Alpha v integrin affinity/specificity and antiangiogenesis effect of a novel tetraaza cyclic peptide derivative, SU015, in various species

The present study was undertaken to define the alpha v beta3 and alpha v beta5 binding potency and specificity of SU015, an integrin antagonist. SU015 inhibited alpha v beta3-mediated human umbilical vein endothelial cell or 293/beta3-transfected CHO cell adhesion to fibrinogen, with IC50 values of 0.21 +/- 0.11 muM and 0.32 +/- 0.02 microM. SU015 demonstrated comparable affinity to alpha v beta5 as compared with alpha v beta3 affinity, as well as a relatively high degree of specificity for human alpha v beta3- and alpha v beta5-mediated functions, as compared with other human integrins, including alphaIIbbeta3 (IC50 >100 microM), alpha5/beta1 (IC50 >100 microM), and alpha4/beta1 (IC50 >100 microM). SU015 demonstrated different degrees of species specificity in blocking alpha v beta3-mediated cellular adhesion, with relatively higher affinity to monkey (IC50 = 0.10 microM) and dog (IC50 = 1.30 microM) endothelial or smooth muscle cell alpha v beta3-mediated adhesion. Additionally, SU015 demonstrated a high degree of alpha v beta3 and alpha v beta5 specificity as compared with alpha4beta1-, alpha5beta1-, or alpha IIb beta3-mediated binding in the above species. In conclusion, SU015 is an alpha v beta3 and alpha v beta5 antagonist with relatively higher potency and specificity as compared with alpha IIb beta3, alpha5beta1, or alpha4beta1 integrins. Additionally, comparable alpha v beta3 and alpha v beta5 affinity for SU015 was demonstrated with human and monkey endothelial cells. These data also suggest that this bicyclic RGD analogue linked to a linker at the bottom leaves the RGD at the top available for binding and allows for conjugation with radioisotope for imaging and radiotherapy.     

Relative binding affinity at metribolone androgenic binding sites of various antiandrogenic agents

Sebaceous glands are androgen sensitive structures with activity reduced by antiandrogens. We characterized the relative binding affinity of cyproterone acetate, 17 alpha-propyl-mesterolone, spironolactone (canrenoic acid), ethisterone and dexamethasone by means of the competitive binding analysis at metribolone (R1881) androgen binding sites. Using the DCC-assay (dextran-coated charcoal absorption) with the Scatchard plot and saturation analysis we quantified R1881 androgen binding sites from sebaceous glands situated in the ventral side of the pinna of the Syrian hamster. As parameters for ligand affinity at these binding sites served the ligand concentration for 50% displacement of 3H-labelled synthetic steroid R1881 in constant concentrations. The in vitro measurements of the used steroids were compared to the biologic sebosuppressive effect in vivo. 17 alpha-Propylmesterolone showed the highest affinity at the androgenic binding site followed by ethisterone, cyproterone acetate and spironolactone. The results, however, do not admit an interpretation about the mode of action of the given substances, which display their biologic activity either after systemic or topic administration. In vivo distribution problems and metabolizing procedures might be due to this discrepancy.     

Relative binding affinity of various progestins and antiprogestins to a rabbit myometrium receptor

Relative binding affinity (RBA) of various progestins and antiprogestins to a cytoplasmic receptor prepared from the myometrium of estrogenized immature female rabbits was investigated. The cytosol was incubated with tritiated promegestone (3H-R5020) in the presence of various concentrations of non-radioactive promegestone (R5020) as standard on the one hand, and several progestins and antiprogestins on the other hand. The incubate was subjected to isoelectric focusing in slabs of polyacrylamide gel and the bound radioactivity in the peak of the receptor was measured, RBA was expressed as a percentage given by the ratio of those concentrations of R5020 and the compound tested which were required for a 50% displacement of 3H-R5020. The RBA's of the progestins tested were in the following order: R5020 greater than norethisterone greater than levonorgestrel greater than progesterone greater than medroxyprogesterone acetate. There was practically no binding to dextronorgestrel, cortisol, testosterone, and estradiol. In the group of antiprogestins, there were no significant differences in the RBA of the compounds RU 486, RU 42633 (monodemethyl derivative of RU 486) and ZK 98.734. Another two derivatives of RU 486, RU 42848 (didemethyl) and RU 42698 (propargyl), had lower RBA's than RU 486. Two 13,17-stereoisomers related to the above antiprogestins (i.e. compounds ZK 98.299 and ZK 115.716) exhibited a decreased RBA in comparison with the compound ZK 98.734.     

Determination of the binding specificity of an integral membrane protein by saturation transfer difference NMR: RGD peptide ligands binding to integrin alphaIIbbeta3.

Saturation transfer difference (STD) NMR is a fast and versatile method to screen compound mixtures in the presence of a receptor for binding affinity and to characterize the ligand's binding epitope. Here we demonstrate that ligand interactions with integral membrane proteins can be investigated by STD NMR if the receptor is embedded into the lipid bilayer of a liposome. The integrin alphaIIbbeta3, also termed GPIIb-IIIa, is a platelet surface glycoprotein that plays a pivotal role in platelet aggregation and that interacts with proteins and peptides presenting the peptide recognition motif RGD. Purified human integrin alphaIIbbeta3 was incorporated into liposomes, and the binding of RGD peptides was analyzed by STD NMR techniques. Cyclo(RGDfV) gave STD NMR effects in the presence of liposomes containing the integrin. The magnitude of the STD effect as a function of the ligand's concentration gave a value for the dissociation constant of 30-60 microM. Adding the weakly binding RGD to the solution of cyclo(RGDfV) resulted in STD effects of the stronger ligand cyclo(RGDfV) only. This demonstrates in agreement with literature that the peptide RGD is a much weaker ligand to the integrin than the peptide cyclo(RGDfV) that largely replaces the RGD peptides from the binding site. The binding epitope of the ligand cyclo(RGDfV) was characterized by STD NMR to contain sections of the D-Phe, the Val methyl groups, Arg alpha, beta, and gamma protons, one Hbeta of Asp, and one Halpha of Gly.     

Protein binding of cefpiramide in the plasma of various species.

The plasma protein binding of cefpiramide in man, dog, rabbit and rat was examined. Cefpiramide was more strongly bound to human plasma than to animal plasma and was exclusively bound to albumin. The characteristics of the protein binding of cefpiramide is one of the factors influencing the extrapolation of drug disposition from animals to man.     

Heterogeneity of cerebral beta-adrenoceptor binding sites in various vertebrate species.

[3H]-Dihydroalprenolol ([3H]-DHA) binds to cerebral membranes of the frog, chick, rat, mouse, rabbit and human with a dissociation equilibrium constant (KD) of about 1 nM and displays binding characteristics indicative of an interaction with beta-adrenoceptors. However, the maximum number of specific binding sites labelled by this beta-adrenoceptor ligand varies substantially between the species with the chick and mouse having the highest, and the frog the lowest density. The structure--activity relationships of adrenergic agents to inhibit specific [3H]-DHA binding suggests that whereas the membrane sites from all the species had similar affinities for non-selective beta-adrenergic agents, several drugs that have been reported to show selectivity for beta1-adrenoceptors demonstrated considerably higher affinities for mammalian rather than avian or amphibian membrane sites. By this pharmacological criteria it is likely that all the beta-adrenoceptor binding sites in frog and chick cerebral tissue have properties resembling beta2-receptors. However, in mammalian cerebral cortex, evidence is presented that beta1- and beta2-adrenoceptors coexist in a ratio of 70%/30% respectively.     

Factors affecting the tissue binding of nicotine in various species.

The binding of [¹⁴C]nicotine to various tissue fractions has been studied in the cat, pigeon and rat using the technique of equilibrium dialysis. Liver homogenates from all three species bound the drug to varying extents though the distribution of radioactivity showed species differences. Cat and rat liver binding appeared to reside predominantly in the microsomal fraction whereas in the pigeon the activity was essentially present in the cytosol. Lung tissue from all three species showed only a small binding capability which was not associated with the microsomes or soluble cell components. Brain homogenates in cat and rat showed a similar small degree of binding but there was a significant degree of binding in pigeon brain homogenate. Cat and pigeon kidney homogenates showed a small degree of tissue binding which appeared to be uniformly distributed in the cell fractions. Rat kidney, however, exhibited an extremely large in vitro binding capability which was apparently associated with the cytosol. The percentage binding was increased with increasing buffer pH over range pH 6–8 corresponding to an increase in the proportion of free base. A Scatchard plot over the concentration range 62–1540 nM gave a linear response, calculations from which indicated a low affinity, high capacity, binding capability for nicotine. In vivo distribution studies in the rat after subcutaneous administration of 0.4 mg/kg [¹⁴C]nicotine revealed a high degree of localisation of radioactivity in the kidney relative to other tissues and subsequent tissue fractionation confirmed the in vitro observations. Plasma from all three species showed no significant binding properties. The nature of the binding entity is not yet known though it can be concentrated by chromatography on Sephadex G-100 and is associated with a fraction of relatively low molecular weight containing little, if any, lipid.     

High affinity binding of3H-paroxetine and3H-imipramine to rat neuronal membranes

Paroxetine is the most potent and one of the most specific serotonin uptake inhibitors. High-affinity³H-paroxetine and³H-imipramine binding was compared in rat neuronal membranes. TheK _d value for³H-paroxetine binding to neuronal membranes was 0.08 nM, which is exactly the same value as with platelet membranes. TheK _d value for³H-imipramine binding to neuronal membranes was about 4 nM, which is higher than theK _d value for³H-imipramine binding to platelet membranes (0.5 nM). The results indicated that the³H-paroxetine binding site is identical in neuronal membranes and in platelet membranes; this binding site is probably located on the serotonin transport mechanism. In addition, part of the highaffinity³H-imipramine binding to neuronal membranes is probably located on the serotonin transport mechanism, but another part is located elsewhere. Furthermore the polypeptides containing the³H-imipramine binding sites may not be identical in neuronal and platelet membranes.     

The integrin specificity of human recombinant osteopontin.

The ability of full-length human recombinant osteopontin (OPN) to support the adhesion of various alphav integrin-expressing cell lines was determined in order to characterize its integrin selectivity. The identity of this protein was assessed by cDNA sequence and mass spectroscopic analysis, and confirmed as full-length OPN. Neither the human embryonic kidney 293 cell line, which expresses the alphavbeta1 integrin, nor the human colonic adenocarcinoma HT-29 cell line, which expresses the alphavbeta5 integrin, were able to adhere to OPN; both of these cell lines are deficient in the beta3 subunit. In contrast, an alphavbeta3 integrin-expressing cell line, SK-MEL-24, was able to adhere to OPN in an arginine-glycine-aspartic acid dependent manner. In addition, this OPN-mediated cellular adhesion was completely blocked with an anti-alphavbeta3 integrin antibody (LM609), confirming that only the alphavbeta3 integrin mediated this cellular adhesion. These data demonstrate that, at least among the alphav integrins, only the alphavbeta3 is able to support cellular adhesion to osteopontin. This finding may have implications for the design of therapeutics targeting OPN-integrin interactions.     

The specificity of high affinity binding of avermectin B1a to mammalian brain

Avermectin B₁a, an anthelmintic with pharmacological properties of paralyzing nematodes and causing an increase in the release of GABA from rat brain synaptosomes, was found to bind to dog brain synaptosomes with an apparent-K_D of 1–2 nM. This high affinity binding was saturable and the density of binding sites was estimated to be 1.54 pmol/mg protein. Results from binding competition among various analogs of the drug suggested that the binding is stereospecific and the affinities to the binding sites correlated well with the anthelmintic activities. The binding sites are unevenly distributed in the dog brain with the highest density found in cerebellum, less in midbrain, hypothalamus and cerebral cortex, and little or no binding in pons and medulla oblongata. Similar specific binding of avermectin B₁a to synaptosomes of adult rat brains was also observed. The binding sites apparently emerged during the first three weeks of post-natal development of the rat brain. Although all the evidence supports the hypothesis that the binding sites may be associated with GABA synapses, the lack of competition between GABA and the drug in their bindings suggests that the drug receptors are not associated with GABA post-synaptic receptors.     

Proadifen-sensitive high affinity binding of 3H-alaproclate to liver membranes

3H-Alaproclate, a selective 5-hydroxytryptamine uptake inhibitor, was found to bind to microsomal membranes from the rat liver with high affinity (KD = 3 nM) and large capacity (Bmax about 2 nmol/g liver). This binding was stereoselective since S-(-)-alaproclate was 30 times more potent than the R-(+)-enantiomer to displace the 3H-labelled racemate. Proadifen (SKF 525A), an inhibitor of cytochrome P-450, displaced the 3H-alaproclate binding with the same, high affinity (Ki = 3 nM) as alaproclate itself. Repeated treatment with phenobarbital sodium (5 x 75 mg/kg intraperitoneally) increased the number of alaproclate binding sites 7-8 times without changing the affinity. However, most of the phenobarbital induced 3H-alaproclate binding was not displaceable by proadifen, showing the presence of at least two different high affinity binding sites. The possible involvement of cytochrome P-450 in the alaproclate binding is discussed.     

Regional distribution of high affinity binding of 3H-adenosine in rat brain

The high and low affinity adenosine binding sites with Kd values ranging respectively from 0.8 to 1.65 microM and from 3.1 to 13.86 microM were demonstrated in the following rat brain areas: cortex, hippocampus, striatum, cerebellum, diencephalon, and pons-medulla. Adenosine receptors involved in the high affinity binding seem to be mainly Ra-type. The analysis of the regional distribution of 3H-Adenosine showed the highest levels of specific binding in striatum and hippocampus; somewhat smaller values in cortex, cerebellum, and diencephalon, and even lower in pons-medulla.     

Quantitative molecular analysis predicts 5-hydroxytryptamine3 receptor binding affinity

A quantitative molecular model was derived to predict drug affinities for 5-hydroxytryptamine3 (5-HT3) receptors. The model was based on the molecular characteristics of a "learning set" of 40 pharmacological agents that had been analyzed previously in radioligand binding studies. Molecules were analyzed for various structural features, i.e., the presence of a benzenoid ring and nitrogen atom, substitutions on the benzenoid ring, the location of the substitutions on the nitrogen, and the molecular characteristics of the most direct pathway from the benzenoid ring to the nitrogen. Weighting factors, based on published 5-HT3 receptor affinity data, were then assigned to each of 10 molecular characteristics. The derived computational model predicts accurately the affinities of the learning set for the 5-HT3 receptor (r = 0.98; p less than 0.001). The computational model was then used to predict the receptor affinities of a "test set" of 40 pharmacological agents. The predicted values for these agents also correlate significantly (r = 0.83; p less than 0.001) with drug affinities for the 5-HT3 receptor, as determined by radioligand binding assays. This first line screening approach allows for the accurate prediction of drug affinities based on molecular characteristics with minimal dependence upon animal tissues or radioactivity.     

High affinity and low affinity ouabain binding sites in the rat heart

Ventricular muscle of rat heart has two classes of receptors which are responsible for the positive inotropic effect of ouabain. Low affinity receptors are apparently related to Na+, K+-ATPase. To determine if high affinity receptors are also sarcolemmal Na+, K+-ATPase of muscle cells, their characteristics were examined. Binding of [3H]ouabain to the high affinity binding site required ATP in the presence of Mg2+ and Na+, was stimulated by Na+ in the presence of Mg2+ and ATP, and was inhibited by K+. Digoxin, digitoxin and cassaine all inhibited [3H]ouabain binding to the high affinity site. Cassaine was about an order of magnitude less potent than the glycosides. These results indicate similarities in high affinity ouabain binding sites in ventricular muscle of rat heart and Na+, K+-ATPase obtained from other sources. Destruction of sympathetic nerve terminals with 6-hydroxydopamine failed to affect the high affinity ouabain binding sites indicating that high affinity sites do not represent the Na+, K+-ATPase in sympathetic nerve terminals. Labeling of Na+, K+-ATPase from [gamma-32P]ATP indicates that high affinity ouabain binding sites account for 25% of the total enzyme molecules present in ventricular muscle of rat heart.     

High affinity binding of Y-25130 for serotonin 3 receptor]

The binding of Y-25130 on serotonin 3 (5-HT3) receptors was evaluated by examining its effect on 3H-BRL 43694 (3H-granisetron) binding to rat cerebral cortex membranes in comparison with those of granisetron, ondansetron and metoclopramide. Y-25130, granisetron, ondansetron, metoclopramide, 5-HT and 2-methyl-5-HT displaced the specific binding of 3H-granisetron to the synaptosomal membranes in a concentration-dependent manner. The rank order of potency for inhibition of 3H-granisetron binding by the test compounds was Y-25130 not equal to granisetron greater than ondansetron much greater than 2-methyl-5-HT not equal to 5-HT not equal to metoclopramide. To determine the manner of interaction between Y-25130 and 5-HT3 receptors, Scatchard analysis of 3H-granisetron specific binding to the membranes of the cerebral cortex in the absence or presence of various concentrations of Y-25130 was performed. It was indicated that Y-25130 exerted a typical competitive-type inhibition of 3H-granisetron binding. The present study indicates that Y-25130 binds competitively to 5-HT3 receptors with high affinity.