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1.
Trtić-Petrović T Jönsson JA 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,814(2):375-384
A technique for determination of drug-protein binding based on a membrane extraction technique termed "equilibrium sampling through membrane (ESTM)" is presented. It involves the establishment of an equilibrium between an aqueous buffer and either a blood plasma sample or a matched buffer, both containing the drug. Analysis of the aqueous buffer in the two cases gives the drug-protein binding. The principle bypasses some sources of systematic error found with common techniques for this measurement based on e.g. ultrafiltration, as it senses the equilibrium conditions without disturbing the sample. The technique is applied to some local anesthetic drugs as model substances and two alternative ways for the evaluation are presented. Results with these evaluation methods are compared with literature values for the drug-protein binding of these compounds. It is found that the drug-protein binding values obtained are lower than literature values, which is attributed to reduced systematic error. 相似文献
2.
Trtić-Petrović T Liu JF Jönsson JA 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2005,826(1-2):169-176
The determination of drug-protein binding and free drug concentration in plasma applying the equilibrium sampling through membrane (ESTM) technique has been studied using supported liquid membrane extraction in a single hollow fibre without any membrane carrier. In the extraction setup, the donor phase (plasma or buffer) was placed in the vial, into which was immersed the hollow fibre with the acceptor phase situated in the lumen. This proposed technique was applied to study the drug-protein binding of five local anaesthetics and two antidepressants as model substances, and the influence of the total drug concentration on the drug-protein binding was investigated. The brief theoretical background for determination of the drug-protein binding under equilibrium conditions is described. The developed method shows a new, improved and simple procedure for determination of free drug concentration in plasma and extent of drug-protein binding. 相似文献
3.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1996,677(1):1-28
In the last few years, continuous progress in instrumental analytical methodology has been achieved with a substantial increase in the number of new, more specific and more flexible methods for ligand-protein assays. In general, the methods used for drug-protein binding studies can be divided into two main groups: separation methods (enabling the calculation of binding parameters, i.e. the number of binding sites and their respective affinity constants) and non-separation methods (describing predominantly qualitative parameters of the ligand-protein complex). This review will be focussed particularly on recent trends in the development of drug-protein binding methods including stereoselective and non-stereoselective aspects using chromatography, capillary electrophoresis and microdialysis as compared to the “conventional approach” using equilibrium dialysis, ultrafiltration or size exclusion chromatography. The advantages and limitations of various methods will be discussed including a focus on “optimal” experimental strategies taking into account in vitro, ex vivo and/or in vivo studies. Furthermore, the importance of some particular aspects concerning the drug binding to proteins (covalent binding of drugs and their metabolites, stereoselective interactions and evaluation of binding data) will be outlined in more detail. 相似文献
4.
Background
Predicting drug-protein interactions from heterogeneous biological data sources is a key step for in silico drug discovery. The difficulty of this prediction task lies in the rarity of known drug-protein interactions and myriad unknown interactions to be predicted. To meet this challenge, a manifold regularization semi-supervised learning method is presented to tackle this issue by using labeled and unlabeled information which often generates better results than using the labeled data alone. Furthermore, our semi-supervised learning method integrates known drug-protein interaction network information as well as chemical structure and genomic sequence data.Results
Using the proposed method, we predicted certain drug-protein interactions on the enzyme, ion channel, GPCRs, and nuclear receptor data sets. Some of them are confirmed by the latest publicly available drug targets databases such as KEGG.Conclusions
We report encouraging results of using our method for drug-protein interaction network reconstruction which may shed light on the molecular interaction inference and new uses of marketed drugs.5.
6.
David S. Hage Stacey A. Tweed 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1997,699(1-2)
Drug-protein binding is an important process in determining the activity and fate of a pharmaceutical agent once it has entered the body. This review examines various chromatographic and electrophoretic methods that have been developed to study such interactions. An overview of each technique is presented along with a discussion of its strengths, weaknesses and potential applications. Formats that are discussed include the use of both soluble and immobilized drugs or proteins, and approaches based on zonal elution, frontal analysis or vacancy peak measurements. Furthermore, examples are provided that illustrate the use of these methods in determining the overall extent of drug-protein binding, in examining the displacement of a drug by other agents and in measuring the equilibrium or rate constants for drug-protein interactions. Examples are also given demonstrating how the same methods, particularly when used in high-performance liquid chromatography or capillary electrophoresis systems, can be employed as rapid screening tools for investigating the binding of different forms of a chiral drug to a protein or the binding of different proteins and peptides to a given pharmaceutical agent. 相似文献
7.
《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1995,666(1):127-137
The potential of on-line dialysis as a sample preparation procedure for compounds highly bound to plasma proteins is evaluated, using non-steroidal anti-inflammatory drugs as model compounds and column liquid chromatography as the separation technique. Different strategies to reduce the degree of drug-protein binding and so increase the analyte recovery are systematically explored and discussed: alteration of the conformation of the binding protein by changing the pH of the sample or by adding an organic solvent, addition of several displacing compounds and combinations of such approaches. A fully automated method is presented for the determination of ketoprofen, ibuprofen, flurbiprofen, fenoprofen and naproxen in human plasma, in which the absolute analyte recoveries are increased from 0–1% (untreated samples) to 40–65%. Relevant analytical data are given to demonstrate the reliability of the proposed procedure. 相似文献
8.
Ultraviolet resonance Raman study of drug binding in dihydrofolate reductase, gyrase, and catechol O-methyltransferase.
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This paper presents a study of the use of ultraviolet resonance Raman (UVRR) spectroscopic methods as a means of elucidating aspects of drug-protein interactions. Some of the RR vibrational bands of the aromatic amino acids tyrosine and tryptophan are sensitive to the microenvironment, and the use of UV excitation radiation allows selective enhancement of the spectral features of the aromatic amino acids, enabling observation specifically of their change in microenvironment upon drug binding. The three drug-protein systems investigated in this study are dihydrofolate reductase with its inhibitor trimethoprim, gyrase with novobiocin, and catechol O-methyltransferase with dinitrocatechol. It is demonstrated that UVRR spectroscopy has adequate sensitivity to be a useful means of detecting drug-protein interactions in those systems for which the electronic absorption of the aromatic amino acids changes because of hydrogen bonding and/or possible dipole-dipole and dipole-polarizability interactions with the ligand. 相似文献
9.
Woebking B Velamakanni S Federici L Seeger MA Murakami S van Veen HW 《Biochemistry》2008,47(41):10904-10914
The ATP-binding cassette transporter MsbA in Gram-negative bacteria can transport antibiotics and toxic ions. However, the key functional regions in MsbA which determine substrate specificity remain to be identified. We recently examined published mutations in the human MsbA homologue ABCB1 that alter multidrug transport in cells and identified mutations that affect the specificity for individual substrates (termed change-in-specificity mutations). When superimposed on the corrected 3.7 A resolution crystal structure of homodimeric MsbA from S almonella typhimurium, these change-in-specificity mutations colocalize in a major groove in each of the two "wings" of transmembrane helices (TMHs) that point away from one another toward the periplasm. Near the apex of the groove, the periplasmic side of TMH 6 in both monomers contains a hotspot of change-in-specificity mutations and residues which, when replaced with cysteines in ABCB1, covalently interact with thiol-reactive drug analogues. We tested the importance of this region of TMH 6 for drug-protein interactions in Escherichia coli MsbA. In particular, we focused on conserved S289 and S290 residues in the hotspot. Their simultaneous replacement with alanine (termed the SASA mutant) significantly reduced the level of binding and transport of ethidium and Taxol by MsbA, whereas the interactions with Hoechst 33342 and erythromycin remained unaffected. Hence, the SASA mutation is associated with a change-in-specificity phenotype analogous to that of the change-in-specificity mutations in ABCB1. This study demonstrates for the first time the significance of TMH 6 for drug binding and transport by MsbA. Based on these data, a possible mechanism for alternating access of drug-binding surfaces in MsbA is discussed. 相似文献
10.
We located the binding sites of doxorubicin (DOX) and N-(trifluoroacetyl) doxorubicin (FDOX) with bovine serum albumin (BSA) and human serum albumins (HSA) at physiological conditions, using constant protein concentration and various drug contents. FTIR, CD and fluorescence spectroscopic methods as well as molecular modeling were used to analyse drug binding sites, the binding constant and the effect of drug complexation on BSA and HSA stability and conformations. Structural analysis showed that doxorubicin and N-(trifluoroacetyl) doxorubicin bind strongly to BSA and HSA via hydrophilic and hydrophobic contacts with overall binding constants of K
DOX-BSA = 7.8 (±0.7)×103 M−1, K
FDOX-BSA = 4.8 (±0.5)×103 M−1 and K
DOX-HSA = 1.1 (±0.3)×104 M−1, K
FDOX-HSA = 8.3 (±0.6)×103 M−1. The number of bound drug molecules per protein is 1.5 (DOX-BSA), 1.3 (FDOX-BSA) 1.5 (DOX-HSA), 0.9 (FDOX-HSA) in these drug-protein complexes. Docking studies showed the participation of several amino acids in drug-protein complexation, which stabilized by H-bonding systems. The order of drug-protein binding is DOX-HSA > FDOX-HSA > DOX-BSA > FDOX>BSA. Drug complexation alters protein conformation by a major reduction of α-helix from 63% (free BSA) to 47–44% (drug-complex) and 57% (free HSA) to 51–40% (drug-complex) inducing a partial protein destabilization. Doxorubicin and its derivative can be transported by BSA and HSA in vitro. 相似文献
11.
The interaction of morin with human serum albumin (HSA) has been investigated by using fluorescence, UV absorption and Fourier transform infrared spectroscopic approaches for the first time. Fluorescence data revealed the presence of a specific binding site on HSA for morin, and the binding affinity was 1.13+/-0.11x10(-5) L Mol(-1) in the physiological condition. The intrinsic fluorescence of morin was conspicuously enhanced in the presence of HSA due to excited-state proton transfer. The binding ability of morin to protein decreased with the increase of the buffer pH from 6.4 to 8.4, which signified that the level of protonation of the hydroxyl groups played an important role during the drug-protein binding process. From the UV absorption spectra of morin in various pH medium, the dissociation behaviors of the hydroxyl groups on the drug molecule were assigned. The second derivative UV absorption spectra of morin after interacting with HSA were used to elucidate the binding mode of morin to protein. The obvious red shift of the UV absorption band I of morin upon binding to HSA further confirmed the formation of HSA-morin complex, and this property was also utilized to estimate the binding constant. The interaction between morin and HSA induced an obvious reduction of the protein alpha-helix and beta-sheet structures. 相似文献
12.
The effect of drug binding to urinary proteins on the diuretic response to furosemide was assessed in normal and nephrotic rats. Nephrosis was induced by treating Sprague-Dawley rats with puromycin aminonucleoside. Binding of furosemide to urinary proteins was found to range from 60 to 95% depending on the concentration of urinary protein. The diuretic response to furosemide reaching the renal tubular lumen was inversely correlated with the degree of proteinuria, a finding that was independent of serum protein concentration of glomerular filtration rate. These data suggest that the binding of furosemide to urinary protein decreases the diuretic effect of furosemide and that drug-protein interactions of this type may also be important in modulating the activity of other lumenally-active drugs or endogenous substances exhibiting a high degree of protein binding. The binding of furosemide to urinary protein may explain the refractoriness of some patients with proteinuria to this agent. 相似文献
13.
Vera-Avila LE García-Salgado E García de Llasera MP Peña-Alvarez A 《Analytical biochemistry》2008,373(2):272-280
Silica glasses doped with 500-700 microg of bovine serum albumin were prepared by the sol-gel method; two pH conditions (pH 5 and 7) were assayed for protein encapsulation. Both biomaterials showed a highly porous structure, with pore sizes in the range 5-28 nm. Columns packed with the ground biogels were on-line coupled to a C18 HPLC column for evaluation of the entrapped protein binding properties using propranolol. Binding capacities (at saturation) were approximately 3.7 and 7.1 microg of propranolol (drug-protein molar ratios 1.4 and 2.7) for the biogels prepared at pH 5 and 7, respectively. The significant difference indicates increased albumin denaturation upon encapsulation at pH 5. A frontal analysis study was then performed in cartridges packed with biogel prepared at pH 7 to evaluate the protein interaction with naproxen at low concentrations (相似文献
14.
The binding interaction between amphotericin B and human serum albumin (HSA) has been studied using surface plasmon resonance (SPR) spectroscopy combined with a fluorescence quenching method to confirm the binding kinetic results. In this paper, the SPR method used to study the drug-protein interaction has been described in detail. The association rate constant, dissociation rate constant and the equilibrium association constant of amphotericin B binding to HSA were obtained using this method. To confirm the feasibility of the SPR method, a fluorescence quenching method was performed to obtain the equilibrium constant. In order to obtain more accurate results, experiment design was used to optimize the fluorescence quenching process. The two equilibrium association constants obtained using the two methods were 4.017 x 10(4) M(-1) (SPR) and 3.656 x 10(4) M(-1) (fluorescence quenching method) respectively. 相似文献
15.
Zhou S 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2003,797(1-2):63-90
Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug-protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug-protein adducts. The methods used for drug-protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug-protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined. 相似文献
16.
M A Quevedo G N Moroni M C Bri?ón 《Biochemical and biophysical research communications》2001,288(4):954-960
The binding of novel nucleoside derivatives (2-7) to the Human Serum Albumin (HSA) was studied using zidovudine (AZT), as standard compound. The applicability of two different techniques to separate unbound drug from drug-protein complex was analyzed: the gel filtration and ultrafiltration methods. Ultrafiltration was found to be an adequate procedure for the separation of unbounded drug from the drug-protein complex. Incubation temperature ranging from 0 to 37 degrees C did not modify considerably the bound fractions. The same effects were observed as HSA concentration was modified. Binding assays of studied compounds to purified 1% (w/v) HSA at 0 degrees C, indicate that bound fraction of 2-7 ranges from 13 to 47%, exhibiting a higher affinity to HSA than AZT (12%), which would introduce some interesting improvements in their pharmacokinetic properties. In addition, by means of displacement studies using HSA site specific drugs such as diazepam and salicylate, it was determined that AZT binds to site I of the HSA molecule, by a mainly entropy driven process (DeltaS = 10.834 cal/mol degrees K), being these observations extensive to 2-7. Some structural basis to explain enhanced affinity of these novel derivatives was also established. 相似文献
17.
A critical role for PfCRT K76T in Plasmodium falciparum verapamil-reversible chloroquine resistance 总被引:6,自引:0,他引:6
Lakshmanan V Bray PG Verdier-Pinard D Johnson DJ Horrocks P Muhle RA Alakpa GE Hughes RH Ward SA Krogstad DJ Sidhu AB Fidock DA 《The EMBO journal》2005,24(13):2294-2305
Chloroquine resistance (CQR) in Plasmodium falciparum is associated with mutations in the digestive vacuole transmembrane protein PfCRT. However, the contribution of individual pfcrt mutations has not been clarified and other genes have been postulated to play a substantial role. Using allelic exchange, we show that removal of the single PfCRT amino-acid change K76T from resistant strains leads to wild-type levels of CQ susceptibility, increased binding of CQ to its target ferriprotoporphyrin IX in the digestive vacuole and loss of verapamil reversibility of CQ and quinine resistance. Our data also indicate that PfCRT mutations preceding residue 76 modulate the degree of verapamil reversibility in CQ-resistant lines. The K76T mutation accounts for earlier observations that CQR can be overcome by subtly altering the CQ side-chain length. Together, these findings establish PfCRT K76T as a critical component of CQR and suggest that CQ access to ferriprotoporphyrin IX is determined by drug-protein interactions involving this mutant residue. 相似文献
18.
Protein chemistry, such as crosslinking and photoaffinity labeling, in combination with modern mass spectrometric techniques, can provide information regarding protein-protein interactions beyond that normally obtained from protein identification and characterization studies. While protein crosslinking can make tertiary and quaternary protein structure information available, photoaffinity labeling can be used to obtain structural data about ligand-protein interaction sites, such as oligonucleotide-protein, drug-protein and protein-protein interaction. In this article, we describe mass spectrometry-based photoaffinity labeling methodologies currently used and discuss their current limitations. We also discuss their potential as a common approach to structural proteomics for providing 3D information regarding the binding region, which ultimately will be used for molecular modeling and structure-based drug design. 相似文献
19.
Sulfamethoxazole (SMX) causes rare hypersensitivity syndrome reactions characterized by fever and multi-organ toxicity. Covalent binding of SMX reactive metabolites to cellular proteins has been demonstrated but the link between cytotoxicity and targets of covalent binding has not been explored. We therefore investigated the relationship between covalent binding of the reactive SMX-hydroxylamine (SMX-HA) metabolite, and its cytotoxicity to a hystiocytic lymphoma (U937) cell line. Incubation of U937 cells with 0-1 mM SMX-HA for 3 h resulted in dose-dependent cytotoxicity, as assessed by tetrazolium dye conversion at 24 h. SMX-HA caused dose-dependent covalent binding to cellular proteins as assessed by immunoblotting with SMX antisera at 3 and 24 h. Covalent binding was predominantly to proteins of approximately 45, 59 and 75 kDa, but other targets were also observed. The relative extent of binding to proteins was significantly different from the relative cytotoxicity at 24 h. Further, cells surviving at 24 h also had extensive covalent binding. Covalent binding was observed under reducing (beta-mercaptoethanol) and non-reducing conditions to plasma membrane and microsomal but not cytosolic proteins. This non-labile covalent binding has not been previously reported. These observations suggest that extensive covalent binding does not necessarily lead to cell death, allowing the accumulation of potentially immunogenic drug-protein conjugates. These observations in whole cells may be relevant to the immunopathogenesis of SMX hypersensitivity syndrome reactions. 相似文献
20.
Recent advances in enantiomer separations by affinity capillary electrophoresis using proteins and peptides 总被引:1,自引:0,他引:1
Enantiomer separations by capillary electrophoresis (CE), using proteins as chiral selectors--affinity capillary electrophoresis (ACE) with free solutions and capillary electrochromatography (CEC)--with protein immobilized capillaries, are reviewed. The separation principle, recent advances in this field and some interesting topics are presented. In ACE, various enantiomer separations have been already reported using either plasma proteins or egg white ones. Miscellaneous proteins were also explored in the last few years. On the contrary, only a limited number of enantiomer separations have been successfully achieved in CEC. CEC is not yet mature enough to date, and further investigations, such as efficiency, durability and reproducibility of capillaries, will be necessary for the use of routine analyses. The study of enantioselective drug-protein binding is important in pharmaceutical developments. Some applications including high-performance CE/frontal analysis (HPCE/FA) are introduced in this paper. 相似文献