Avidin binding of radiolabeled biotin derivatives

Three N-acyl derivatives of biotinylethylenediamine were prepared: I, biotinylamidoethyl-3-(3-[125I]iodo-4-hydroxyphenyl)propionamide; II, biotinylamidoethyl-[3H]acetamide; and III, biotinylamidoethyl-3-(3,5-[125I]diiodo-4-hydroxyphenyl)propionamid e. Each compound was combined with a large excess of avidin, yielding 1:1 molar complexes. Aside from a small fraction of each complex that dissociated more rapidly, the dissociation half-lives of these complexes were: I, 41 days; II, 4.4 days; and III, 148 days. The iodo- (mono or di) hydroxyphenylpropionyl moieties of I and III, therefore, contribute significantly to the binding strength of these compounds toward avidin. We also formed 4:1 complexes of I, II, and III with avidin (compound in excess), each of which exhibited biphasic dissociation, with initial half-lives of 4, 3.2, and 24 days, respectively. Thus, I or especially III potentially can be used as a sensitive tracer in quantitative studies with avidin.     

D-Ala2, (F5) Phe4]-dynorphin 1-13-NH2 (DAFPHEDYN): a potent analog of dynorphin 1-13

Intracerebroventricular administration of the dynorphin analog, [D-Ala2,(F5)Phe4]-dynorphin 1-13-NH2 (DAFPHEDYN) in rats produced diuresis and profound analgesia. Both effects were antagonized by central administration of naltrexone or naloxone. Intravenous administration of 10, 25, and 50 mg/kg of DAFPHEDYN failed to induce diuresis. The increased potency of DAFPHEDYN was apparent from the failure of an equal dose of the parent compound (dynorphin 1-13) to produce diuresis and the failure of [D-Ala2]-dynorphin 1-13-NH2 to produce analgesia. Radioligand binding studies indicated the DAFPHEDYN retains the same degree of kappa selectivity as the parent compound (dynorphin 1-13) though a drop in affinity occurred. DAFPHEDYN may be of significant interest because it retains the essential pharmacology of the parent compound and exhibits marked in vivo potency.     

Synthesis and biological characterization of novel hybrid 7-[[2-(4-phenyl-piperazin-1-yl)-ethyl]-propyl-amino]-5,6,7,8-tetrahydro-naphthalen-2-ol and their heterocyclic bioisosteric analogues for dopamine D2 and D3 receptors

In a recent preliminary communication we described the development of a series of hybrid molecules for the dopamine D2 and D3 receptor subtypes. The design of these compounds was based on combining pharmacophoric elements of aminotetralin and piperazine molecular fragments derived from known dopamine receptor agonist and antagonist molecules. Molecules developed from this approach exhibited high affinity and selectivity for the D3 receptor as judged from preliminary [(3)H]spiperone binding data. In this report, we have expanded our previous finding by developing additional novel molecules and additionally evaluated functional activities of these novel molecules in the [(3)H]thymidine incorporation mitogenesis assay. The binding results indicated highest selectivity in the bioisosteric benzothiazole derivative N6-[2-(4-phenyl-piperazin-1-yl)-ethyl]-N6-propyl-4,5,6,7-tetrahydro-benzothiazole-2,6-diamine (14) for the D3 receptor whereas the racemic compound 7-([2-[4-(2,3-dichloro-phenyl)-piperazin-1-yl]-ethyl]-propyl-amino)-5,6,7,8-tetrahydro-naphthalen-2-ol (10c) showed the strongest potency. Mitogenesis studies to evaluate functional activity demonstrated potent agonist properties in these novel derivatives for both D2 and D3 receptors. In this regard, compound 7-[[4-(4-phenyl-piperazin-1-yl)-butyl]-prop-2-ynyl-amino]-5,6,7,8-tetrahydro-naphthalen-2-ol (7b) exhibited the most potent agonist activity at the D3 receptor, 10 times more potent than quinpirole and was also the most selective compound for the D3 receptor in this series. Racemic compound 10a was resolved; however, little separation of activity was found between the two enantiomers of 10a. The marginally more active enantiomer (-)-10a was examined in vivo using the 6-OH-DA induced unilaterally lesioned rat model to evaluate its activity in producing contralateral rotations. The results demonstrated that in comparison to the reference compound apomorphine, (-)-10a was quite potent in inducing contralateral rotations and exhibited longer duration of action.     

Extension of the single amino acid chelate concept (SAAC) to bifunctional biotin analogues for complexation of the M(CO)3(+1) Core (M = Tc and Re): syntheses, characterization, biotinidase stability, and avidin binding

Biotin and avidin form one of the most stable complexes known (K(D) = 10(-15) M(-1)) making this pairing attractive for a variety of biomedical applications including targeted radiotherapy. In this application, one of the pair is attached to a targeting molecule, while the other is subsequently used to deliver a radionuclide for imaging and/or therapeutic applications. Recently, we reported a new single amino acid chelate (SAAC) capable of forming stable complexes with Tc(CO)3 or Re(CO)3 cores. We describe here the application of SAAC analogues for the development of a series of novel radiolabeled biotin derivatives capable of forming robust complexes with both Tc and Re. Compounds were prepared through varying modification of the free carboxylic acid group of biotin. Each 99mTc complex of SAAC-biotin was studied for their ability to bind avidin, susceptibility to biotinidase, and specificity for avidin in an in vivo avidin-containing tumor model. The radiochemical stability of the 99mTc(CO)3 complexes was also investigated by challenging each 99mTc-complex with large molar excesses of cysteine and histidine at elevated temperature. All compounds were radiochemically stable for greater than 24 h at elevated temperature in the presence of histidine and cysteine. Both [99mTc(CO)3(L6)]+1 [TcL6; L6 = biotinylamidopropyl-N,N-(dipicolyl)amine] and [99mTc(CO)3(L12a)]+1 (TcL12; L12 = N,N-(dipicolyl)biotinamido-Boc-lysine; TcL12a; L12a = N,N-(dipicolyl)biotinamide-lysine) readily bound to avidin whereas [99mTc(CO)3(L9)]+1 [TcL9; L9 = N,N-(dipicolyl)biotinamine] demonstrated minimal specific binding. TcL6 and TcL9 were resistant to biotinidase cleavage, while TcL12a, which contains a lysine linkage, was rapidly cleaved. The highest uptake in an in vivo avidin tumor model was exhibited by TcL6, followed by TcL9 and TcL12a, respectively. This is likely the result of both intact binding to avidin and resistance to circulating biotinidase. Ligand L6 is the first SAAC analogue of biotin to demonstrate potential as a radiolabeled targeting vector of biotin capable of forming robust radiochemical complexes with both 99mTc and rhenium radionuclides. Computational simulations were performed to assess biotin-derivative accommodation within the binding site of the avidin. These calculations predict that deformation of the surface domain of the binding pocket can occur to accommodate the transition metal-biotin derivatives with negligible changes to the inner-beta-barrel, the region most responsible for binding and retaining biotin and its derivatives. The biological activity and biodistribution of the technetium complexes TcL6, TcL9, and TcL12a were examined in an avidin tumor model. In the avidin bead tumor localization model, TcL6 demonstrated the most favorable localization with a 7:1 ratio of avidin bead implanted muscle versus normal muscle, while TcL9 exhibited a 2:1 ratio. However, TcL9 displayed no specificity for avidin.     

A robust protocol to map binding sites of the 14-3-3 interactome: Cdc25C requires phosphorylation of both S216 and S263 to bind 14-3-3

Modern proteomic techniques have identified hundreds of proteins that bind 14-3-3s, the most widespread eukaryotic phosphoserine/threonine sensors, but accurate prediction of the target phospho-sites is difficult. Here we describe a systematic approach using synthetic peptides that tests large numbers of potential binding sites in parallel for human 14-3-3. By profiling the sequence requirements for three diverse 14-3-3 binding sites (from IRS-1, IRSp53 and GIT2), we have generated enhanced bioinformatics tools to score sites and allow more tractable testing by co-immunoprecipitation. This approach has allowed us to identify two additional sites other than Ser216 in the widely studied cell division cycle (Cdc) protein 25C, whose function depends on 14-3-3 binding. These Ser247 and Ser263 sites in human Cdc25C, which were not predicted by the existing Scansite search, are conserved across species and flank the nuclear localization region. Furthermore, we found strong interactions between 14-3-3 and peptides with the sequence Rxx[S/T]xR typical for PKC sites, and which is as abundant as the canonical Rxx[S/T]xP motif in the proteome. Two such sites are required for 14-3-3 binding in the polarity protein Numb. A recent survey of >200 reported sites identified only a handful containing this motif, suggesting that it is currently under-appreciated as a candidate binding site. This approach allows one to rapidly map 14-3-3 binding sites and has revealed alternate motifs.     

Avidin binding of biotinylated analogues of substance P

We report the bioactivities of three biotinylated analogues of Substance P, [alpha-biotinyl-Lys3]Substance P-(3-11), [alpha-biotinyl-Arg1]Substance P, [epsilon-biotinyl-Lys3]Substance P, as well as the bioactivities of their complexes with avidin on guinea pig ileum. The rate of dissociation of an [alpha-biotinyl-Arg1]Substance P-avidin complex has been determined in the presence of 2-(4'-hydroxyazobenzene) benzoic acid and [3H]biotin. The biphasic dissociation of a 4:4 complex between [alpha-biotinyl-Arg1]Substance P and avidin led us to postulate the existence of two sets of binding sites, with the following rates of dissociation, at 4 degrees C, kappa-1 = 6 x 10(-4) x s-1 and kappa-1 = 1.2 x 10(-5) x s-1. We have confirmed this non-equivalence of the four binding sites of avidin by nuclear magnetic resonance using a spin-echo technique. The [alpha-biotinyl-Arg1]Substance P-avidin may be used for the affinity chromatography of Substance P receptors and the decreased affinity of this complex may be taken as an advantage since it can be displaced under mild conditions, i.e. by biotin-containing buffer.     

The differential loss of [3H]pirenzepine vs [3H] (-) quinuclidinylbenzilate binding to soluble rat brain muscarinic receptors indicates that pirenzepine binds to an allosteric state of the muscarinic receptor

[3H]Pirenzepine [( 3H]PZ) and [3H] (-)Quinuclidinylbenzilate [( 3H] (-)QNB) specific binding to soluble rat brain muscarinic cholinergic receptors was assessed as a function of time subsequent to receptor solubilization. The soluble brain muscarinic receptor is stable at 4 degrees C when assayed by [3H] (-)QNB binding (t 1/2 = 80 hrs). In contrast the pirenzepine state of the receptor decays rapidly (t 1/2 = 3.0 hrs). Prior occupation of the receptor with [3H] (-)QNB or [3H]PZ increases the receptor stability by two to five fold (t 1/2 QNB greater than 1,000 hrs; t 1/2 PZ = 6.5 hrs). These data indicate that pirenzepine binds to an allosteric state of the muscarinic receptor and that caution should be employed in the assignment of receptor subtypes based solely upon the binding of ligands which recognize unique conformational states.     

Rabbit antibodies against the human milk sialyloligosaccharide alditol of LS-tetrasaccharide a (NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH)

The sialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4Glc (LS-tetrasaccharide a), a minor component of human milk, is obtained in relatively large quantities from autohydrolysates of the major milk disialyloligosaccharide, NeuAc alpha 2-3Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc (disialyllacto-N-tetraose). Rabbits immunized with an oligosaccharide-protein conjugate prepared from keyhole limpet hemocyanin and LS-tetrasaccharide a produce antibodies directed against the corresponding oligosaccharide alditol. The anti-LS-tetrasaccharide a sera bind 3H-labeled LS-tetrasaccharide a in a direct-binding radioimmunoassay on nitrocellulose filters. The specificities of these antibodies are determined by comparing inhibitory activities of structurally related oligosaccharides. Strong hapten-antibody binding (Ka greater than 10(6) M-1) requires sialic acid linked alpha 2-3 to the nonreducing terminal galactose residue of reduced lacto-N-tetraose (Gal beta 1-3GlcNAc beta 1-3Gal beta 1-4GlcOH). Specificities of antibodies prepared against keyhole limpet hemocyanin conjugates of LS-tetrasaccharide b (Gal beta 1-3[NeuAc alpha 2-6]GlcNAc beta 1-3Gal beta 1-4Glc) and LS-tetrasaccharide c (NeuAc alpha 2-6Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc) differ only slightly from rabbit antibodies prepared against the corresponding bovine serum albumin conjugates described previously [D. F. Smith and V. Ginsburg (1980) J. Biol. Chem. 255, 55-59].     

Structural insights of the MLF1/14-3-3 interaction

Myeloid leukaemia factor 1 (MLF1) binds to 14-3-3 adapter proteins by a sequence surrounding Ser34 with the functional consequences of this interaction largely unknown. We present here the high-resolution crystal structure of this binding motif [MLF1(29-42)pSer34] in complex with 14-3-3ε and analyse the interaction with isothermal titration calorimetry. Fragment-based ligand discovery employing crystals of the binary 14-3-3ε/MLF1(29-42)pSer34 complex was used to identify a molecule that binds to the interface rim of the two proteins, potentially representing the starting point for the development of a small molecule that stabilizes the MLF1/14-3-3 protein-protein interaction. Such a compound might be used as a chemical biology tool to further analyse the 14-3-3/MLF1 interaction without the use of genetic methods. Database Structural data are available in the Protein Data Bank under the accession number(s) 3UAL [14-3-3ε/MLF1(29-42)pSer34 complex] and 3UBW [14-3-3ε/MLF1(29-42)pSer34/3-pyrrolidinol complex] Structured digital abstract ? 14-3-3 epsilon?and?MLF1?bind?by?x-ray crystallography?(View interaction) ? 14-3-3 epsilon?and?MLF1?bind?by?isothermal titration calorimetry?(View Interaction:?1,?2).     

Synthesis and nicotinic acetylcholine receptor binding affinities of 2- and 3-isoxazolyl-8-azabicyclo[3.2.1]octanes

A series of epiboxidine homologues, 2- and 3-isoxazole substituted 8-azabicyclo[3.2.1]octane derivatives was synthesized and evaluated as potential ligands for neuronal nicotinic acetylcholine receptors in [(3)H]cytisine labeled rat brain. The 2beta-isoxazolyl-8-azabicyclo[3.2.1]octane 9b (K(i)=3 nM) was the most potent compound of the series with a binding affinity twice that of nicotine. The 3beta-isoxazolyl-8-azabicyclo[3.2.1]octane 15b (K(i)=148 nM) exhibited moderate affinity while the corresponding 2alpha- and 3alpha-isomers exhibited micromolar binding affinity.     

(+/-)-[3H]Epinephrine and (-)[3H]dihydroalprenolol binding to beta1- and beta2-noradrenergic receptors in brain, heart, and lung membranes.

(+/-)-[3H]Epinephrine binds to beta-receptors in calf cerebellar and rat lung membranes in the presence of 1.0 mM pyrocatechol and 1.0 microM phentolamine, with dissociation constants at 4 degrees C of 11 nM and 24 nM, respectively. (+/-)-[3H]Epinephrine associates to equilibrium within 20 min in both tissues, and over 50% of the binding is rapidly dissociable. Inhibition of binding by agonists and antagonists is highly stereoselective, and the structure-activity relationships of adrenergic agents in inhibiting (+/-)-[3H]epinephrine binding suggest an interaction with beta2 type noradrenergic receptors. (-)-Isoproterenol has an apparent Ki of 2 nM, (-)-epinephrine is 1.5 to 3 times weaker, and (-)-norepinephrine is 30 to 60 times weaker. Salbutamol and terbutaline, selective beta2-agonists, are potent inhibitors of binding, as are several nonspecific antagonists. Properties of the sites labeled by (+/-)-[3H]epinephrine in calf cerebellum and rat lung are closely similar. (-)-[3H]Dihydroalprenolol binding in calf cerebellum and rat lung also shows beta2 characteristics. Antagonists have similar potencies in inhibiting (-)-[3H]dihydroalprenolol and (+/-)-[3H]epinephrine binding in both tissues, but agonists are in general more potent inhibitors of (+/-)-[3H]epinephrine. Sodium and lithium selectively lower the affinity of (+/-)-[3H]epinephrine at its binding sites and the affinities of agonists, but not antagonists, at the (-)-[3H]dihydroalprenolol site. Specific (+/-)-[3H]epinephrine binding was not detectable in calf cortex and rat heart, where (-)-[3H]dihydroalprenolol binding suggests a beta1-receptor. A physiological significance of (+/-)-[3H]epinephrine binding is suggested by the strong correlation for agonists and antagonists between affinities in inhibiting binding, and in stimulating or inhibiting a beta-receptor-coupled adenylate cyclase in frog erythrocytes.     

Biological activity of 24,24-difluoro-1 alpha, 25-dihydroxyvitamin D3 and 1 alpha, 25-dihydroxyvitamin D3-26,23-lactone in inducing differentiation of human myeloid leukemia cells

Vitamin D compounds added to the culture medium induce differentiation of human myeloid leukemia cells (HL-60 cells) by binding to a specific cytosol receptor protein. This system provides a biologically relevant and technically simple assay to examine the relationship between molecular structure and biological activity of vitamin D compounds. Using this culture system, the biological activity of 24,24-F2-1 alpha,25(OH)2D3 and 1 alpha,25(OH)2D3-26,23-lactone was assayed. 24,24-F2-1 alpha,25(OH)2D3 was four to seven times more potent than 1 alpha,25(OH)2D3 in inducing phagocytosis and C3 rosette formation of HL-60 cells, though both compounds bound equally well to the cytosol receptor, suggesting that the defuorination at the 24-carbon position may stimulate membrane permeability of the compound. 1 alpha,25(OH)2D3-26,23-lactone, on the other hand, was only 1/200th as active as 1 alpha,25(OH)2D3. The binding affinity of the lactone for the cytosol receptor was identical with that of 1 alpha (OH)D3, suggesting that the lactone formation between the 26 and 23 positions masks the function of the 25-hydroxyl group. The binding affinity of vitamin D3 derivatives to the specific cytosol receptor of HL-60 cells was well correlated with that of intestinal cytosol protein specifically bound to 1 alpha,25(OH)2D3.     

Pharmacology of [3H]R(+)-7-OH-DPAT binding in the rat caudate-putamen

Dopamine D3 receptors may be involved in drug addiction and in disorders such as schizophrenia and Parkinson's disease. To determine the pharmacological properties of dopamine D3 receptors in the rat caudate-putamen, we have investigated R(+)-[3H]7-hydroxy-N,N-di-n-propyl-2-aminotetralin ([3H]R(+)-7-OH-DPAT) binding to membrane preparations from the rat caudate-putamen. Kinetic analyses showed that [3H]R(+)-7-OH-DPAT binding reached equilibrium in approximately 1 h and that both association and dissociation curves were composed of at least two components. Likewise, saturation curves showed at least two binding components with a combined Bmax value of about 600 fmol/mg protein, which is three times higher than what is present in the subcortical limbic area. Competition curves were performed with agonists such as R(-)-propylnorapomorphine, dopamine, PD 128907, quinpirole, and bromocriptine, and antagonists such as haloperidol, raclopride, clozapine, GR 218231x, remoxipride, and U99194A. These experiments revealed that [3H]R(+)-7-OH-DPAT binding could be resolved into three specific binding sites (R1-R3) and one nonspecific binding site, with R1-R2 probably representing D3 receptor binding and the minor R3 representing D2 receptor binding. The low affinities of (+/-)-8-OH-DPAT and 1,3-di(2-tolyl)guanidine to inhibit [3H]R(+)-7-OH-DPAT binding indicate negligible involvement of 5-HT1A or sigma binding sites, respectively. The pharmacological profile of [3H]R(+)-7-OH-DPAT (2 nM) binding in the caudate-putamen was similar to that of dopamine on [125I]iodosulpride binding in the cerebellar lobule X, which contain D3 but not D2 receptors. Mg2+ increased and GTP and Na+ decreased the binding of [3H]R(+)-7-OH-DPAT, suggesting a coupling of endogenous D3 receptors to G proteins. Taken together, these results suggest that dopamine D3 receptors display multiple agonist binding states, and that D3 receptors are present in high concentrations in the rat caudate-putamen. These results may have implications for the physiological and pathological roles of dopamine D3 receptors in the brain.     

Biotinylated human beta-endorphins as probes for the opioid receptor

The reaction of human beta-endorphin and biotinyl N-hydroxysuccinimide with or without spacer arm, afforded a series of products that were separated by high performance liquid chromatography (HPLC). Liquid secondary ion mass spectrometry of the biotinylated products and their tryptic digests produced abundant protonated molecular ions (MH+), which specified the number and location of biotinylation. Between 1 and 4 biotinyl residues were incorporated per human beta-endorphin molecule, at Lys-9, -19, -24, -28, and -29, but not at the amino-terminal Tyr-1. Three HPLC fractions were isolated for receptor binding studies with monobiotinylation of Lys-9 (B1 beta and B1X beta; X = C6 spacer arm), Lys-19 (B1 gamma), and a mixture of Lys-24, Lys-28, and Lys-29 derivatives (B1 alpha, BX1 alpha). All derivatives displayed tight binding to avidin, and no dissociation from avidin was detectable over several hours at 0 degrees C for the derivatives (BX1 alpha) tested. IC50 values for binding to mu and delta opioid receptor sites were 3-8 times higher for monobiotinylated derivatives than for the parent human beta-endorphin (IC50,mu = 1.5 nM, IC50,delta = 1.3 nM). Association with avidin decreased opioid receptor affinities for the C6 spacer derivative biotinylated at position Lys-9, which is close to the (1-5) enkephalin receptor region. In contrast, avidin did not affect or even increased apparent affinities to mu and delta sites for derivatives biotinylated at the alpha-helical part of the molecule (Lys-19, -24, -28, and -29). Thus, when bound to avidin, the biotinylated human beta-endorphin derivatives with spacer arm (BX1 alpha), substituted near the carboxyl terminal (Lys-24, -28, and -29), displayed mu binding affinities equal to and delta binding affinities only four times lower than underivatized human beta-endorphin. Biotinylated human beta-endorphins also bound to low affinity nonopioid binding sites on NG-108-15 cells; however, affinities to these sites were considerably reduced when derivatives were bound to avidin. The ability of biotinylated human beta-endorphin to cross-link the mu and delta opioid receptors to avidin allows application of the biotin-avidin system as a molecular probe of the opioid receptor.     

(+/-)-2-(3-Piperidyl)-1,2,3,4-tetrahydroisoquinolines as a new class of specific bradycardic agents

A series of (+/-)-2-(3-piperidyl)-1,2,3,4-tetrahydroisoquinolines were prepared and their bradycardic activities were examined in isolated guinea-pigs' right atria and in anesthetized rats. Modifications on the benzyl moiety of the parent compound, 1, led to the identification of compound 11e as a potent and specific bradycardic agent.     

Synthesis and biologic activities of 11 beta-substituted estradiol as potential antiestrogens

The effect of attachment of a dimethylaminoethoxy or a dimethylaminopropoxy group at the 11 beta-position of estradiol (E2) on its relative binding affinity (RBA) to estrogen receptor (ER) and intrinsic biologic activity is described. The binding of 11 beta-[2-(N,N-dimethylamino) ethoxy]estra-1,3,5(10)-triene-3,17 beta-diol (4) and 11 beta-[3-(N,N- dimethylamino)propoxy]estra-1,3,5(10)-triene-3,17 beta-diol (5) to the ER from immature rat uterine tissue was measured relative to that of [3H]E2 by a competitive binding assay. It was found that the 11 beta-substituted E2 analogs have considerably lower RBA to ER than the corresponding parent compound. The intrinsic activity of compounds 4 and 5 were studied in terms of uterotrophic and antiuterotrophic activity. It was found that the uterotrophic activity of these compounds was drastically reduced compared with E2. However, no antiuterotrophic activity was observed in these compounds at dosages ranging from 1 to 100 micrograms/rat/d.     

Photoaffinity Labeling with 2-[2-(4-Azido-3-[125I]-Iodophenyl)ethylamino]adenosine and Autoradiography with 2-[2-(4-Amino-3-[125I]Iodophenyl)ethylamino]adenosine of A2a Adenosine Receptors in Rat Brain

Abstract: The A_2a adenosine receptor agonist 2-[2-(4-amino-3-iodophenyl)ethylamino]adenosine is a potent coronary vasodilator. The corresponding radioiodinated ligand, [¹²⁵I]APE, discriminates between high- and low-affinity conformations of A_2a adenosine receptors. In this study, [¹²⁵I]APE was used for rapid (24-h) autoradiography in rat brain sections. The pattern of [¹²⁵I]APE binding is consistent with that expected of an A_2a-selective radioligand. It is highest in striatum, nucleus accumbens, and olfactory tubercle, with little binding to cortex and septal nuclei. Specific [¹²⁵I]APE binding to these brain regions is abolished by 1 µ M 2- p -(2-carboxyethyl)phenethylamino-5'- N -ethylcarboxamidoadenosine (CGS-21680) but is little affected by 100 n M 8-cyclopentyl-1,3-dipropylxanthine. Conversion of [¹²⁵I]APE to the corresponding arylazide results in [¹²⁵I]AzPE. The rank-order potency of compounds to compete for [¹²⁵I]AzPE binding in the dark is CGS-21680 > d -( R )- N ⁶-phenylisopropyladenosine > N ⁶-cyclopentyladenosine, indicating that it also is an A_2a-selective ligand. Specific photoaffinity labeling by [¹²⁵I]AzPE of a single polypeptide (42 kDa) corresponding to A_2a adenosine receptors is reduced 55 ± 4% by 100 µ M guanosine 5'- O -(3-thiotriphosphate) and 91 ± 1.3% by 100 n M CGS-21680. [¹²⁵I]APE and [¹²⁵I]AzPE are valuable new tools for characterizing A_2a adenosine receptors and their coupling to GTP-binding proteins by autoradiography and photoaffinity labeling.     

Interaction of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitroso urea hydrochloride with nucleic acids and proteins

The binding of the 14C-labelled-ethylene and -pyrimidine moieties of 3-[(4-amino-2-methyl-5-pyrimidinyl)methyl]-1-(2-chloroethyl)-1-nitrosourea hydrochloride (ACNU) to the biological macromolecules was studied with the AH-130 hepatoma-bearing rats, suspension of AH-130 cells, and isolated nucleic acids and proteins. In all systems examined, a significant level of the binding of the [14C]ethylene of ACNU to nucleic acids, probably due to alkylation, was observed. In contrast, the extent of the binding of the [14C]-pyrimidine was negligible. When a compound lacking the 4-amino group of ACNU (deamino-ACNU) was used for the binding study, relatively higher binding of this compound than that of ACNU to [14C]lysine was observed. It was revealed, therefore, that the low binding of ACNU to proteins could be due to instantaneous depletion of an isocyanate-intermediate, according to the formation of an intramolecularly carbamoylated product with the amino-group on the pyrimidine ring of ACNU molecule during incubation. This could be the molecular basis for the low carbamoylating activity of ACNU in vivo and in vitro, and the antitumor action of ACNU would be dependent on its alkylating activity only.     

Complete protection of antisense oligonucleotides against serum nuclease degradation by an avidin-biotin system.

It has been recently demonstrated that a complex of avidin, a cationic protein, and a monobiotinylated antisense oligonucleotide for the GLUT1 glucose transporter mRNA is taken up by cells in vitro and by organs in vivo via absorptive-mediated endocytosis. In the present study, a GLUT1 biotinylated oligonucleotide-avidin construct showing complete protection against serum 3'-exonuclease-mediated degradation is described. 21-mer antisense oligonucleotides complementary to nucleotides 162-182 and 161-181 of the bovine GLUT1 glucose transporter mRNA were synthesized with a 6-aminodeoxyuridine at positions 3 and 20, respectively, biotinylated with NHS- or NHS-XX-biotin to yield near 5'- or near 3'-biotinylated oligonucleotide (bio-DNA), and 5'- and 3'-end radiolabeled. Serum induced a rapid degradation of unprotected (no avidin) [5'-32P]-5'-bio-DNA (> 95% at 30 min). Avidin partially protected this construct (approximately 31% of intact 21-mer oligo remained at 1 h). Similar results were obtained with the [3'-32P]-5'-bio-DNA; however, no degradation products of varying size were observed, confirming that the degradation is mediated primarily by a 3'-exonuclease. Incubation of the [5'-32P]-3'-bio-DNA with serum showed a rapid conversion to the 20- and 19-mer forms (t1/2 approximately 13 min). Conversely, avidin totally protected this construct against the serum 3'-exonuclease. In conclusion, avidin fully protects antisense oligonucleotides biotinylated at the near 3'-terminus against serum 3'-exonuclease degradation, and this property may be useful for avidin-mediated drug delivery of oligonucleotides to tissues in vivo or to cultured cells in vitro.     

17β-acylurea derivatives of 4-azasteroids as inhibitors of testosterone 5α-reductase

A series of 17 beta-acylurea-4-aza-5 alpha-androstan-3-one derivatives has been assayed in vitro as inhibitors of testosterone 5 alpha-reductase, using the particulate fraction of human hyperplastic prostate and rat prostate as enzyme sources. The most active derivatives were 1-[4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carbonyl]- 1,3-dicyclohexylurea (compound 1) and 1-[4-methyl-3-oxo-4-aza-5 alpha-androstane-17 beta-carbonyl]- 1,3-diisopropylurea (compound 3) which demonstrated IC50 values of 41 and 55 nM for the human enzyme and of 83 and 53 nM for the rat enzyme, respectively. Neither compound showed any relevant binding affinity to the rat prostate androgen receptor (IC50 of approximately 100 and 84 microM). When given orally in immature castrated rats together with subcutaneous testosterone propionate (TP) for 7 consecutive days, compound 3 (laboratory code FCE 26073), at 3 mg/kg/day, significantly decreased the ventral prostate growth promoting effect of TP by 40-50%, whereas compound 1 was ineffective up to the dose of 10 mg/kg/day.