Human Alpha 1-acid Glycoprotein Research Articles

Potential Clinically Relevant Effects of Sialylation on Human Serum AAG-Drug Interactions Assessed by Isothermal Titration Calorimetry: Insight into Pharmacoglycomics?

Human serum alpha-1 acid glycoprotein is an acute-phase plasma protein involved in the binding and transport of many drugs, especially basic and lipophilic substances. It has been reported that the sialic acid groups that terminate the N-glycan chains of alpha-1 acid glycoprotein change in response to certain health conditions and may have a major impact on drug binding to alpha-1 acid glycoprotein. The interaction between native or desialylated alpha-1 acid glycoprotein and four representative drugs-clindamycin, diltiazem, lidocaine, and warfarin-was quantitatively evaluated using isothermal titration calorimetry. The calorimetry assay used here is a convenient and widely used approach to directly measure the amount of heat released or absorbed during the association processes of biomolecules in solution and to quantitatively estimate the thermodynamics of the interaction. The results showed that the binding of drugs with alpha-1 acid glycoprotein were enthalpy-driven exothermic interactions, and the binding affinity was in the range of 10-5-10-6 M. Desialylated alpha-1 acid glycoprotein showed significantly different binding with diltiazem, lidocaine, and warfarin compared with native alpha-1 acid glycoprotein, whereas clindamycin showed no significant difference. Therefore, a different degree of sialylation may result in different binding affinities, and the clinical significance of changes in sialylation or glycosylation of alpha-1 acid glycoprotein in general should not be neglected.

Insight into intermolecular binding mechanism of apatinib mesylate and human alpha-1-acid glycoprotein: combined multi-spectroscopic approaches with in silico

Apatinib mesylate (APM), an oral tyrosine kinase inhibitor, has a good anti-tumor activity in the treatment of various cancers, particularly in advanced non-small cell lung cancer. In this study, the intermolecular binding mechanism between APM and human alpha-1-acid glycoprotein (HAG) was investigated by combining multi-spectroscopic approaches with in silico techniques. The findings revealed that APM gave rise to the fluorescence quenching of HAG by forming a ground-state complex between APM and HAG with a stoichiometric ratio of 1:1, and APM has a moderate affinity for HAG as the binding constant of APM and HAG of approximately 105 M−1, which was larger than the APM-HAG complex. The findings from thermodynamic parameter analysis indicated that the dominant driving forces for the formation of the APM-HAG complex were van der Waals forces, hydrogen bonding and hydrophobic interactions, which were also verified with site-probe studies and molecular docking. The findings from in silico study indicated that APM inserted into the opening of the hydrophobic cavity of HAG, leads to a slight conformational change in the HAG, which was verified by circular dichroism (CD) measurements, that was, the beta sheet level of HAG decreased. Additionally, the results of synchronous and 3D fluorescence spectroscopies confirmed the decline in hydrophobicity of the microenvironment around Trp and Tyr residues. Moreover, some common metal ions such as Cu2+, Mg2+, Fe3+, Ca2+, and Zn2+ could cause the alteration in the binding constant of APM with HAG, leading to the change in the efficacy of APM. It will be expected that these study findings are to provide useful information for further understanding pharmacokinetic and structural modifications of APM. Communicated by Ramaswamy H. Sarma

Effect of the sialic acid residues upon the binding of beta blocker propranolol to human serum alpha-1 acid glycoprotein

Effect of the sialic acid residues upon the binding of beta blocker propranolol to human serum alpha-1 acid glycoprotein

Potential use of Cypridina luciferin for quantifying alpha 1-acid glycoprotein in human serum.

Alpha 1-acid glycoprotein (AGP) is an acute phase protein in mammals, including humans. The amount of AGP in human serum varies in response to certain diseases; thus, many efforts have been made to develop methods for quantifying human AGP. We recently discovered that luminescence occurs merely by mixing Cypridina luciferin with human AGP under human serum-free neutral or basic buffer conditions. In this study, we tested an application of Cypridina luciferin for quantifying AGP contained in human serum. Our luminescence spectrum measurements of Cypridina luciferin with human serum samples showed that the maximum emission wavelength with human serum (480nm) differed from that with human AGP (464nm) due to the abundant presence of endogenous human serum albumin (HSA). Furthermore, the luminescence intensities of Cypridina luciferin with human AGP in HSA-depleted human serum were consistent with those in a human serum-free basic buffer, but those in human serum were not. These results indicated that depletion of HSA in human serum was required to use Cypridina luciferin for quantifying AGP in human serum. Additionally, we found that the luminescence intensity of Cypridina luciferin with bovine AGP was approximately tenfold lower than that with human AGP.

Derivatization of sialylated glycopeptides plus based sialoglycopeptides enrichment using cation exchange media

Glycosylation is one of the most important post-translational modifications. However, the characterizations of glycopeptides, especially the negatively charged sialoglycopeptides that are associated with various diseases, remain challenging, due to the co-existence with high abundant peptides and the low ionization efficiency of sialoglycopeptides resulting from the carboxyl groups. Therefore, it is essential to develop an efficient enrichment method for sialoglycopeptides. Here, we present a novel derivatization-based enrichment method that can (i) identify linkage isomers of sialic acids by generating mass difference, (ii) unify the net charge of peptides into zero, and (iii) introduce positive charges to sialoglycopeptides by conjugating quaternary ammonium with sialic acid. The derivatization, termed derivatization of sialylated glycopeptides plus (DOSG+), enables efficient enrichment through electrostatic interaction using weak cation exchange (WCX) media. DOSG+ -based WCX enrichment was validated and optimized with samples derived from bovine fetuin. Peptides were removed efficiently (recovery rate <1%). The signal intensity of a selected model sialoglycopeptide was increased by ∼30% (suggesting recovery rate >100%). The method was employed on human alpha-1 acid glycoprotein (AGP), and recombinant human erythropoietin (EPO), demonstrating the application of DOSG+ -based WCX enrichment on complexed N-linked and O-linked sialoglycopeptides. The method is simple, efficient, and targets small-scale sialoglycopeptide enrichment.

Comprehending binding features between ibrutinib and Human Alpha-1 acid glycoprotein: Combined experimental approaches and theoretical simulations

Human alpha-1 acidic glycoprotein (HAG) is one of the proteins widely present in the blood, and the level of HAG in patients with cancer and inflammation is significantly increased. As one of transport proteins in the blood, the ability of HAG to bind with a drug, especially alkaline drugs, affects significantly the drug content at the target site, which in turn affects the efficacy of the drug. In this study, the interaction mechanism between HAG and the first generation Bruton's tyrosine kinase (BTK) inhibitor namely ibrutinib was explored by a combination of multi-spectroscopic techniques and theoretical calculations. The findings revealed that the quenching and binding constants of the HAG-ibrutinib system both reduced as the temperature rose, demonstrating that ibrutinib quenched the intrinsic fluorescence of HAG in a static manner. It was confirmed that HAG and ibrutinib formed a 1:1 complex with moderate affinity due to the binding constant of around 105 M−1 and accompanied by Förster resonance energy transfer. It was verified by thermodynamic parameter analysis and competition assays as well as molecular simulation that the existence of hydrogen bonds, van der Waals forces, and hydrophobic forces in the complexation of HAG and ibrutinib.The findings from theoretical calculations including molecular docking and theoretical calculation simulation confirmed that ibrutinib bound to the barrel hydrophobic pocket of HAG with a binding energy of −41.9 kJ∙mol−1, and the the binding constant of around 105 M−1 and the contribution of each residue in the complexation of ibrutinib and HAG. Additionally, it can be confirmed that metal ions affected the binding interaction of ibrutinib with HAG, among them, some promoted binding while others inhibited it.

Glycoprotein analysis using lectin microcolumns and capillary electrophoresis: Characterization of alpha1-acid glycoprotein by combined separation methods

Separations based on combinations of 2.1 mm I.D. high-performance affinity microcolumns and capillary electrophoresis were developed and used to characterize the glycoforms of an intact glycoprotein. Human alpha1-acid glycoprotein (AGP) was used as a model analyte due to its heterogeneous glycosylation resulting from variations in its degree of branching, fucosylation, and number of sialic acids. Three separation formats were examined based on microcolumns that contained the lectins concanavalin A (Con A) or Aleuria aurantia lectin (AAL). These microcolumns were used with one another or in combination with capillary electrophoresis. N-Glycan analysis of the non-retained and retained AGP fractions was carried out by using PNGase F digestion and nanoflow electrospray ionization mass spectrometry. Con A microcolumns were found to selectively enrich AGP that contained bi-antennary N-glycans, while AAL microcolumns retained AGP with fucose-containing N-glycans. Results from these separation methods indicated that fucosylation of the N-linked glycans was more abundant when a high degree of branching was present in AGP. Sialic acid residues were more abundant when higher degrees of branching and more fucose residues were present in AGP. The separation and analysis methods that were developed could be used with relatively small amounts of AGP and can be adapted for use with other intact glycoproteins.

A Method for Overcoming Plasma Protein Inhibition of Tyrosine Kinase Inhibitors

FMS-like tyrosine kinase 3 (FLT3) is the most frequently-mutated gene in acute myeloid leukemia and a target for tyrosine kinase inhibitors (TKI). FLT3 TKI have yielded limited improvements to clinical outcomes. One reason for this is TKI inhibition by endogenous factors. We characterized plasma protein binding of FLT3 TKI, specifically staurosporine-derivatives (STS-TKI) by alpha-1-acid glycoprotein (AGP); simulating its effects upon drug efficacy. Human AGP inhibits the anti-proliferative activity of STS-TKI in FLT3-ITD-dependent cells, with IC50 shifts higher than clinically achievable. This is not seen with non-human plasma. Mifepristone co-treatment, with its higher AGP affinity, improves TKI activity despite AGP, yielding IC50s predicted to be clinically effective. In a mouse model of AGP drug inhibition, mifepristone restores midostaurin activity. This suggests combinatorial methods for overcoming plasma protein inhibition of existing TKIs for leukemia as well as providing a platform for investigating the drug-protein interaction space for developing more potent small-molecule agents.

On the binding reaction of loratadine with human serum acute phase protein alpha 1-acid glycoprotein

Loratadine is an important anti-allergic drug. It is a second generation antihistamine drug used to treat allergic rhinitis, hay fever and urticaria. Human serum alpha 1-acid glycoprotein (AG) is an important acute phase protein and its serum concentration is found to increase in inflammation and acute response.The binding interaction between loratadine and AG is studied using spectroscopy and molecular docking techniques. The results obtained from fluorescence quenching experiments demonstrated that the fluorescence intensity of AG is quenched by loratadine. Loratadine was found to bind AG with the binding constant of ≈104 at 298 K. The Gibb’s free energy change was found to be negative for the interaction of loratadine with AG indicating the binding process is spontaneous. Binding of loratadine with AG induced ordered structures in the protein. Hydrogen bonding and hydrophobic interactions were the main bonding forces between AG–loratadine as revealed by molecular docking results. This study suggests the importance of binding of anti-allergic drug to AG spatially in the diseases where the plasma concentration of AG increases many folds and interaction with this protein becomes significant. This study will help in design of drug dosage and adjustment accordingly to achieve optimal treatment outcome. Communicated by Ramaswamy H. Sarma

Studies of binding by 2-imidazolines to human serum albumin and alpha1-acid glycoprotein by high-performance affinity chromatography

2-Imidazoline drugs are used in a variety of applications, such as the treatment of hypertension and opioid withdrawal. It is known these drugs bind to serum proteins and have significant variations within this class of compounds in the overall level of this binding. However, little specific information is available on the interactions of these compounds with the two major transport proteins for many drugs, human serum albumin (HSA) and alpha1-acid glycoprotein (AGP). This study examined binding by 2-imidazolines to these proteins by using 25 mm × 2.1 mm i.d. high-performance affinity microcolumns that contained HSA or AGP. The drugs that were examined were antazoline, clonidine, dexmedetomidine, lofexidine, moxonidine, phentolamine, and tizanidine, which represented a wide range of structures and pharmaceutical applications. The major metabolite of lofexidine, N-(2-aminoethyl)-2-(2,6-dichlorophenoxy) propenamide (LADP), was also examined. All these 2-imidazolines were found to have weak-to-moderate binding to HSA, with global affinities that ranged from 1.62 × 102 to 1.07 × 104 M−1 at pH 7.4 and 37 °C. These compounds had stronger binding with AGP, with global affinities constants ranging from 3.80 × 102 to 1.85 × 104 M−1. No stereoselectivity was observed by HSA for the enantiomers of dexmedetomidine, lofexidine, or LADP. However, AGP did show some stereoselectivity for lofexidine and LADP but not for dexmedetomidine. These results provide a better understanding of interactions of 2-imidazoline with HSA vs AGP in the circulation and of how this binding can change between drugs within this class of compounds.

Derivatization of Sialylated Glycopeptides (DOSG) Enabling Site-Specific Isomeric Profiling Using LC-MS/MS.

Glycosylation is a ubiquitous and important post-translational modification, functioning as a critical role in many biological processes. Among all the monosaccharides, sialic acids are of utmost importance, due to their relatively high abundance and association with various diseases. Elucidation of linkage isomers on sialic acids remains challenging. Various chemical derivatization techniques have been developed to identify the linkage isomers of sialic acids on released N-glycans, such as esterification and alkylamidation. Here, we present an efficient, effective, and stable derivatization method that can discriminate α2,3- and α2,6-linked sialic acids on glycopeptides by generating mass difference of the isomers. A similar chemical method has been developed for released N-glycans. We adapted and optimized the method to be effectively utilized for the derivatization of glycopeptides. The method also efficiently derivatized the carboxylic groups on peptides in a controlled way, which was confirmed by MS/MS data. The efficient derivatization of the peptide backbone carboxylic groups prompts ca. 4.6-fold increase in signal. The method was employed to characterize sialylated glycopeptides derived from bovine fetuin, human alpha-1 acid glycoprotein (AGP), and human erythropoietin (EPO). The method effectively and reliably allowed the characterization of sialylated N- and O-glycopeptide isomers. For example, 11 sialylated glycans including 28 isoforms were well profiled on Asn72 of AGP. The method is facile and highly reproducible that targets site- and sialic acid linkage-specific profiling of glycosylation.

Luminescence of Cypridina Luciferin in the Presence of Human Plasma Alpha 1-Acid Glycoprotein

The enzyme Cypridina luciferase (CLase) enables Cypridina luciferin to emit light efficiently through an oxidation reaction. The catalytic mechanism on the substrate of CLase has been studied, but the details remain to be clarified. Here, we examined the luminescence of Cypridina luciferin in the presence of several proteins with drug-binding ability. Luminescence measurements showed that the mixture of human plasma alpha 1-acid glycoprotein (hAGP) and Cypridina luciferin produced light. The total value of the luminescence intensity over 60 s was over 12.6-fold higher than those in the presence of ovalbumin, human serum albumin, or bovine serum albumin. In the presence of heat-treated hAGP, the luminescence intensity of Cypridina luciferin was lower than in the presence of intact hAGP. Chlorpromazine, which binds to hAGP, showed an inhibitory effect on the luminescence of Cypridina luciferin, both in the presence of hAGP and a recombinant CLase. Furthermore, BlastP analysis showed that hAGP had partial amino acid sequence similarity to known CLases in the region including amino acid residues involved in the drug-binding ability of hAGP. These findings indicate enzymological similarity between hAGP and CLase and provide insights into both the enzymological understanding of CLase and development of a luminescence detection method for hAGP.

Characteristics of Dolutegravir and Bictegravir Plasma Protein Binding: a First Approach for the Study of Pharmacologic Sanctuaries.

This study aimed to characterize in vitro dolutegravir (DTG) and bictegravir (BIC) binding. They had a preferential binding to human serum albumin (HSA) with two classes of albumin sites. Human alpha-1-acid glycoprotein (HAAG) binding of DTG and BIC showed an atypical nonlinear binding. The low-affinity site on HSA, the main plasma binding protein, suggests that the high protein binding rate should not impair passive diffusion.

Isomeric Separation of N-Glycopeptides Derived from Glycoproteins by Porous Graphitic Carbon (PGC) LC-MS/MS.

Protein glycosylation is involved in many biological processes and physiological functions. Despite the recent advances in LC-MS/MS methodologies, the profiling of site-specific glycosylation is one of the major analytical challenges of glycoprotein analysis. Herein, we report that the separation of glycopeptide isomers on porous graphitic carbon (PGC)-LC was significantly improved by elevating the separation temperature under basic mobile phases. These findings permitted the isomeric separation of glycopeptides resulting from highly specific enzymatic digestions. The selectivity for different glycan types was studied using bovine fetuin, asialofetuin, IgG, ribonuclease B, and alpha-1 acid glycoprotein (AGP) by PGC-LC-MS. Comprehensive structural isomeric separation of glycopeptides was observed by high-resolution MS and confirmed by MS/MS. The specific structures of the glycopeptide isomers were identified and confirmed through exoglycosidase digestions. Glycosylation analysis of human AGP revealed the potential use of PGC-LC-MS for extensive glycoprotein analysis for biomarker discovery. This newly developed separation technique was shown as a reproducible and useful analytical method to study site-specific isomeric glycosylation.

Insights on the interaction mechanism of brigatinib to human α-1-acid glycoprotein: Experimental and computational approaches

Brigatinib, a multi-target kinase inhibitor, is primarily used to treat anaplastic lymphoma kinase (ALK)-positive patients with advanced non-small cell lung cancer (NSCLC) who have previously received crizotinib or are resistant to crizotinib. In this study, we focused on elucidating the interaction mechanism between brigatinib and human alpha-1-acid glycoprotein (HAG) through experimental and computational approaches. Steady-state fluorescence and UV–vis spectroscopy measurements revealed that brigatinib could quench the intrinsic fluorescence of HAG in a static quenching manner and formed the brigatinib-HAG complex with the stoichiometric ratio of 1:1. The findings revealed that brigatinib had a stronger affinity on HAG due to higher binding constant of 2.91 × 105 M−1 at 298 K. It can be proved from thermodynamic parameter analysis that brigatinib spontaneously bound to HAG in the means of enthalpy driven, the main forces for stabilizing brigatinib-HAG complexes were hydrogen bonding and hydrophobic interactions. The experimental results also indicated that the binding interaction induced micro-environmental changes around tryptophan residues and the alteration in secondary structure of HAG. The presence of metal ions like Mg2+, Zn2+, Ca2+, Ni2+ and Co2+ affects the binding interaction and thus change the therapeutic efficacy of brigatinib. Molecular docking results suggested that brigatinib was embedded to the hydrophobic cavity of HAG. The experimental and computational results certified that hydrogen bonding and hydrophobic interaction as well as electrostatic energy and van der Waals forces plays a leading role in the binding process.

Qualitative and quantitative characterization of sialylated N-glycans using three fluorophores, two columns, and two instrumentations

Sialylation can influence the stability, half-life, and immunogenicity of glycoproteins, but sialylated N-glycans are known to be difficult to analyze. Human alpha1-acid glycoprotein (AGP) is reported to have glycans that consist of sialylated N-glycans. The N-glycan profiling of AGP is qualitatively and quantitatively investigated here by UPLC and LC-ESI-MS/MS. Three fluorescent tags (AB, AA, and ProA) and two separation columns (HILIC and AEX-HILIC) were adopted to confirm and compare each analytical characteristic. The results of AA were comparable to those of the well-established AB. The qualification of ProA was notable due to its superior fluorescence intensity and ionization efficiency, and ProA showed smaller quantitative or larger-sized fragments in LC-ESI-MS/MS compared to AB and AA. However, the MS quantification of ProA was distorted because the increased sialylation level decreased the LC-ESI-MS/MS ionization efficiency. HILIC had better peak separability, AEX-HILIC had an advantage in UPLC sialylation profiling, and each isomeric glycan could be identified by both columns in LC-ESI-MS/MS. In conclusion, ProA is favored for UPLC and LC-ESI-MS/MS detection but not reliable for MS quantification. This study firstly demonstrates the qualification and quantification of sialylated N-glycans by comparing the commonly used analytical conditions with different fluorescent tags, columns, and instruments.

Glycoform Analysis of Alpha1-Acid Glycoprotein by Capillary Electrophoresis Using Electrophoretic Injection.

Human alpha1-acid glycoprotein (AGP) is an acute phase glycoprotein that has a heterogeneous glycosylation pattern. This pattern can change in certain diseases, which has resulted in interest in using AGP glycoforms as potential biomarkers for these diseases. This report describes a method that uses capillary electrophoresis to characterize and analyze AGP glycoforms both in purified samples of AGP and in human serum. This method uses static and dynamic coatings of poly(ethylene oxide) that are applied to a silica capillary for separation of AGP glycoforms in the reversed-polarity mode of CE and in the presence of negligible electroosmotic flow. Electrophoretic injection is performed onto such capillaries by using a stationary stacking interface between the sample and running buffer. In addition, acidic precipitation and desalting are used to allow for the isolation and the analysis of AGP from only 65μL of serum. Up to eleven AGP glycoform bands can be reproducibly separated by this method, with the difference in migration time between neighboring bands being 12- to almost 60-fold larger than the standard deviation for the migration time of any given band. A limit of detection down to about 2nM per glycoform band can be obtained by this method for AGP in serum based on absorbance detection and without the need for further sample modification or labeling.

Combinatorial Approaches to Overcome Plasma Protein Inhibition of FLT3 Tyrosine Kinase Inhibitors

Background: The FMS-like tyrosine kinase 3 (FLT3) is the most frequently mutated gene in acute myeloid leukemia (AML) and also results in poor prognosis for adult and pediatric patients, and thus represents an attractive target for tyrosine kinase inhibitors (TKI). The activity of FLT3-targeted TKI is inhibited to varying extents by plasma protein binding. Staurosporine (STS)-derived TKI are inhibited almost exclusively by the plasma protein alpha-1 acid glycoprotein (AGP), an acute-phase reactant. We studied the impact of AGP binding on the other STS-derivatives and report the development of a novel method to overcome this binding.Methods: We assayed the impact of human AGP upon the activity of the STS-derived TKI (midostaurin, lestaurtinib, TTT-3002) against proliferation of the FLT3-ITD dependent cell line MOLM-14 and upon the parent compound (staurosporine) against the non-FLT3-dependent cell line HL-60. These experiments were repeated, co-incubating with drugs that competitively bind AGP to identify those that may restore TKI activity.Results: The TKI are inhibited in a linear AGP-dependent manner (fold change increase of IC50 per mg/dL AGP: midostaurin 3.00-fold, lestaurtinib 11.73-fold, TTT-3002 0.33-fold) across the range of AGP concentrations observed in human plasma. These results correspond to the drug-protein binding constants for the TKI: midostaurin 12.6 µM-1, lestaurtinib 49.2 µM-1, TTT-3002 1.41 µM-1, all validated by competitive fluorescence displacement of the AGP-binding dye, 1-anilino-8-naphthalenesulfonate. These results predict that in vivo IC50 values for these FLT3 TKI will be significantly higher than those measured under typical (10% FCS) in vitro culture conditions: midostaurin 4.7 µM, lestaurtinib 4.8 µM, TTT-3002 34 nM. By comparison, activity of the parent compound, staurosporine, against HL60 is completely inhibited by AGP. Assays using bovine plasma, serum or purified AGP do not demonstrate similar inhibition of FLT3 TKI. We are developing a murine model to overcome this experimental limitation.We have developed a mathematical model describing the interactions of AGP with FLT3 TKI using classical mass action relationships that match experimental results and furthermore describe the effects of competitive plasma protein binding by unrelated agents. These models predict that disinhibition of TKI may be achievable in vivo, and define the properties of such “rescue” agents.Mifepristone binds AGP (2-10 fold greater than STS-derived TKI) and has no independent effect upon FLT3-dependent cell growth. Co-treatment with mifepristone restores the IC50 of TTT-3002 from 12 nM with AGP to < 0.1 nM. Disinhibition is seen for lestaurtinib (IC50 shift reduced from >1000-fold to 50-fold) and midostaurin (300-fold reduced to 80-fold). This results in predicted in vivo IC50 that are clinically relevant, and serve as a proof-of-principle for this method. Using this principle we have screened a library of FDA-approved compounds for the ability to rescue TKI activity despite the presence of potentially inhibitory plasma proteins. This screen has identified 40 potential agents that may displace STS-derived TKI from AGP, and an additional 90 agents that may restore TKI activity through off-target effects. Several agents have already been validated in vitro, and found to decrease the IC50 of midostaurin and other TKI to clinically achievable ranges despite the presence of inhibiting proteins.Conclusions: The failure of FLT3 TKI in previous clinical trials has been linked to a lack of plasma drug activity. This work provides biochemical confirmation of this effect. Furthermore, these results indicate that this is a property of the class as a whole, including midostaurin. Indeed, for midostaurin, the predicted in vivo IC50 is higher than steady state levels suggesting that in clinical trials it likely acts through non-FLT3 mechanisms. Disinhibition of TKIs by mifepristone suggests a novel combinatorial approach restore TKI activity using unrelated compounds. We are currently examining other agents for similar synergy. By improving TKI potency in the face of inhibitory plasma protein binding, such combinations would be expected to improve their clinical efficacy by reducing the dosages necessary to thoroughly inhibit FLT3. Finally, this report provides a method for predicting at least one factor that affects the success or failure of FLT3 TKI in clinical trials. DisclosuresNo relevant conflicts of interest to declare.

Characterization of solution-phase drug-protein interactions by ultrafast affinity extraction.

A number of tools based on high-performance affinity separations have been developed for studying drug-protein interactions. An example of one recent approach is ultrafast affinity extraction. This method has been employed to examine the free (or non-bound) fractions of drugs and other solutes in simple or complex samples that contain soluble binding agents. These free fractions have also been used to determine the binding constants and rate constants for the interactions of drugs with these soluble agents. This report describes the general principles of ultrafast affinity extraction and the experimental conditions under which it can be used to characterize such interactions. This method will be illustrated by utilizing data that have been obtained when using this approach to measure the binding and dissociation of various drugs with the serum transport proteins human serum albumin and alpha1-acid glycoprotein. A number of practical factors will be discussed that should be considered in the design and optimization of this approach for use with single-column or multi-column systems. Techniques will also be described for analyzing the resulting data for the determination of free fractions, rate constants and binding constants. In addition, the extension of this method to complex samples, such as clinical specimens, will be considered.

A truncated form of human alpha 1-acid glycoprotein is useful as a molecular tool for insect glycobiology

N-glycosylation is an important posttranslational modification that results in a variety of biological activities, structural stability, and protein-protein interactions. There are still many mysteries in the structure and function of N-glycans, and detailed elucidation is necessary. Baculovirus expression system (BES) is widely used to produce recombinant glycoproteins, but it is not suitable for clinical use due to differences in N-glycan structure between insects and mammals. It is necessary to develop adequate model glycoproteins for analysis to efficiently alter the insect-type N-glycosylation pathway to human type. The previous research shows the recombinant alpha 1-acid glycoprotein (a1AGP) secreted from silkworm cultured cells or larvae is highly glycosylated and expected to be an excellent research candidate for the glycoprotein analysis expressed by BES. Therefore, we improved the a1AGP to be a better model for studying glycosylation. The modified a1AGP (a1AGPΔ) recombinant protein was successfully expressed and purified by using BES, however, the expression level in silkworm cultured cells and larvae were lower than that of the a1AGP. Subsequently, we confirmed the detailed profile of N-glycan on the a1AGPΔ by LS/MS analysis the N-glycan structure at each glycosylation site. These results indicated that the recombinant a1AGPΔ could be usable as a better model glycoprotein of N-glycosylation research in BES.