Dataset Information

Tritium Labeling of Neuromedin S by Conjugation with [³H]N-Succinimidyl Propionate.

ABSTRACT: The human neuropeptide neuromedin S (NMS) consists of 33 amino acids. The introduction of tritium atoms into NMS has not been described so far. This represents a gap for using [³H]NMS in radioreceptor binding assays or in tracking and monitoring their metabolic pathway. Two approaches for the incorporation of tritium into NMS were explored in this study: (1) halogenation at the His-18 residue followed by catalyzed iodine-127/tritium exchange and (2) conjugation of tritiated N-succinimidyl-[2,3-³H₃]propionate ([³H]NSP) to at least one of the three available primary amines of amino acids Ile-1, Lys-15, and Lys-16 in the peptide sequence. Although iodination of histidine was achieved, subsequent iodine-127/deuterium exchange was unsuccessful. Derivatization at the three possible amino positions in the peptide using nonradioactive NSP resulted in a mixture of unconjugated NSM and 1- to 3-conjugations at different amino acids in the peptide sequence. Each labeling position in the mixture was assigned following detailed LC-MS/MS analysis. After separating the mixture, it was shown in an in vitro fluorometric imaging plate reader (FLIPR) and in a competitive binding assay that the propionyl-modified NMS derivatives were comparable to the unlabeled NMS, regardless of the degree of labeling and the labeling position(s). A molecular simulation with NMS in the binding pocket of the protein neuromedin U receptor 2 (NMUR₂) confirmed that the possible labeling positions are located outside the binding region of NMUR₂. Tritium labeling was achieved at the N-terminal Ile-1 using [³H]NSP in 7% yield with a radiochemical purity of >95% and a molar activity of 90 Ci/mmol. This approach provides access to tritiated NMS and enables new investigations to characterize NMS or corresponding NMS ligands.

SUBMITTER: Edelmann MR

PROVIDER: S-EPMC9851027 | biostudies-literature | 2023 Jan

REPOSITORIES: biostudies-literature

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Tritium Labeling of Neuromedin S by Conjugation with [3H]N-Succinimidyl Propionate.

Edelmann Martin R MR Erny Johannes J Guba Wolfgang W Hierl Markus M

ACS omega 20230104 2

The human neuropeptide neuromedin S (NMS) consists of 33 amino acids. The introduction of tritium atoms into NMS has not been described so far. This represents a gap for using [3H]NMS in radioreceptor binding assays or in tracking and monitoring their metabolic pathway. Two approaches for the incorporation of tritium into NMS were explored in this study: (1) halogenation at the His-18 residue followed by catalyzed iodine-127/tritium exchange and (2) conjugation of tritiated N-s ...[more]

PMID: 36687043

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Project description:BackgroundPatients with T cell deficiency <10% of normal proliferation are indicated to receive immune reconstruction by hematopoietic stem cell transplantation (HSCT). This study aimed to investigate whether non-radioactive assays can be used to quantitatively detect the lymphocyte proliferation <10% of normal as radioactive [3H]-thymidine."MethodsRadioactive [3H]-thymidine, non-radioactive carboxyfluorescein diacetate succinimidyl ester (CFSE), and Ki-67 protein expressions were used to measure the lymphocyte proliferation as calculated using the stimulation index (SI), subtraction percentage, and proliferation index (FlowJo software). Normal references were established for comparison in the absence of parallel healthy controls.ResultsNormal ranges of mitogen-stimulated lymphocyte proliferation were established as a SI of 15-267 (CSFE 47-92%, Ki-67 42-79%) with phytohemagglutinin (PHA) 5 μg/ml stimulation; 19-139 (CFSE 62-83%, 45-74% Ki-67) with concanavalin-A (ConA) 5 μg/ml stimulation; 7-53 (CFSE 6-23%, Ki-67 10-24%) with pokeweed mitogen (PWM) 0.1 ug/ml stimulation; 3-28 (CFSE 4-10%, Ki-67 5-14%) with candida 10 ug/ml stimulation; and 2-27 (CFSE 6-41%, Ki-67 6-30%) with bacille Calmette-Guerin (BCG) 0.02 ng/ml stimulation. The normalized CFSE-proliferation index was between 2.1 and 3.0. Although there was no significant correlation between these three assays in the healthy controls, the SI value for <10% [3H]-thymidine proliferation in those with T cell deficiency was compatible with CFSE- and Ki-67-stained lymphocyte percentages, and validated in patients with IL2RG, RAG1, and ZAP70 mutations. When calculating [3H]-thymidine <10% of normal lymphocyte proliferation, the threshold of parallel controls was more reliable than previously established normal references.ConclusionThe large quantitative value of radioactive [3H]-thymidine was more easily recognizable than that for non-radioactive CFSE and Ki-67. Even though the correlation was not significant, those identified to have <10% of normal proliferation by [3H]-thymidine could be consistently detected by CFSE and Ki-67, and consequently indicated for HSCT.

Project description:IntroductionLabeling single domain antibody fragments (sdAbs) with 18F is an attractive strategy for immunoPET. Earlier, we developed a residualizing label, N-succinimidyl 3-((4-(4-fluorobutyl)-1H-1,2,3-triazol-1-yl)methyl)-5-(guanidinomethyl)benzoate ([18F]RL-I), synthesized via a click reaction for labeling sdAbs with 18F, that has attractive features but suffered from modest radiochemical yields and suboptimal hydrophobicity. Herein, we have evaluated the potential utility of an analogous agent, N-succinimidyl 3-(1-(2-(2-(2-(2-[18F]fluoroethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)-5-(guanidinomethyl)benzoate ([18F]SFETGMB; [18F]RL-III) designed to address these limitations.Methods[18F]RL-III was synthesized by the click reaction between 3-((2,3-bis(tert-butoxycarbonyl)guanidino)methyl)-5-ethynylbenzoate and 1-azido-2-(2-(2-(2-[18F]fluoroethoxy)ethoxy)ethoxy)ethane and subsequent deprotection. The anti-HER2 sdAbs 5F7 and 2Rs15d were labeled by conjugation with [18F]RL-III and compared in a paired-label fashion to the sdAbs labeled using N-succinimidyl 4-guanidinomethyl-3-[125I]iodobenzoate ([125I]SGMIB) or N-succinimidyl 3-guanidinomethyl-5-[125I]iodobenzoate (iso-[125I]SGMIB). The 18F-labeled sdAbs were evaluated in vitro using HER2-expressing breast and ovarian carcinoma cells (BT474/BT474M1 and SKOV-3) and in vivo in athymic mice bearing subcutaneous SKOV-3 or BT474 xenografts. PET imaging of athymic mice bearing either subcutaneous BT474 or intracranial BT474M1Br-Fluc xenografts after administration of [18F]RL-III-5F7 also was performed.ResultsRadiochemical yields for the synthesis of Boc2-[18F]RL-III (21.5 ± 3.4%) were significantly higher than reported for Boc2-[18F]RL-I. The overall radiochemical yields for the synthesis of [18F]RL-III-2Rs15d and [18F]RL-III-5F7 from aqueous [18F]fluoride were 1.7 ± 0.7% and 3.8 ± 2.3%, respectively. Both sdAbs, labeled using [18F]RL-III, retained affinity and immunoreactivity to HER2. Uptake and internalization of [18F]RL-III-5F7 in HER2-expressing cells was higher than that seen for [18F]RL-III-2Rs15d. Although different xenograft models were used, [18F]RL-III-2Rs15d showed relatively high uptake in a number of normal tissues, while uptake of [18F]RL-III-5F7 was mainly in tumor and kidneys with minimal background activity. Concordant with the necropsy experiments, microPET imaging with [18F]RL-III-5F7 in the BT474 subcutaneous model demonstrated clear delineation of the tumor (12.2 ± 5.1% ID/g) with minimal background activity except in kidneys. A tumor uptake (max) of 0.98%ID/g and a tumor-to-normal brain ratio of 9.8:1 were observed for [18F]RL-III-5F7 in the intracranial model.ConclusionsAlthough higher radiochemical yields than that reported for [18F]RL-I were obtained, considerable improvements are needed for this method to be of practical utility. Despite clear tumor delineation with [18F]RL-III-5F7 as early as 1 h, high activity levels in the kidneys remain a concern.

Dataset Information

Tritium Labeling of Neuromedin S by Conjugation with [³H]N-Succinimidyl Propionate.

Publications

Tritium Labeling of Neuromedin S by Conjugation with [<sup>3</sup>H]<i>N</i>-Succinimidyl Propionate.

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