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Phenothiazine-Biaryl-Containing Fluorescent RGD Peptides.

ABSTRACT: Cyclic RGD peptides are well-known ligands of integrins. The integrins ?V ?3 and ?5 ?1 are involved in angiogenesis, and integrin ?V ?3 is abundantly present on cancer cells, thus representing a therapeutic target. Hence, synthetic and biophysical studies continuously are being directed towards the understanding of ligand-integrin interaction. In this context, the development of versatile synthetic strategies to obtain fluorescent building blocks that can add molecular diversity and modular spectral characteristics while not compromising binding affinity or selectivity is a relevant task. An on-resin intramolecular Suzuki-Miyaura cross-coupling (SMC) between l- or d-7-bromotryptophan (7BrTrp) and a phenothiazine (Ptz) boronic acid affords fluorescent cyclic RGD pseudopeptides, c(RGD(W/w)Ptz). Ring closure by SMC establishes a phenothiazine-indole moiety with axial chirality. An array of eight novel compounds has been synthesized, among them one fluorescent compound with good affinity to integrin ?V ?3 . The fluorescence properties of the analogues can be efficiently tuned depending on the substituents in Ptz moiety even for fluorescence emission in the visible (red) spectral range.

SUBMITTER: Ghabraie E

PROVIDER: S-EPMC7540173 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Phenothiazine-Biaryl-Containing Fluorescent RGD Peptides.

Ghabraie Elmira E Kemker Isabell I Tonali Nicolo N Ismail Mohamed M Dodero Veronica I VI Sewald Norbert N

Chemistry (Weinheim an der Bergstrasse, Germany) 20200818 52

Cyclic RGD peptides are well-known ligands of integrins. The integrins αV β3 and α5 β1 are involved in angiogenesis, and integrin αV β3 is abundantly present on cancer cells, thus representing a therapeutic target. Hence, synthetic and biophysical studies continuously are being directed towards the understanding of ligand-integrin interaction. In this context, the development of versatile synthetic strategies to obtain fluorescent ...[more]

PMID: 32297686

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Project description:This study sought to evaluate FITC-conjugated cyclic RGD peptides (FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2) as fluorescent probes for in vitro assays of integrin ?v?3/?v?5 expression in tumor tissues. FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were prepared, and their integrin ?v?3/?v?5 binding affinity was determined using the displacement assay against (125)I-echistatin bound to U87MG glioma cells. IC50 values of FITC-Galacto-RGD2, FITC-3P-RGD2, and FITC-RGD2 were calculated to be 28 ± 8, 32 ± 7, and 89 ± 17 nM, respectively. The integrin ?v?3/?v?5 binding affinity followed a general trend: FITC-Galacto-RGD2 ? FITC-3P-RGD2 > FITC-RGD2. The xenografted tumor-bearing models were established by subcutaneous injection of 5 × 10(6) tumor cells into shoulder flank (U87MG, A549, HT29, and PC-3) or mammary fat pad (MDA-MB-435) of each athymic nude mouse. Three to six weeks after inoculation, the tumor size was 0.1-0.3 g. Tumors were harvested for integrin ?v?3/?v?5 staining, as well as hematoxylin and eosin (H&E) staining. Six human carcinoma tissues (colon cancer, pancreatic cancer, lung adenocarcinoma, squamous cell lung cancer, gastric cancer, and esophageal cancer) were obtained from recently diagnosed cancer patients. Human carcinoma slides were deparaffinized in xylene, rehydrated with ethanol, and then used for integrin ?v?3/?v?5 staining, as well as H&E staining. It was found that the tumor staining procedures with FITC-conjugated cyclic RGD peptides were much simpler than those with the fluorescence-labeled integrin ?v?3 antibodies. Since FITC-RGD2, FITC-3P-RGD2, and FITC-Galacto-RGD2 were able to co-localize with the fluorescence-labeled integrin ?3 antibody, their tumor localization and tumor cell binding are integrin ?v?3-specific. Quantification of the fluorescent intensity in five xenografted tumors (U87MG, MDA-MB-435, A549, HT29, and PC-3) and six human carcinoma tissues revealed an excellent linear relationship between the relative integrin ?v?3/?v?5 expression levels determined with FITC-Galacto-RGD2 and those obtained with the fluorescence-labeled anti-human integrin ?3 antibody. There was also an excellent linear relationship between the tumor uptake (%ID/g) of (99m)Tc-3P-RGD2 (an integrin ?v?3/?v?5-targeted radiotracer) and the relative integrin ?v?3/?v?5 expression levels from the quantification of fluorescent intensity in the tumor tissues stained with FITC-Galacto-RGD2. These results suggest that FITC-conjugated cyclic RGD peptides might be useful to correlate the in vitro findings with the in vivo imaging data from an integrin ?v?3/?v?5-targeted radiotracer. The results from this study clearly showed that the FITC-conjugated cyclic RGD peptides (particularly FITC-3P-RGD2 and FITC-Galacto-RGD2) are useful fluorescent probes for assaying relative integrin ?v?3/?v?5 expression levels in tumor tissues.

Project description:Direct bromination of the tyrosine residues of peptides and antibodies with bromine-76, to create probes for PET imaging, has been reported. For peptides that do not contain tyrosine residues, however, a prosthetic group is required to achieve labeling via conjugation to other functional groups such as terminal ?-amines or lysine ?-amines. The goal of this study was to develop new strategies for labeling small peptides with Br-76 using either a direct labeling method or a prosthetic group, depending on the available functional group on the peptides. A new labeling agent, N-succinimidyl-3-[(76)Br]bromo-2,6-dimethoxybenzoate ([(76)Br]SBDMB) was prepared for cyclic RGD peptide labeling. N-succinimidyl-2, 6-dimethoxybenzoate was also used to pre-attach a 2, 6-dimethoxybenzoyl (DMB) moiety to the peptide, which could then be labeled with Br-76. A competitive cell binding assay was performed to determine the binding affinity of the brominated peptides. PET imaging of U87MG human glioblastoma xenografted mice was performed using [(76)Br]-BrE[c(RGDyK)](2) and [(76)Br]-BrDMB-E[c(RGDyK)](2). An ex vivo biodistribution assay was performed to confirm PET quantification. The mechanisms of bromination reaction between DMB-c(RGDyK) and the brominating agent CH(3)COOBr were investigated with the SCRF-B3LYP/6-31G* method with the Gaussian 09 program package. The yield for direct labeling of c(RGDyK) and E[c(RGDyK)](2) using chloramine-T and peracetic acid at ambient temperature was greater than 50%. The yield for [(76)Br]SBDMB was over 60% using peracetic acid. The conjugation yields for labeling c(RGDfK) and c(RGDyK) were over 70% using the prosthetic group at room temperature. Labeling yield for pre-conjugated peptides was over 60%. SDMB conjugation and bromination did not affect the binding affinity of the peptides with integrin receptors. Both [(76)Br]Br-E[c(RGDyK)](2) and [(76)Br]BrDMB-E[c(RGDyK)](2) showed high tumor uptake in U87MG tumor bearing mice. The specificity of the imaging tracers was confirmed by decreased tumor uptake after co-administration of unlabeled dimeric RGD peptides. The energy barrier of the transition state of bromination for the dimethoxybenzoyl group was about 9 kcal/mol lower than that for the tyrosine residue. In conclusion, the newly developed N-succinimidyl-2, 6-dimethoxybenzoate molecule can be used either for one step labeling through pre-conjugation or as the precursor for a Br-76 labeled prosthetic group for indirect labeling. Radiobromination on a dimethoxybenzoyl group has selectivity over radiobromination on tyrosine. The energy barrier difference of the transition states of bromination between the dimethoxybenzoyl group and the tyrosine residue may account for the reaction selectivity when both groups are present in the same molecule.

Dataset Information

Phenothiazine-Biaryl-Containing Fluorescent RGD Peptides.

Publications

Phenothiazine-Biaryl-Containing Fluorescent RGD Peptides.

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