Drug Binding Properties Research Articles

Non-P-glycoprotein multidrug resistance in cell lines which are defective in the cellular accumulation of drug.

Non-Pgp mdr related to a defect in drug accumulation has now been documented in a number of different cell lines exposed to certain cytotoxic agents. In studies conducted thus far most isolates have been obtained after selection in either adriamycin or mitoxantrone. The work in this area is in its early stages and very little is known about the molecular events which contribute to this mode of drug resistance. At the present time no protein with drug binding properties comparable to Pgp has been identified in non-Pgp mdr isolates. Evidence based on the finding that all isolates do not respond in the same way to reversal agents such as verapamil suggests the possibility that more than one mechanism may exist for non-Pgp mdr. Future studies may thus reveal that cells contain a multiplicity of genes which upon transcriptional activation can function to alter drug transport processes and thus contribute to the development of mdr. Identifying and characterizing these genes will be important since they may function in transport systems of normal cells. The exact identify of proteins which contribute to non-Pgp mdr remains to be determined. One protein designated P190 has been found to be overexpressed in cell lines of human promyelocytic leukemia, lung and adenocarcinoma treated with adriamycin. The protein also is increased in some clinical samples from patients undergoing chemotherapy. P190 which has a minor sequence homology with Pgp can bind ATP and may thus contribute to the energy dependent drug efflux systems found in cells containing this protein. Transfection studies with a P190 cDNA should determine whether this protein actually contributes to drug resistance. Many other protein changes have been detected in non-Pgp mdr cells but the importance of these in resistance also remains to be determined. In some systems a particular protein change can be identified in multiple independent isolates suggesting a correlation between the development of resistance and the presence of this cellular alteration. Experiments conducted thus far on the mechanism of non-Pgp mdr are intriguing. Studies utilizing fluorescence microscopy to follow the fate of daunomycin suggests that the drug passes to the interior of the cell and eventually localizes in the Golgi apparatus. Drug located at this site may move directly into an efflux pathway for rapid extrusion from the cell. Evidence also indicates that as drug leaves the Golgi some may be sequestered into other organelles such as lysosomes or mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Thermodynamic stability and drug-binding properties of oligodeoxyribonucleotide duplexes containing 3-deazaadenine:thymine base pairs.

We have used ultraviolet melting techniques to study the effect on stability of incorporating the nucleoside analogue 2'-deoxy-3-deazaadenosine (d3cA) into the duplex 5'-d(CGCAATCG)-3'-d(GCGTTAGC). Our results demonstrate that the successive replacement of dA by d3CA increasingly destabilises the duplex. The destabilising effect of this analogue is considerably enhanced as the pH is lowered and the results are consistent with protonation of 3-deazaadenine (probably at N-1) contributing to duplex destablisation. Surprisingly, the incorporation of d3CA does not significantly affect the binding of distamycin-A.

Quantum chemical study on the interaction of some bisphosphonates and Ca2+: The role of molecular electrostatic potentials in the prediction of binding geometry

Molecular electrostatic potentials have been used to model the calcium binding properties of some bisphosphonate drugs, which are used to treat various bone diseases. The mechanism of action involves the binding of bisphosphonates to the bone surface, where calcium plays an important role. Electrostatic potential maps derived from ab initio partial charges have been compared with both the crystal structure and the fully optimized ab initio structure of (dichloro)methylene-bisphosphonate-calcium ion complex. Molecular electrostatic potentials can correctly predict the calcium binding geometry of bisphosphonate-type compounds and this type of information can be used in the practical drug design work.

Model Study of Hepatocyte Targeting Polymeric Super Molecular Assembly as Drug Carrier

Abstract The drug binding properties of lactose carrying polystyrene (PVLA), which could be endocytosed into hepatocytes due to its interaction with receptors of asialoglycoprotein, were examined by UV and NMR spectroscopies. The results obtained with a hydrophilic drug model, 8-anilino-naphthalene-1-sulphonate (ANS), and with a hydrophobic drug model, 1,6-diphenyl-hexatriene (DPH), indicated the PVLA can be used as a hepatocyte targeting drug carrier.

Effect of Plasmodium berghei infection and chloroquine on the hepatic drug metabolizing system of mice

The hepatic microsomal mixed-function oxidase (MFO) system was markedly impaired during Plasmodium berghei infection in mice. Cytochrome P-450 and other mono-oxygenases, viz. aniline hydroxylase, aminopyrine- N-demethylase and benzo( a)pyrene hydroxylase, were significantly decreased while microsomal heme showed a four-fold increase at peak parasitemia (> 50%). Oral treatment with chloroquine (16 mg kg −1 body wt for 4 days) of P. berghei-infected mice cleared the parasitemia within 72 h and almost normalized the altered levels of MFO indices, a week after cessation of treatment. The findings were further supported by the isoenzymic profile and drug-binding properties of terminal mono-oxygenase, cytochrome P-450.

Influence of inflammation on serum concentration, molecular heterogeneity and drug binding properties of canine alpha-1-acid glycoprotein.

The concentration and the heterogeneity of alpha-1-acid glycoprotein (alpha-1-AGP) and oxprenolol binding were determined in serum of healthy dogs and dogs with inflammatory disease. In inflammation, an increase in the mean alpha-1-AGP concentration from 0.47 to 2.85 g/l was accompanied by a reduction in the mean free oxprenolol fraction from 25% to 6%. alpha-1-AGP concentration and oxprenolol binding were inversely correlated. The heterogeneity of canine alpha-1-AGP remained essentially unchanged in dogs with inflammation and, in both these dogs and the controls, between five and seven forms with different isoelectric points and one single concanavalin A-reactive form were detected. It is concluded that in dogs, as in humans, oxprenolol binds to serum alpha-1-AGP. Changes in serum binding of oxprenolol during inflammation result from a change in the serum concentration of alpha-1-AGP rather than a change of molecular heterogeneity.

60] Preparation and use of iodinated calmodulin for studies of calmodulin-binding proteins

Publisher Summary A modification of calmodulin that has been particularly useful in studies of calmodulin and its role in the cell is iodination. Iodination of calmodulin has been employed to establish radioimmunoassays to detect and measure calmodulin in biological tissues. Iodination of calmodulin under carefully controlled conditions also allows the introduction of an isotopic label with retention of drug binding, protein binding, and enzyme activator properties. This chapter summarizes some observations that are important for the reproducible use of iodinated calmodulin in studies of calmodulin-binding proteins and calmodulin activator activities. Vertebrate calmodulin can be readily iodinated by methods utilizing chloramine-T, lactoperoxidase, or Bolton-Hunter reagent. The chapter shows the results obtained when a partially purified bovine brain phosphodiesterase preparation and purified chicken gizzard myosin light-chain kinase were tested for their ability to interact with iodinated calmodulin. The results are consistent with the phosphodiesterase activator assays.

Drug-binding properties of rat α-foetoprotein. Specificities of the phenylbutazone-binding and warfarin-binding sites

Rat alpha-foetoprotein (alpha-FP) strongly binds the drugs warfarin and phenylbutazone, as does albumin; however, the binding sites for the two drugs seemed to be different. This possibility and the specificity of this/these drug-binding site(s) of rat alpha-FP were investigated by competitive protein-binding experiments with a variety of drugs, representing different pharmacological groups, and biomolecules that are strongly bound by the foetal protein and that are suspected to play a specific role during foetal development. The binding mechanisms were further investigated by using comparisons between computer-derived theoretical displacement curves and experimental points in order to distinguish different possible binding models. The results indicate: that warfarin and phenylbutazone are bound at two distinct sites on rat alpha-FP and that a negative modulatory effect is exerted between the two sites; that the degree of specificity of these two drug-binding sites is different, since the warfarin-binding site appears to be specific for the binding of coumarinic and anthranilic drugs whereas that for phenylbutazone is able to bind substances of very varied chemical structure and is more hydrophobic; that the phenylbutazone-binding site is the site that binds oestrogens that thyroid hormones and, probably, fatty acids and bilirubin are bound at (an)other site(s) but exert negative modulatory effects on phenylbutazone binding. The nature of the different binding areas of rat alpha-FP is compared with that of those already proposed for albumin. The potential risks of toxicity of such interactions between drugs and/or biomolecules on foetal development are also discussed.

Immunohistochemistry and effect of vinblastine on intracisternal microtubules of the canine neurons

The immuno-histochemical and drug-binding properties of intracisternal microtubules (IMs) in the RER of canine neurons were studied with antitubulin IgG and vinblastine. Specific fluorescence against antitubulin IgG was not detected in the RER in which the IMs were aggregated but was observed in the cytoplasm outside the RER. Injection of vinblastine into the sympathetic ganglion did not change the size, shape or arrangement of IMs in the RER, although typical honeycomb crystals were formed near IM-containing RER. These findings show that the IM does not possess antigenicity to antitubulin IgG and has no vinblastine-binding site. Thus it is suggested that the IM may be a defective or modified microtubule.

Dihydropyridine-Sensitive Ca2+ Channels

[3H]Desmethoxyverapamil, (S/-)[3H]bepridil, and d-cis-[3H]diltiazem bind to skeletal muscle transverse-tubule (T-tubule) membranes with affinity constants in the range of 2-50 nM and with Bmax values of the order of 50-80 pmol/mg of protein. These drugs are mutually competitive and modulate allosterically the 1,4-dihydropyridine binding. The molecular size of the native [3H]nitrendipine receptor of T-tubule membranes has been determined using the radiation inactivation technique. The [3H]nitrendipine receptor was found to have a Mr of 210,000 +/- 20,000. Photoaffinity labelling using (+)[3H]PN 200-110 indicates the presence in the dihydropyridine receptor of a polypeptide (or polypeptides) of Mr 170,000 +/- 5,000. The 3-[(3-cholamidopropyl)-dimethylammonio]-1 propane sulfonate (CHAPS) solubilization of the T-tubule membranes preserved the binding properties of the different drugs to the calcium channel. The dihydropyridine-sensitive calcium channel has been substantially purified by a combination of ion exchange chromatography, lectin affinity chromatography, and gel filtration. The purified material contained polypeptides of apparent molecular weights of 142,000, 32,000, and 33,000, which copurified with (+)[3H]PN 200-110 binding activity. Two stages in which there is an increased binding of [3H]nitrendipine have been observed during chick myogenesis in ovo. The first one occurs during embryonic life and has the same properties as in the in vitro development. The second stage occurs near hatching and corresponds to a large increase in the number of nitrendipine receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Drug-binding properties of human α-foetoprotein

The drug-binding properties of human alpha-foetoprotein (alpha FP) were investigated by a fluorescence-spectral method. Human alpha FP was shown to bind to albumin's site I marker (warfarin, phenylbutazone), site II marker (L-tryptophan), but not site III marker (cholic acid, digoxin). The binding of human alpha FP towards lower alcohols was examined, and this binding seems to depend partly on the hydrophobicity of the ligands. The binding of human alpha FP is discussed in comparison with human serum albumin or rat alpha FP.

Drug-binding properties of rat alpha 1-foetoprotein. Binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid.

As part of an investigation into whether alpha 1-foetoprotein (alpha 1-FP) plays the same transport role in foetal serum as albumin does in the adult, the binding properties of both proteins were compared with respect to the binding of a series of compounds known to be bound by albumin's specific drug-binding sites. The binding of warfarin, phenylbutazone, azapropazone, diazepam, digitoxin and cholic acid by rat alpha 1-FP and serum albumin was studied by equilibrium dialysis at 4 degrees C. Rat alpha 1-FP was shown to have neither albumin's high-affinity site II (diazepam as marker) nor its site III (digitoxin and cholic acid as markers). High-affinity binding by alpha 1-FP was found for the specific markers (warfarin, phenylbutazone, azapropazone) of albumin's drug-binding site I. However, instead of albumin's one high-affinity site/molecule, a mean value of 0.5 site/molecule was obtained with rat alpha 1-FP. Charcoal treatment at neutral pH of rat serum albumin did not affect its measured binding properties, but treatment of the alpha 1-FP led to an increased affinity for warfarin, phenylbutazone and azapropazone without a change in the measured number of sites, indicating competition for binding at this site by (an) endogenous ligand(s). These results are discussed in terms of the structures of the two proteins and with respect to the physiological implications of the differences found.

An allosteric model for benzodiazepine receptor function

FREDERICK J. EHLERT*, WILLIAM R. ROESKE, KELVIN W. GEE and HENRY I. YAMAMURA# Departments of Pharmacology, Biochemistry, Psychiatry and Internal Medicine, and the Arizona Research Laboratories, University of Arizona Health Sciences Center, Tucson, AZ 85724, U.S.A. The assignment of specific 3H-labeled ligand binding to a pharmacologically relevant receptor is not a trivial matter. Nevertheless, evidence has accumu- lated during the past few years that benzodiazepines mediate their pharmacological effects by interacting with neuronally localized receptors within the brain. An intriguing question is how the recognition of a benzodiazepine by its receptor initiates a chain of events leading to the pharmacological response, e.g. prevention of seizures, tranquility and sedation. The conventional approach has been to rationalize the initial recognition step within the context of a lock-and-key model. This hypothesis can readily account for the pharmacological specificity and stereospecificity of receptors as being a consequence of their geometry. It is with this recognition process that the implications of receptor binding data have their most immediate application. In addition, ligand binding methods can yield some insight into the activation of the receptor-effector system. It has been pointed out that part of the intrinsic binding energy of an agonist is used to induce a conforma- tional change in the receptor [1]; in other words, the observed affinity of a drug for a receptor depends on both the bimolecular association of the ligand with the receptor and the subsequent conformational change of the ligand-receptor complex. Although this idea is not a new one, the importance of this phenomenon in regard to interpreting binding data is not generally recognized. It is useful to consider how the binding properties of drugs for other neurotransmitter receptors are affected by the intrinsic activity of the ligand. With regard to beta-adrenergic and muscarinic receptors, there are striking differences in the way agonists and antagonists bind to these receptors [2-4]. In general, agonists tend to display heterogeneous binding properties [5]. The magnitude of this heterogeneity can be estimated by computing the relative differ- ences in the affinities of a drug for subclasses of the receptor or, in some instances, by estimating the relative densities of the high and low affinity states of the receptor. Strong correlations between the * Present address: Department of Pharmacology, School of Medicine, University of California, Los Angeles, CA 90024, U.S.A. t Address correspondence to: Henry I. Yamamura, Ph.D., Department of Pharmacology, Arizona Health Sci- ences Center, Tucson, AZ 85724, U.S.A. heterogeneity of binding and the efficacy ligand have been noted for muscarinic and beta- adrenergic receptors [2-4]. Also, the negatively cooperative effects of guanine nucleotides on binding properties show a strong correlation with efficacy [5, 6]. For opiate receptors, a correlation between the influence of Na + on binding and efficacy opiate analogues has been noted [7]. It is not our intent to assess the models that have been proposed to account for these phenomena but merely to emphasize the fundamental principle providing basis for these relationships: the intrinsic activity of an agonist is related to its ability to induce a confor- mational change in the receptor. During the course of our studies of the benzodi- azepine receptor binding activity of the beta-car- boline compounds which are structurally unrelated to benzodiazepines (see Fig. 1), we noticed certain correlations between the influence of gamma-ami- nobutyric acid (GABA) on binding and the intrinsic activity of the drug [8, 9]. These differences have been rationalized in terms of an allosteric model for benzodiazepine receptor function which views the benzodiazepine receptor as a heterotropic site on the GABA receptor-chloride ionophore complex [10], The model is an attempt to define the rela- tionships between the behavioral effects of benzo- diazepines and the influence of GABA on binding. Presently, it is our goal to describe the model in greater detail and to demonstrate that it can account for most of the pharmacological effects of ligands that interact specifically with the recognition site of the benzodiazepine receptor. We shall begin our discussion by reviewing some of the complex binding characteristics of the benzodiazepine receptor. Benzodiazepine receptor heterogeneity Initially, it seemed as if benzodiazepine receptors were homogeneous [11-13]. This assumption was based on the results of numerous binding studies which have shown that the equilibrium binding iso- therms for [3H]diazepam and [3H]flunitrazepam ([3H]FLU) in the range of concentrations studied were consistent with the simple Langmuir isotherm [11-15]. Also, when the specific binding of [3H]FLU or [3H]diazepam in low concentrations is inhibited by other nonlabeled benzodiazepines, the competi- tion curves usually have Hill coefficients of approx- imately 1. However, evidence soon emerged which suggested that benzodiazepine receptors are not sim- ply a homogeneous class of binding sites. In 1979, 2375

A comparative study of some physico-chemical properties of human serum albumin samples from different sources—II: The characteristics of the NB transition and the binding behaviour with regard to warfarin and diazepam

A comparative study of the NB transition and the drug binding properties of human serum albumin samples from various sources has been carried out with the help of circular dichroism and equilibrium dialysis. It was found that when warfarin was used as a marker the midpoint pH and the cooperative nature of the NB transition that occurs in the albumin around physiological pH varied with the albumin sample. The midpoint pH was found to be related to the cooperative nature of the albumin samples. A similar relationship has been found for allosteric proteins. However, when diazepam was used as a marker molecule for the N-B transition, variations in the midpoint pH and cooperative nature of the NB transition disappeared. This is attributed to the strong allosteric effect of diazepam on binding. The affinity of warfarin for albumin depends strongly on the sample, but this is not the case with diazepam. The cooperative binding properties found for the albumin samples are compared with those found for the albumin in serum. After a discussion it is concluded that the cooperative binding properties of a particular albumin sample should be taken into account when that sample is used in binding studies.

Isolation and identification of a trypsin-resistant fragment of human serum albumin with bilirubin- and drug-binding properties

Extensive digestion of human serum albumin with trypsin at pH 8.8 yields essentially one main fragment which is resistant to further tryptic degradation. The fragment has been characterized by amino acid analysis, N- and C-terminal analyses, cyanogen bromide digestion, electrophoresis, ultracentrifugation and gelfiltration, and circular dichroism measurements. The results indicate that the main fragment consists of the amino acids 182 585. Repeated digestion did not degrade the isolated fragment further. The fragment mainly retains the secondary and tertiary structure of intact human serum albumin as well as its capacity to bind bilirubin and diazepam. The localization of the binding sites for these substances is discussed.

Polypeptides similar to the alpha and beta subunits of tubulin are exposed on the neuronal surface.

THE finding by Borisy and Taylor1 that nervous tissue was the source of large amounts of soluble tubulin, the subunit protein of microtubules, has greatly facilitated the biochemical study of microtubular structure. From 10% to 20% of the total protein of developing brain is in the form of soluble tubulin2,3 and most studies have focused on this form of the protein. In 1970, however, Feit and Barondes demonstrated a large amount of colchicine binding activity, a term which has become associated with the presence of tubulin, in particulate fractions of brain4—fractions expected to be free of microtubule-derived tubulin. Recent studies indicate that polypeptides with the same size and drug-binding properties as soluble tubulin are present in synaptosomal plasma membranes5–7. These studies suggest a membrane-associated form of the protein which is insoluble in non-chaotropic buffers. I here report experiments which provide additional evidence for membrane-associated tubulin-like proteins and also suggest that a portion of this polypeptide(s) may be exposed on the external surface of the cell.

Studies on the Tryptophan and Drug-binding Properties of Human Serum Albumin Fragments by Affinity Chromatography and Circular Dichroism Measurements

Abstract After cyanogen bromide digestion of human serum albumin, three fragments, A, B, and C, are obtained. Two different types of affinity columns of Sepharose with l-tryptophan were prepared, one with a free carboxyl group and one with a free amino group. Only Fragment C showed affinity to the Sepharose column with the carboxyl group of tryptophan free, while the column having the amino group free did not extract any fragment from the cyanogen bromide digest. Fragments A, B, and C were separated and studied by circular dichroism. Large parts of the native structure are preserved in the fragments. Human serum albumin contains 45 to 50% α helix, and totally about 35% is found in the fragments. Fragment A contains about 42% α helix and about 15 to 22% β structure, B contains 22 to 26% α helix and 9 to 14% β structure, and C contains 28 to 36% α helix and 11 to 32 % β structure, the significance of which is discussed. The binding of l-tryptophan and drugs to the different fragments was studied by circular dichroism and equilibrium dialysis; only Fragments A and C were found to be active. The apparent association constants, Ka, for the binding of l-[14C]tryptophan at pH 9.0 and 10° were 0.16 x 104 m-1 and 0.17 x 104 m-1 for Fragments A and C, respectively. The lone tryptophan residue of human serum albumin is found in Fragment C, the importance of which is discussed.