP1. We found that Arf GAP1 has a similar affinity for Arf1.GTP as another Arf GAP, ASAP1, but the catalytic rate is approximately 0.5% that of ASAP1. Coatomer stimulated Arf GAP1 activity; however, different from that predicted from the current model, coatomer affected the K(m) and not the k(cat) values. Effects of most mutations in Arf GAP1 paralleled those in ASAP1. Mutation of an arginine that aligned with an arginine presumed to be catalytic in ASAP1 abrogated activity. Peptide from the cytoplasmic tail of cargo proteins inhibited Arf GAP1; however, the unrelated Arf GAP ASAP1 was also inhibited. The curvature of the lipid bilayer had a small effect on activity of Arf GAP1 under the conditions of our experiments. We conclude that coatomer is an allosteric regulator of Arf GAP1. The relevance of the results to the two models of Arf GAP1-mediated regulation of Arf1 is discussed.

Estimation of serum protein binding of compounds metabolized in serum using matrix inhibition

It is difficult to evaluate the serum protein binding of compounds that are metabolized in rat serum, even when using ultrafiltration. Protein binding was estimated using matrix inhibition, a method that uses the change in metabolic velocity achieved by changing the free fraction of a compound in the incubation mixture by diluting the serum with phosphate buffered saline. The T(1/2) of phenyl nicotinate, benzyl nicotinate, octyl nicotinate, hexyl nicotinate, butyl nicotinate and [(3)H] compound A were 0.165, 0.780, 2.62, 3.94, 5.22 and 135 min, respectively, with protein binding values of 82.1%, 91.6%, 98.8%, 98.5%, 85.5% and 96.9%. The protein binding value of compound A estimated by ultrafiltration was 93.4%, indicating that the two methods give similar values. The matrix inhibition method is thus applicable for the evaluation of compounds metabolized in serum, and provides a simple, useful method to determine protein binding.

68Ga- and 111In-labelled DOTA-RGD peptides for imaging of αvβ3 integrin expression

alphavbeta3 integrins are important cell adhesion receptors involved in angiogenic processes. Recently, we demonstrated using [(18)F]Galacto-RGD that monitoring of alphavbeta3 expression is feasible. Here, we introduce (68)Ga- and (111)In-labelled derivatives and compare them with [(18)F]Galacto-RGD. For radiolabelling, cyclo(RGDfK(DOTA)) was synthesised using SPPS. For in vitro characterisation determination of partition coefficients, protein binding, metabolic stability, alphavbeta3 affinity and cell uptake and for in vivo characterization, biodistribution studies and micro positron emission tomography (PET) imaging were carried out. For in vivo and in vitro studies, human melanoma M21 (alphavbeta3 positive) and M21-L (alphavbeta3 negative) cells were used. Both tracers can be synthesised straightforward. The compounds showed hydrophilic properties and high metabolic stability. Up to 23% protein-bound activity for [(68)Ga]DOTA-RGD and only up to 1.4% for [(111)In]DOTA-RGD was found. Cell uptake studies indicate receptor-specific accumulation. This is confirmed by the biodistribution data. One hour p.i. accumulation in alphavbeta3-positive tumours was 2.9 +/- 0.3%ID/g and in alphavbeta3-negative tumours 0.8 +/- 0.1%ID/g for [(68)Ga]DOTA-RGD ([(111)In]DOTA-RGD: 1.9 +/- 0.3%ID/g and 0.5 +/- 0.2%ID/g; [(18)F]Galacto-RGD: 1.6 +/- 0.2%ID/g and 0.4 +/- 0.1%ID/g). Thus, tumour uptake ratios were comparable. Due to approx. 3-fold higher blood pool activities for [(68)Ga]DOTA-RGD, tumour/blood ratios were higher for [(111)In]DOTA-RGD and [(18)F]Galacto-RGD. However, microPET studies demonstrated that visualisation of alphavbeta3-positive tumours using [(68)Ga]DOTA-RGD is possible. Our data indicate that [(68)Ga]DOTA-RGD allows monitoring of alphavbeta3 expression. Especially, the much easier radiosynthesis compared to [(18)F]Galacto-RGD would make it an attractive alternative. However, due to higher blood pool activity, [(18)F]Galacto-RGD remains superior for imaging alphavbeta3 expression. Introduction of alternative chelator systems may overcome the disadvantages.

Identification of unknown protein complex members by radiolocalization and analysis of low‐abundance complexes resolved using native polyacrylamide gel electrophoresis

Identification of unknown binding partners of a protein of interest can be a difficult process. Current strategies to determine protein binding partners result in a high amount of false-positives, requiring use of several different methods to confirm the accuracy of the apparent association. We have developed and utilized a method that is reliable and easily substantiated. Complexes are isolated from cell extract after exposure to the radiolabeled protein of interest, followed by resolution on a native polyacrylamide gel. Native conformations are preserved, allowing the complex members to maintain associations. By radiolabeling the protein of interest, the complex can be easily identified at detection levels below the threshold of Serva Blue, Coomassie, and silver stains. The visualized radioactive band is analyzed by MS to identify binding partners, which can be subsequently verified by antibody shift and immunoprecipitation of the complex. By using this method we have successfully identified binding partners of two proteins that reside in different locations of a cellular organelle.

An Acetylation Site in the Middle Domain of Hsp90 Regulates Chaperone Function

Heat-shock protein 90 (Hsp90) chaperones a key subset of signaling proteins and is necessary for malignant transformation. Hsp90 is subject to an array of posttranslational modifications that affect its function, including acetylation. Histone deacetylase (HDAC) inhibitors and knockdown of HDAC6 induce Hsp90 acetylation and inhibit its activity. However, direct determination of the functional consequences of Hsp90 acetylation has awaited mapping of specific sites. We now demonstrate that Hsp90 K294 is acetylated. Mutational analysis of K294 shows that its acetylation status is a strong determinant of client protein and cochaperone binding. In yeast, Hsp90 mutants that cannot be acetylated at K294 have reduced viability and chaperone function compared to WT or to mutants that mimic constitutive acetylation. These data suggest that acetylation/deacetylation of K294 plays an important role in regulating the Hsp90 chaperone cycle.

Determination of plasma protein binding of diltiazem in horses by ultrafiltration

Determination of plasma protein binding of diltiazem in horses by ultrafiltration

250 ORAL Presurgical treatment of metastatic renal cell carcinoma patients with bevacizumab and erlotinib: preliminary efficacy and biomaker data

Sunitinib® (SU11248) is a highly potent tyrosine kinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR). Radiolabeled inhibitors of RTKs might be useful tools for monitoring RTKs levels in tumour tissue giving valuable information for anti-angiogenic therapy. We report here the synthesis of a 125I-labeled derivative of sunitinib® and its first radiopharmaceutical characterization.The non-radioactive reference compound 5-iodo-sunitinib 4 was prepared by Knoevenagel condensation of 5-iodo-oxindole with the corresponding substituted 5-formyl-1H-pyrrole. In a competition binding assay against VEGFR-2 a binding constant (Kd) of 16 nM for 4 was found. The ability of 4 to inhibit tyrosine kinase activity was demonstrated on RTK expressing cells suggesting this radiotracer as a useful tool for monitoring VEGFR expression. 5-[125I]lodo-sunitinib, [125I]-4 was obtained via destannylation of the corresponding tributylstannyl precursor with [125I]NaI in the presence of H2O2 in high radiochemical yield (>95%) and radiochemical purity (<98%) after HPLC purification. Determination of human plasma protein binding at time intervals of 0; 1; 2; 4 and 24 h suggested a low non-specific binding of 5-10%. Preliminary biodistribution studies of [125I]-4 in healthy CD-1 mice showed a relatively high uptake in VEGFR-2 rich tissues like kidney and lung followed by rapid washout (9.6 and 9.7; 4.5 and 3.8% ID/g of kidney and lung at 1 and 4 h, respectively).

Determination of Compound Binding to Plasma Proteins

The pharmacokinetic and pharmacodynamic properties of a compound are profoundly affected by the extent of its binding to plasma proteins. Consequently, the determination of the plasma protein binding properties of a compound is essential during drug development and is increasingly required during lead prioritization. The protocols described in this unit detail the techniques of ultrafiltration and equilibrium dialysis for determination of plasma protein binding. Equilibrium dialysis is the more robust of the two techniques, whereas ultrafiltration is the more rapid. Two versions of the equilibrium dialysis technique are provided: the traditional approach and a higher-throughput 96-well plate version.

Pharmacokinetics and bioavailability of the isoflavone biochanin A in rats

Biochanin A (BCA) is a dietary isoflavone present in legumes, most notably red clover, and in many herbal dietary supplements. BCA has been reported to have chemopreventive properties and is metabolized to the isoflavone genistein (GEN), BCA conjugates, and GEN conjugates. The metabolites may contribute to the chemopreventive effects of BCA. The absorption, metabolism, and disposition of BCA have not been determined in rats. Our objective was to evaluate the pharmacokinetics and metabolism of BCA in rats. Male Sprague-Dawley rats were administered BCA by intravenous injection (1 and 5 mg/kg), by intraperitoneal injection (5 and 50 mg/kg), and orally (5 and 50 mg/kg). Plasma and bile samples were enzymatically hydrolyzed in vitro to determine conjugate concentrations for BCA and GEN. Equilibrium dialysis was used to determine protein binding. The BCA and GEN concentrations in plasma, urine, and bile were determined by liquid chromatography-tandem mass spectrometry (LC/MS/MS). The pharmacokinetic parameters of BCA were analyzed by noncompartmental analysis. Significant levels of BCA conjugates and GEN conjugates were detected in plasma and bile. Both BCA and GEN were found to have a high clearance and a large apparent volume of distribution; the bioavailability of both was poor (<4%). Reentry peaks were evident after oral administration of both BCA and GEN, suggesting enterohepatic cycling. The free fraction of BCA in rat plasma was 1.5%. A 2-compartment model that included both linear and nonlinear clearance terms and enterohepatic recirculation best described the plasma data. This represents the first evaluation of the dose-dependent pharmacokinetics and metabolism of BCA in rats.

Determination of Drug Binding to Plasma Proteins Using Competitive Equilibrium Binding to Dextran-Coated Charcoal

A method for determination of drug binding to plasma proteins, which is based on the comparison of drug affinities to plasma proteins and dextran-coated charcoal, is described. The method is free of nonspecific binding feature. The fractions unbound obtained by the charcoal method are in good agreement with values from a traditional ultrafiltration method for various low and highly bound compounds. The method presently requires drug concentrations much less than that of plasma proteins. A possibility of using the method to determine protein binding at an arbitrary drug concentration is discussed. A mechanism of nonspecific binding of drug to ultrafiltration membranes, which yields a good agreement with experimental observations, is suggested.

Table 4. Sample results from test tube method of determining protein binding to ion exchangers: Table 4.

Table 4. Sample results from test tube method of determining protein binding to ion exchangers: Table 4.

Sample results from test tube method of determining protein binding to ion exchangers

Sample results from test tube method of determining protein binding to ion exchangers

Determination of human plasma protein binding of baicalin by ultrafiltration and high‐performance liquid chromatography

A simple and selective HPLC assay was developed and utilized for determination of human plasma protein binding of baicalin. The method involved solid-phase extraction and reversed-phase chromatographic separation with a mobile phase of acetonitrile-0.02 mol/L phosphate buffer (pH 2.5; 25:75, v/v) and UV detection at 276 nm. The standard curve for baicalin was linear over the concentration range 0.1-20 microg/mL and the limit of detection was 0.02 microg/mL. The absolute recovery was greater than 76%. The intra-day and inter-day variations were less than 10%. Ultrafiltration technique was applied to determining the plasma protein binding of baicalin in human plasma. Results show the plasma protein binding of baicalin was in the range 86-92% over all the concentrations studied and the protein binding association constant was determined to be 1.21 x 10(5) L/mol at 4 degrees C.

In vitro plasma protein binding determination of flunarizine using equilibrium dialysis and liquid chromatography–tandem mass spectrometry

A highly sensitive method was developed and validated for determining the free fraction of flunarizine in human plasma. Equilibrium dialysis was used for the separation of free (unbound) drug and liquid chromatography/tandem mass spectrometry (LC–MS/MS) was used for quantitation. Post-dialysis plasma or buffer samples of 0.2 mL were extracted using a liquid–liquid extraction procedure and analyzed using a high performance liquid chromatography electrospray tandem mass spectrometer system. The compounds were eluted isocratically on a Supelco Supelcosil ABZ + Plus column, ionized using a positive ion atmospheric pressure electrospray ionization source, and analyzed using multiple reaction monitoring. The ion transitions monitored were m/ z 405 → 203 for flunarizine and m/ z 409 → 207 for flunarizine-d4 (internal standard, IS). The chromatographic run time was 3.5 min per injection, with retention times of 2.1 min for both flunarizine and IS. The calibration curve for flunarizine was linear over the concentration range of 0.25–2000 ng/mL ( r 2 > 0.9989) in the combined matrix of human plasma and isotonic sodium phosphate buffer (1:1, v/v) with the lower limit of quantitation of 0.25 ng/mL. The inter-assay coefficient of variability (CV) for the quality control samples was less than 13.5%, and the inter-assay percent nominal was greater than 98.2%. In vitro protein binding of flunarizine was determined at concentrations of 5, 10 and 100 μg/mL using the validated method. Flunarizine was extensively bound to plasma protein with a 0.083 ± 0.005% overall percent free drug in plasma and a CV value less than 7.8%. This validated method will be used for the ex vivo assessment of flunarizine protein binding in human plasma from a drug–drug interaction clinical study.

DNA sequence determinants of nuclear protein binding to the c-Ha-ras antioxidant/electrophile response element in vascular smooth muscle cells: identification of Nrf2 and heat shock protein 90β as heterocomplex components

The antioxidant/electrophile response element (ARE/EpRE) is a cis-acting element involved in redox regulation of c-Ha-ras gene. Protein binding to the ARE/EpRE may be credited to deoxyribonucleic acid sequence; therefore, studies were conducted to evaluate the influence of internal and flanking regions to the 10-bp human c-Ha-ras ARE/EpRE core (hHaras10) on nuclear protein binding in oxidant-treated vascular smooth muscle cells. A protein doublet bound to an extended oligonucleotide comprising the ARE/EpRE core in genomic context (hHaras27), whereas a single complex bound to hHarasl0. Protein binding involved specific interactions of 25- and 23-kDa proteins with hHarasl0, and binding of 80-, 65-, and 55-kDa proteins to hHaras27. Competition assays with hNQO1 and rGSTA2 confirmed the specificity of deoxyribonucleic acid-protein interactions and indicated preferred binding of p25 and p23 to the c-Ha-ras ARE/EpRE. "NNN" sequences within the core afforded unique protein-binding profiles to the c-Ha-ras ARE/EpRE. In addition, Nrf2 and heat shock protein 90beta (p80) were identified as components of the c-Ha-ras ARE/EpRE heterocomplex. We conclude that both internal bases and flanking sequences regulate nuclear protein recruitment and complex assembly on the c-Ha-ras ARE/EpRE.

A modified method for determining protein binding capacity of plant polyphenolics using radiolabelled protein.

A modified radiochemical protein binding method for determining the protein binding capacity of plant polyphenolics (tannins) is described. Purified tannin or unfractionated plant extracts were immobilised on filter paper discs and incubated with the 125I-labelled bovine serum albumin. Protein bound to the disc was proportional to the amount of tannin applied to the disc, although at high concentrations of polyphenolics the discs became saturated and the relationship was no longer applicable. The method was validated using purified procyanidin from Sorghum grain and has been applied to crude polyphenolic extracts from maple, white oak, black oak, walnut and tulip poplar leaves. Specific chemical assays for the determination of proanthocyanidins (acid butanol method) and hydrolysable tannins (modified potassium iodate method) were employed to validate the new protein binding method with the complex plant extracts.

Determination of interstitial rocuronium concentrations in the muscle tissue of anesthetized dogs by microdialysis

Introduction: The objective was to establish and validate a microdialysis technique for the quantification of interstitial concentrations of the neuromuscular blocker, rocuronium, in the muscle tissue of dogs under steady-state conditions. Methods: The standard and combined retrodialysis approaches were used for in vivo microdialysis probe calibration. After induction of anesthesia with pentobarbital (30 mg/kg), the left femoral vein was cannulated and blood drawn for protein binding determination. Microdialysis probes were inserted in the muscle and calibrated in vivo, using vecuronium as the calibrator. Each dog received a short 2-min infusion followed by a 120-min infusion of rocuronium via the right jugular vein and three microdialysis samples were collected at steady-state during a 2-h period. Samples were stored at −70 °C until HPLC analysis. Results: Using combined retrodialysis, rocuronium unbound interstitial ( C ISFu) and venous plasma ( C pssuv) concentrations are in good agreement; with a ratio C ISFu/ C pssuv of 100±11%. Using standard retrodialysis, this ratio was 47±7%. Conclusion: Combined retrodialysis is a more reliable and accurate technique for quantitative assessment of rocuronium interstitial concentrations especially for lengthy anesthetic procedures. These findings have potential implications, as drug concentrations in the site of action would be more relevant for concentration–effect relation of muscle relaxants.

Semi-automatic high-throughput determination of plasma protein binding using a 96-well plate filtrate assembly and fast liquid chromatography–tandem mass spectrometry

A semi-automatic, high-throughput method has been developed to rapidly assess plasma protein binding of new chemical entities in drug discovery phase. New chemical entities are mixed with plasma and the unbound fractions are separated from the bound fraction by ultrafiltration in a 96-well filtrate assembly. The unbound fractions are then analyzed by fast liquid chromatography–tandem mass spectrometry (LC–MS/MS). Sample handling is automated by a robotic system. Employing a cocktail approach where multiple new chemical entities are allowed to bind to plasma proteins in the same well has further increased the throughput. We have validated the method with 12 commercially available compounds. The plasma protein binding data obtained by this method are comparable with the literature values. This method enables the determination of protein binding for 32 compounds in one single experiment instead of 1–2 compounds using the conventional methods.

Validation of a simple liquid chromatography assay for creatine suitable for pharmacokinetic applications, determination of plasma protein binding and verification of percent labeled claim of various creatine products

Creatine has been quantified in various tissues by a range of methodologies. This paper reports on the development and validation of a simplified HPLC assay to determine plasma creatine, plasma protein binding of creatine, creatine in microdialysate and creatine in over-the-counter products. An isocratic, reversed-phase (C 18) HPLC assay, using potassium phosphate monobasic (pH 4) as a mobile phase, was validated in human plasma and microdialysis perfusion fluid (normal saline). The lower limit of quantification for the assay was 1 mg l −1 in saline and 5 mg l −1 in plasma. The RSD was below 6% and accuracy was below 12% in both matrices. Protein binding in human plasma was found to be negligible (<10%). Over-the-counter creatine monohydrate products tested contained 100% creatine monohydrate. This assay was found to be suitable for pharmacokinetic studies and the assessment of plasma creatine and skeletal muscle microdialysate.

Molecular determination of agouti-related protein binding to human melanocortin-4 receptor.

Agouti-related protein (AGRP) is an endogenous antagonist of the melanocortin-4 receptor (MC4R) that functions in the hypothalamic control of feeding behavior. Our previous studies have suggested that in addition to exoloops 2 and 3, several transmembrane domains of MC4R may be important for AGRP binding. However, the detailed molecular basis of MC4R domains in AGRP binding is presently unclear. The present studies were designed to determine the specific contribution of MC4R exoloops and transmembrane domains to AGRP binding by using chimeric receptor constructs of the human melanocortin-1 receptor (hMC1R), a receptor that is not inhibited by AGRP, and the human MC4R (hMC4R), a receptor that is potently inhibited by AGRP. Our results indicate that substitutions of the second and third extracellular loops of the MC4R with homologous domains of the MC1R dramatically decreased AGRP 87-132 binding affinity, but did not affect AGRP 110-117 binding affinity. In contrast, cassette substitutions of the third or fourth transmembrane domain of the MC4R with the homologous domain of the MC1R resulted in a substantial decrease of AGRP 87-132 binding affinity and loss of AGRP 110-117 binding affinity. These data suggest that the AGRP fragment 110-117 has no binding sites at exoloops of hMC4R and that transmembrane domains of MC4R may play an important role in AGRP 110-117 binding and function, whereas the exoloops do not. The second and third extracellular loops of MC4R are important for AGRP 87-132 N-terminal binding, whereas the third and fourth transmembrane domains of hMC4R are crucial for AGRP 110-117 binding.