Radioiodination of a photoactivatable heterobifunctional reagent

The N-hydroxysuccinimide ester of 4-azidosalicylic acid, a photoactivable heterobifunctional reagent, can be radioiodinated. The low efficiency (3%) of the radioiodination by a previously published method (I. Ji and T. H. Ji, 1982, Anal. Biochem. 121, 286-289) has been increased to 63% by substituting the solvent, acetone, with others such as aqueous acetonitrile, dimethylformamide, or dimethyl sulfoxide. The resulting 125I reagent was used for derivatizing human choriogonadotropin. The radioactive hormone derivative was crosslinked to the alpha beta dimer upon photolysis.     

3-([3-125I]iodo-4-hydroxyphenyl)propionyl carbohydrazide,a new radioiodination reagent for ultrasensitive detection and determination of periodate-oxidized nucleoside derivatives and other carbonyl compounds

A new radioiodination reagent for the identification and quantitation of periodate-oxidized ribonucleosides was developed. The reagent, 3-([3-¹²⁵I]iodo-4-hydroxyphenyl)propionyl carbohydrazide, was prepared by radioiodination of 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester in the presence of chloramine T, followed by reduction of the latter with sodium arsenite and treatment of the radioiodinated ester with an excess of carbohydrazide. The reagent reacted quantitatively with periodate-oxidized nucleosides to form ¹²⁵I-labeled morpholine derivatives which were separated by thin-layer chromatography and quantitated by liquid scintillation counting. The reagent was found to react also with other carbonyl compounds and thus may find more general application in the qualitative and quantitative ultramicroanalysis of aldehydes and ketones.     

Evaluation of chemical, physical, and biologic properties of tumor-targeting radioiodinated quinazolinone derivative

Our group is developing a novel technology, enzyme-mediated cancer imaging and therapy (EMCIT), that aims to entrap radioiodinated compounds within solid tumors for noninvasive tumor detection and therapy. In this approach, a water-soluble, radioiodinated prodrug is hydrolyzed in vivo to a highly water-insoluble compound by an enzyme overexpressed extracellularly by tumor cells. We have synthesized and characterized the water-soluble prodrug, 2-(2'-phosphoryloxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]5, which is readily hydrolyzed by alkaline phosphatase, an enzyme expressed by many tumor cell lines, to a water-insoluble drug, 2-(2'-hydroxyphenyl)-6-[(125)I]iodo-4-(3H)-quinazolinone [(125)I]1. In the course of our study, we discovered that ammonium 2-(2'-phosphoryloxyphenyl)-6-tributylstannyl-4-(3H)-quinazolinone, an intermediate in the radioiodination of the prodrug, exists as two isomers (3 and 4) whose radioiodination leads, respectively, to [(125)I]6 and [(125)I]5. These prodrugs have different in vitro and in vivo biologic activities. Compound 6 is not hydrolyzed by alkaline phosphatase (ALP), whereas 5 is highly soluble (mg/mL) in aqueous solution and is rapidly dephosphorylated in the presence of ALP to 1, a water-insoluble molecule (ng/mL). Mouse biodistribution studies indicate that [(125)I]6 has high uptake in kidney and liver and [(125)I]5 has very low uptake in all normal organs. Compounds 3 and 6 are converted, respectively, to 4 and 5 after incubation in DMSO. The stability of 5 in human serum is high. The minimum ALP concentration needed to hydrolyze 5 is much greater than the ALP level in the blood of patients with cancer, and the latter should not affect the pharmacokinetics of the compound. Incubation of 5 with viable human and mouse tumor-cell lines--but not with normal human cells and mouse tissues--leads to its hydrolysis and the formation of large crystals of 1. We expect that 5 will also be hydrolyzed in vivo by tumor cells that express phosphatase activity extracellularly and anticipate the specific precipitation of radioiodinated 1 within tumor cell clusters. This should lead to high tumor-to-normal-tissue ratios and enable imaging (SPECT/PET) and radionuclide therapy of solid tumors.     

Biotin reagents in antibody pretargeting. 6. Synthesis and in vivo evaluation of astatinated and radioiodinated aryl- and nido-carboranyl-biotin derivatives

An investigation has been conducted to prepare and evaluate several radiohalogenated biotin derivatives as part of our studies to develop reagents for carrying (211)At in cancer pretargeting protocols. The primary goal of the investigation was to determine the in vivo stability and distribution properties of astatinated biotin derivatives. In addition to astatination, the biotin derivatives were radioiodinated for in vitro and in vivo comparison. Biodistributions were conducted in athymic mice, with sacrifice times of 1, 4, and 24 h to correspond to 9%, 32%, and 90% of (211)At decay (t(1/2) = 7.21 h). In the investigation, two biotin derivatives, 1a and 2a, were synthesized which had structures that contain a biotin moiety, a biotinidase-blocking moiety, an ether linker moiety, and an aryl stannane moiety for radiohalogenation. Biotin derivatives 1a and 2a were radiolabeled with (125/131)I to give [(125)/(131)I]1b or [(125)I]2b and with (211)At to give [(211)At]1c or [(211)At]2c. In vivo studies demonstrated that co-injected [(125)I]2b and [(131)I]1b had very similar tissue distributions in athymic mice. Co-injection of [(211)At]2c and [(125)I]2b provided data that indicated that rapid deastatination occurred in vivo. A second set of biotin derivatives, 3a, 4a, and 5a, were synthesized which had structures that contain a biotin moiety, a biotinidase-blocking moiety, and an anionic nido-carborane moiety for radiohalogenation. The biotin derivatives 4a and 5a contained an aryl moiety not present in 3a, and 5a had a trialkylamine functionality not present in 3a or 4a. Biotin derivative 3a was radioiodinated, but was not further investigated. Biotin derivatives 4a and 5a were radiolabeled with (211)At and (125)I to produce [(125)I]4b/[(211)At]4c and [(125)I]5b/[(211)At]5c. Comparison of [(125)I]4b and (separately) [(125)I]5b with [(131)I]1b showed that the nido-carborane containing biotin derivatives were retained in blood and tissue more than the aryl iodide derivative. In vivo evaluations of [(211)At]4c/[(125)I]4b and (separately) [(211)At]5c/[(125)I]5b indicated that some deastatination occurred in these compounds, but it was much less than observed for the aryl derivative [(211)At]2c. While the nido-carborane containing biotin derivatives provide a significant improvement in astatine stability over biotin derivatives previously studied, additional derivatives need to be prepared and studied to further improve the in vivo stability and blood/tissue clearance of these compounds.     

Synthesis and characterization of styrylchromone derivatives as beta-amyloid imaging agents

Several promising agents have been synthesized and evaluated for in vivo imaging probes of beta-amyloid plaques in Alzheimer's disease (AD) brain. Recently, we have developed flavone derivatives, which possess the basic structure of the 2-phenylchromone, as useful candidates for amyloid imaging agents. In an attempt to further develop novel tracers, we synthesized and evaluated a series of 2-styrylchromone derivatives, which replace the 2-phenyl substituent of flavone backbone with the 2-styryl. A series of radioiodinated styrylchromone derivatives were designed and synthesized. The binding affinities for amyloid plaques were assessed by in vitro binding assay using pre-formed synthetic Abeta(1-40) aggregates. The new series of styrylchromone derivatives showed high binding affinity to Abeta aggregates at the K(d) values of 32.0, 17.5 and 8.7nM for [(125)I]6, [(125)I]9, and [(125)I]12, respectively. In biodistribution studies using normal mice, [(125)I]6 and [(125)I]9 examined in normal mice displayed high brain uptakes with 4.9 and 2.8%ID/g at 2min post injection. The radioactivity washed out from the brain rapidly (1.6 and 1.0%ID/g at 60min post injection for [(125)I]6 and [(125)I]9, respectively). But [(125)I]12 did not show marked brain uptake, and the washout rate from the brain was relatively slow throughout the time course (1.1 and 1.4%ID/g at 2 and 30min post injection, respectively). Although additional modifications are necessary to improve the brain uptake and rapid clearance of non-specifically bound radiotracer, the styrylchromone backbone may be useful as a backbone structure to develop novel beta-amyloid imaging agents.     

The influence of radioiodination on the adsorption of IgG and serum albumin to polystyrene

The adsorption of radioiodinated rabbit IgG and bovine serum albumin (BSA) to polystyrene tubes was investigated. Adsorption isotherms where the proportion of the protein bound was relatively constant over a range of intermediate protein concentrations, and where the proportion bound was protein dependent, were obtained. To investigate the effects of radioiodination, proteins labeled to give a wide range of substitution ratios (0.03 to 3.7 125I/protein molecule) were employed. While labeling did not appear to affect BSA adsorption, the kinetics of IgG binding were altered in a number of ways. The proportion bound in the concentration independent region was decreased even at substitution ratios less than or equal to 0.2. In addition, while all preparations of iodinated BSA, and IgG preparations with less than or equal to 1.6 125I/IgG, gave bimodal adsorption isotherms, with more heavily labeled IgG (greater than or equal to 2.5 125I/IgG) the apparent high affinity binding to the plastic surface was abolished. These results indicate that radioiodination substantially alters the kinetics of the binding of IgG to polystyrene. In addition, the results obtained are discussed with respect to previous relevant and often apparently contradictory findings.     

Synthesis and radioiodination of some daunorubicin and doxorubicin derivatives

Daunorubicin and doxorubicin are efficient agents for cancer treatment. Their clinical efficacy is, however, hampered by their indiscriminant toxicity. This problem may be circumvented by encapsulating the drugs in liposomes and selectively targeting the tumor cells using tumor targeting agents. Furthermore, the antitumor effect could be enhanced by attaching the Auger electron emitter, (125)I, to daunorubicin and doxorubicin derivatives. In this context a number of ester, amide, and amine derivatives of daunorubicin and doxorubicin were synthesized. Benzoic acid ester derivatives of daunorubicin were synthesized by nucleophilic esterification of the 14-bromodaunorubicin with the potassium salt of the corresponding benzoic acid, resulting in good yields. Nicotinic acids and benzoic acids, activated with a succinimidyl group, were coupled to the amino group of daunorubicin to give the corresponding amide derivatives. Amine derivatives were obtained by the reductive amination of aromatic aldehydes with daunorubicin hydrochloride. The stannylated ester and amide derivatives were used as precursors for radioiodination. Radiolabeling with (125)I was performed using chloramine-T as an oxidant. The optimized labeling resulted in high radiolabeling yields (85-95%) of the radioiodinated daunorubicin and doxorubicin derivatives. Radioiodination of the amines was conducted at the ortho position of the activated phenyl rings providing moderate radiochemical yields (55-75%).     

Radioiodinated aza-diphenylacetylenes as potential SPECT imaging agents for beta-amyloid plaque detection

Two new iodinated fluoro- and hydroxy-pegylated aza-diphenylacetylene derivatives, 1 and 2, targeting beta-amyloid (Abeta) plaques have been successfully prepared. In vitro binding carried out in tissue homogenates prepared from postmortem AD brains with [(125)I]IMPY (6-iodo-2-(4'-dimethylamino)phenyl-imidazo[1,2-a]pyridine) as the radioligand indicated good binding affinities (K(i)=9.2 and 16.8 nM for 1 and 2, respectively). Brain penetrations of the corresponding radioiodinated ligands, evaluated in the normal mice, showed good initial brain penetrations (3.55% and 5.67% ID/g for [(125)I]1 and [(125)I]2 at 2 min post-injection). The washout from normal mice brain was relatively fast (0.33% and 0.91% ID/g at 2h post-injection). The specific binding of these radioiodinated ligands to beta-amyloid plaques was clearly demonstrated using film autoradiography of AD brain sections. Taken together, these preliminary results strongly suggest that these novel iodinated aza-diphenylacetylenes may be potentially useful for imaging Abeta plaques in the living human brain.     

Novel imaging agents for β-amyloid plaque based on the N-benzoylindole core

We report the synthesis and evaluation of a series of N-benzoylindole derivatives as novel potential imaging agents for β-amyloid plaques. In vitro binding studies using Aβ(1-40) aggregates versus [(125)I]TZDM showed that all these derivatives demonstrated high binding affinities (K(i) values ranged from 8.4 to 121.6 nM). Moreover, two radioiodinated compounds [(125)I]7 and [(125)I]14 were prepared. Autoradiography for [(125)I]14 displayed intense and specific labeling of Aβ plaques in the brain sections of AD model mice (C57, APP/PS1) with low background. In vivo biodistribution in normal mice exhibited sufficient initial brain uptake for imaging (2.19% and 2.00%ID/g at 2 min postinjection for [(125)I]7 and [(125)I]14, respectively). Although additional modifications are necessary to improve brain uptake and clearance from the brain, the N-benzoylindole may be served as a backbone structure to develop novel β-amyloid imaging probes.     

Dibenzothiazoles as novel amyloid-imaging agents

Novel dibenzothiazole derivatives were synthesized and evaluated as amyloid-imaging agents. In vitro quantitative binding studies using AD brain tissue homogenates showed that the dibenzothiazole derivatives displayed high binding affinities with K(i) values in the nanomolar range (6.8-36 nM). These derivatives are relatively lipophilic with partition coefficients (logP oct) in the range of 1.25-3.05. Preliminary structure-activity relationship studies indicated dibenzothiazole derivatives bearing electron-donating groups exhibited higher binding affinities than those bearing electron-withdrawing groups. A lead compound was selected for its high binding affinity and radiolabeled with [(125)I] through direct radioiodination using sodium [(125)I] iodide in the presence of Chloramine T. The radioligand (4-[2,6']dibenzothiazolyl-2'-yl-2-[(125)I]-phenylamine) displayed moderate lipophilicity (logP oct, 2.70), very good brain uptake (3.71+/-0.63% ID/g at 2 min after iv injection in mice), and rapid washout from normal brains (0.78% and 0.43% ID/g at 30 and 60 min, respectively). These studies indicated that lipophilic dibenzothiazole derivatives represent a promising pharmacophore for the development of novel amyloid-imaging agents for potential application in Alzheimer's disease and related neurodegenerative disorders.     

Nepsilon-(3-[*I]Iodobenzoyl)-Lys5-Nalpha-maleimido-Gly1-GEEEK ([*I]IB-Mal-D-GEEEK): a radioiodinated prosthetic group containing negatively charged D-glutamates for labeling internalizing monoclonal antibodies

Novel methods are needed for the radiohalogenation of cell-internalizing proteins and peptides because rapid loss of label occurs after lysosomal processing when these molecules are labeled using conventional radioiodination methodologies. We have developed a radiolabeled prosthetic group that contains multiple negatively charged D-amino acids to facilitate trapping of the radioactivity in the cell after proteolysis of the labeled protein. N(epsilon)-(3-[(125)I]iodobenzoyl)-Lys(5)-N(alpha)-maleimido-Gly(1)-GEEEK ([(125)I]IB-Mal-D-GEEEK) was synthesized via iododestannylation in 90.3 +/- 3.9% radiochemical yields. This radioiodinated agent was conjugated to iminothiolane-treated L8A4, an anti-epidermal growth factor receptor variant III (EGFRvIII) specific monoclonal antibody (mAb) in 54.3 +/- 17.7% conjugation yields. In vitro assays with the EGFRvIII-expressing U87MGDeltaEGFR glioma cell line demonstrated that the internalized radioactivity for the [(125)I]IB-Mal-D-GEEEK-L8A4 conjugate increased from 14.1% at 1 h to 44.7% at 24 h and was about 15-fold higher than that of directly radioiodinated L8A4 at 24 h. A commensurately increased tumor uptake in vivo in athymic mice bearing subcutaneous U87MGDeltaEGFR xenografts (52.6 +/- 14.3% injected dose per gram versus 17.4 +/- 3.5% ID/g at 72 h) also was observed. These results suggest that [(125)I]IB-Mal-d-GEEEK is a promising reagent for the radioiodination of internalizing mAbs.     

Radioiodination of new EGFR inhibitors as potential SPECT agents for molecular imaging of breast cancer

In our search for the development of novel SPECT radioligands for EGFR positive tumours, new potentially irreversible tyrosine kinase (TK) inhibitors are being explored. The radioiodination of N-{4-[(3-chloro-4-fluorophenyl) amino]quinazoline-6-yl}-3-bromopropionamide, a novel EGFR-TK inhibitor synthesised in our laboratory, was accomplished via halogen exchange. Purification by RP-HPLC gave [125I]-N-{4-[(3-chloro-4-fluorophenyl)amino]quinazoline-6-yl}-3-iodopropionamide with a radiochemical purity higher than 95% and a high specific activity. In vitro studies indicate that both iodinated quinazoline and its bromo precursor inhibit A431 cell growth and also possess higher potency than the parent quinazoline to inhibit the EGFR autophosphorylation. In vivo stability studies suggest metabolization of the radioiodinated quinazoline indicating a short biological half-life. The in vitro results point out that these quinazoline derivatives could be promising candidates for SPECT imaging of EGFR positive tumours provided that they are selectively modified in order to achieve better in vivo radiochemical stability.     

Characterization of ovarian gonadotropin receptor. Monomer and associated form of the receptor

We have purified luteinizing hormone/human choriogonadotropin (hCG) receptor from rat ovary by sequential affinity column on wheat germ lectin-Sepharose and hCG-Sepharose chromatography. The purified receptor, previously identified as a single protein on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (Kusuda, S., and Dufau, M.L. (1986) J. Biol. Chem. 261, 16161-16168), was further characterized by radioiodination with 1,3,4,6-tetrachloro-3 alpha, 6 alpha-diphenylglycouril, and column chromatography on wheat germ lectin-Sepharose. Autoradiography of SDS-PAGE analysis under reducing conditions showed a single radiolabeled band of Mr = 80,000. The radioiodinated receptors treated with peptide:N-glycosidase F migrated at Mr = 54,000. Treatment with neuraminidase alone caused only a minor reduction in molecular weight, and subsequent treatment with endo-alpha-N-acetyl-D-galactosaminidase had little further effect on the receptor. When the radioiodinated receptor was analyzed by fast protein liquid chromatography, a single broad peak was eluted with Mr of approximately 350,000. The higher Mr of radioiodinated receptors than that of native receptors (Mr = 190,000 dimeric form) could be due to the aggregation of labeled molecules. These complexes dissociated into the monomeric form in the presence of SDS. To determine whether the monomers can bind hormone, the purified unlabeled receptors resolved with SDS were electroblotted to nitrocellulose membranes and incubated with 125I-hCG. Autoradiograms of the blots showed a band of monomer (Mr = 78,000) as well as one of dimer (Mr approximately 150,000). These studies have demonstrated that the luteinizing hormone/hCG receptors are predominantly N-linked glycosylated and suggest that the native receptor is a dimer of identical hormone binding subunits associated by noncovalent interactions. Although the individual subunits can bind hormone, it is conceivable that the dimeric form is necessary for signal transduction.     

Molecular mapping of pulmonary endothelial membrane glycoproteins of the intact rabbit lung

To study the biochemical characteristics of endothelium in vivo, we radioiodinated endothelial membrane proteins of the perfused rabbit lung using a water soluble analog of the Bolton-Hunter reagent, 125I-sulfosuccinimidyl (hydroxyphenyl) propionate (125I-s-SHPP). This technique led to a 10-fold increase in specific activity of radioiodinated lung membrane protein compared with our previously reported method using lactoperoxidase and glucose oxidase-catalyzed radioiodination. Tissue autoradiography confirmed that radioiodination was largely confined to the endothelium. Perfusion pressure, wet-to-dry weight ratios, and the morphological appearance of the lungs were within normal limits, indicating that the procedure does not cause apparent lung injury. Lectin binding to a crude membrane fraction of 125I-s-SHPP labeled lung led to isolation of several putative endothelial membrane proteins. Immunoprecipitation studies with appropriate antibodies enabled the identification of radioiodinated angiotensin-converting enzyme and beta 2-microglobulin associated major histocompatibility complex class I molecules in the membrane fraction. This technique will be useful for studying biochemical responses of the endothelium in vivo to a variety of pharmacological and physiology stimuli.     

(125)I]EYF: a new high affinity radioligand to neuropeptide FF receptors

[(125)I]EYF ([(125)I]EYWSLAAPQRFamide), a new radioiodinated probe derived from a peptide present in the rat Neuropeptide FF precursor (EFWSLAAPQRFamide, EFW-NPSF) was synthesized and its binding characteristics investigated on sections of the rat spinal cord and on membranes of mouse olfactory bulb. In both tissues, [(125)I]EYF binding was saturable and revealed a very high affinity interaction with a single class of binding sites in rat and mouse (K(D) = 0.041 and 0.019 nM, respectively).Competition studies showed that [(125)I]EYF bound to one class of binding sites exhibiting a high affinity for all the different peptides the precursor could generate (NPA-NPFF, SPA-NPFF, NPFF, EFW-NPSF, QFW-NPSF) with the exception of NPSF which displayed a low affinity.Autoradiographic studies demonstrated that [(125)I]EYF binding sites were fully inhibited by a synthetic Neuropeptide FF agonist (1DMe) in all areas of the rat brain. The density of [(125)I]EYF binding sites was high in the intralaminar thalamic nuclei, the parafascicular thalamic nucleus and in the superficial layers of the dorsal horn.Non specific binding reached 5-10% of the total binding in all brain areas. Similarly, in mouse brain experiments, the non-specific binding was never superior to 10%.These findings demonstrate that putative neuropeptides generated by the Neuropeptide FF precursor and containing the NPFF or NPSF sequences should bind to the same receptor. Furthermore, these data indicate that [(125)I]EYF is a useful radiolabeled probe to investigate the NPFF receptors; its major advantages being its high affinity and the very low non-specific binding it induces.     

Identification of high affinity receptors for human monocyte chemoattractant protein-1 on human monocytes

The binding of human monocyte chemoattractant protein-1 (MCP-1) to human monocytes was studied. MCP-1 was radioiodinated with Iodo-beads (Pierce Chemical Co., Rockford, IL) without significant loss of biologic activity. 125I-MCP-1 binding to PBMC occurred within 5 min at 0 degrees C and the binding was inhibited by unlabeled MCP-1 dose dependently but not by neutrophil attractant/activation protein-1 or FMLP. 125I-MCP-1 bound to monocytes; no significant binding to either neutrophils or lymphocytes was observed. Scatchard plot analysis indicated that monocytes had a minimum of 1700 +/- 600 binding sites per cell with a Kd of 1.9 +/- 0.2 x 10(-9) M. For analysis of binding by flow cytometry, MCP-1 was biotinylated. In contrast to radioiodination, biotinylation resulted in loss of activity; potency was 10-fold less, but the efficacy was retained. Detection by flow cytometry of bound biotinylated MCP-1 with avidin-FITC confirmed results obtained with 125I-MCP-1. Biotinylated MCP-1 bound to monocytes but not to lymphocytes; and the binding was inhibited by a 100-fold excess of unlabeled MCP-1.     

Synthesis and evaluation of radioiodinated cholesteryl ethers as lipoprotein probes.

Two novel cholesteryl ether derivatives were synthesized and radioiodinated: (1) [125I]cholesteryl m-iodobenzyl ether (125I-CIBE) and (2) [125I]cholesteryl 12-(m-iodophenyl)dodecyl ether (125I-CIDE). These radioiodinated ethers were incorporated into low-density lipoprotein (LDL) by incubating the compounds (solubilized in saline with Tween-20) with isolated LDL or with whole plasma. Such LDL preparations were taken up by cultured fibroblasts in a receptor-dependent manner similar to that of radioiodinated LDL. Upon injection into guinea pigs, 125I-CIBE-labeled guinea pig LDL cleared from the plasma similarly to radioiodinated guinea pig LDL. The primary sites of 125I-CIBE uptake were the adrenal and the liver, and the compound was stable to both hydrolysis and deiodination over 24 h. In summary, 125I-CIBE and 125I-CIDE, like previously described tritiated cholesteryl ethers, appear to be potentially useful tracers of cholesteryl ester uptake. Moreover, these radioiodinated probes have the advantage of being more easily quantitated in tissue samples as well as being detectable by noninvasive scintigraphic imaging.     

Kinetic evidence for tandemly arranged ligand binding sites in melanocortin 4 receptor complexes

The melanocortin 4 receptor (MC(4)R) binding of the peptide analogue of melanocyte stimulating hormone, [(125)I]NDP-MSH, and the low molecular weight radionucleid 1-(D-1,2,3,4-tetrahydroisoquinoline-3-carboxy-D-4-(125)iodophenylalanyl)-4-cyclohexyl-4-[(1,2,4-triazol-1-yl)methyl]piperidine trifluoroacetate ([(125)I]THIQ) were compared. Kinetic analysis indicated heterogeneity in the binding of both radioligands, the binding apparently proceeding to two tandemly arranged interconnected mutually dependent binding sites. Steric considerations and BRET analysis of Rluc and GFP tagged receptors proposed that these sites are located on different subunits of receptor dimers, which form receptor complexes. According to the minimal model proposed, ligand binding proceeds consecutively to the two binding sites of the dimer. After binding of the first ligand conformational transformations of the complex occur, which is followed by binding of the second ligand. When both receptor units have bound [(125)I]NDP-MSH, the radioligand can be released only from one unit. The [(125)I]NDP-MSH bound to the remaining unit stays practically irreversibly bound due to a very slow retransformation rate of the transformed complex. The considerably faster binding of [(125)I]THIQ did not allow accurate kinetic differentiation of the two binding sites. However, addition of NDP-MSH as well as a fragment of the human agouti protein, hAGRP(83-132) to the preformed [(125)I]THIQ-MC(4)R complex drastically retarded the release of [(125)I]THIQ from the complex, blocking conformational transformations in the complex by binding into the second binding site. The consecutive binding of ligands to the MC(4)R dimers has substantial impact on the apparent ligand potencies, when determined in competition with the two different radioligands applied herein; the apparent potencies of the same ligand differing up to three orders of magnitude when assayed in competition with [(125)I]NDP-MSH or [(125)I]THIQ.     

Biotin reagents for antibody pretargeting. 7. Investigation of chemically inert biotinidase blocking functionalities for synthetic utility

An investigation was conducted to evaluate three biotin derivatives designed to block biotinidase cleavage of the biotinamide bond. Difficulties in multistep syntheses of molecules containing tert-butyl protected hydroxymethyl and carboxylate groups positioned alpha to a biotinamide bond led to the investigation of alternative biotinidase-blocking moieties that do not require protection and deprotection. The targeted biotin derivatives contained serine-O-methyl ether, 2-aminobutyric acid, and valine moieties conjugated to the biotin carboxylate functionality. Those derivatives were further modified with a radioiodinated aryl ring to study their biotinidase stability. As a comparison to previously studied biotin derivatives, radioiodinated versions of biotin conjugates that contained (a) no biotinidase stabilizing group, (b) an N-methyl (sarcosine) stabilizing group, (c) an alpha-carboxylate (aspartate) stabilizing group and hydroxymethyl (serine) stabilizing group were also prepared and tested. When tested in human serum, all of the radioiodinated biotinidase-stabilized biotin derivatives had <1% biotinamide cleavage. Thus, under the conditions studied, all of the tested biotinidase blocking moieties appeared to be equal with regards to protection from biotinidase cleavage. Further testing of the biotin derivatives included a HPLC assay to determine their relative dissociation from recombinant streptavidin (rSAv). The dissociation of cyanocobalamin (CN-Cbl) adducts of biotin-serine-O-methyl ether, biotin-aminobutyric acid, and biotin-valine were compared with the CN-Cbl adduct of biotin-sarcosine. The relative rates of dissociation found were biotin-sarcosine-CN-Cbl > biotin-valine-CN-Cbl > biotin-serine-O-methyl ether-CN-Cbl > biotin-aminobutyric acid-CN-Cbl. Due to the high cost of serine-O-ethyl ether (and its N-Boc derivative) and difficulty in syntheses of its biotin derivatives, that adduct is not an attractive candidate for application to compounds used in vivo. The higher lipophilicity and diminished binding of the biotin-valine adduct also makes its use in vivo less attractive. Thus, the biotin-aminobutyric acid adduct appears to be the best candidate for incorporation into biotin derivatives used in vivo, as it simplifies the synthetic procedures, has low cost, and provides effective blocking of biotinidase while retaining high binding affinity.     

LOCALIZATION OF POLYSOME-BOUND ALBUMIN AND SERINE DEHYDRATASE IN RAT LIVER CELL FRACTIONS

The polysomes involved in albumin and serine dehydratase synthesis were identified and localized by the binding to rat liver polysomes of anti-rat serum albumin and anti-serine dehydratase [¹²⁵I]Fab dimer and monomer. Techniques were developed for the isolation of undegraded free and membrane-bound polysomes and for the preparation of [¹²⁵I]Fab monomers and dimers from the IgG obtained from the antisera to the two proteins, rat serum albumin and serine dehydratase. The distribution of anti-rat serum albumin [¹²⁵I]Fab dimer in the polysome profile is in accordance with the size of polysomes that are expected to be synthesizing albumin. By direct precipitation, it has been demonstrated that nascent chains isolated from the membrane-bound polysomes by puromycin were precipitated by anti-rat serum albumin-IgG at a level of 5–6 times those released from free polysomes. Anti-rat serum albumin-[¹²⁵I]Fab dimer reacted with membrane-bound polysomes almost exclusively compared to the binding of nonimmune, control [¹²⁵I]Fab dimer; a significant degree of binding of anti-rat serum albumin-[¹²⁵I]Fab to free polysomes was also obtained. The [¹²⁵I]Fab dimer made from normal control rabbit serum does not react with polysomes from liver at all and this preparation will not interact with polysomes extracted from tissues that do not synthesize rat serum albumin. Both anti-serine dehydratase-[¹²⁵I]Fab monomer and dimer react with free and bound polysomes from livers of animals fed a chow diet or those fed a high 90% protein diet and given glucagon. In the latter instance, however, it is clear that the majority of the binding occurs to the bound polysomes. Furthermore, the specificity of this reaction may be further shown by the use of kidney polysomes that do not normally synthesize serine dehydratase. When these latter polysomes are isolated, even after the addition of crude and purified serine dehydratase, no reaction with anti-serine dehydratase-Fab fragments could be demonstrated. These results indicate that the reaction of the Fab fragments are specific for polysomes that synthesize rat serum albumin or rat liver serine dehydratase. Furthermore, they demonstrate that even with this high degree of specificity, some polysomes in the fraction labeled "free" are in the process of synthesizing rat serum albumin while bound polysomes to a significant, if not major, degree are the site of the synthesis of rat liver serine dehydratase.