Labeling and receptor affinity of an ultra-short somatostatin analogue Thz-Phe-D-Trp-Lys-Thr-DOTA,Journal of Peptide Science

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Labeling and receptor affinity of an ultra-short somatostatin analogue Thz-Phe-D-Trp-Lys-Thr-DOTA
Journal of Peptide Science ( IF 1.8 ) Pub Date : 2021-07-21 , DOI: 10.1002/psc.3361
Anzhelika O Fedotova ₁ , Bayirta V Egorova ₁ , Galina A Posypanova ₂ , Nikolay A Titchenko ₁ , Derenik S Khachatryan _{2,

3} , Anton V Kolotaev _{2,

3} , Vasiliy N Osipov ₄ , Stepan N Kalmykov _{1,

2}

Affiliation

Somatostatin analogues play an important role in the therapy of neuroendocrine tumors by binding to somatostatin receptors on the surface of cancer cells. In this work, we analyze the receptor-binding affinity and in vitro stability of a novel ultra-short somatostatin analogue Thz-Phe-D-Trp-Lys-Thr-DOTA (DOTA-P4). This conjugate is successfully radiolabeled with ⁴⁴Sc, ⁹⁰Y, ¹⁵²Eu, and ²⁰⁷Bi, characterized and validated by thin layer and high-performance liquid chromatography. The optimum conditions for M-DOTA-P4 labeling are found. In vitro stability studies are performed in saline, in the presence of serum proteins, and with biologically relevant metal cations. All complexes demonstrate no cation release in vitro within 4–24 h. The conformations of DOTA-conjugates are studied by circular dichroism spectroscopy. The circular dichroism spectra of DOTA-P4 conjugates show a negative peak at 225 nm, which may correspond to the required β-sheet conformation. The binding to somatostatin receptors of types 2 and 5 is performed with the IMR-32 cells at 4°C, with non-specific binding representing 26% of the total binding. A two-line approximation of the Scatchard plot results in the apparent dissociation constants of 0.10 and 2.25 nM. It is shown that the chelator position with respect to the amino acid sequence significantly affects the labeling conditions with cations of different ionic radii. For the first time, the binding of a linear type ultra-short peptide conjugate with DOTA to somatostatin receptors is demonstrated. The obtained results are promising for experiments with DOTA-P4 in vivo in mice with inoculated tumors.

中文翻译：

超短生长抑素类似物 Thz-Phe-D-Trp-Lys-Thr-DOTA 的标记和受体亲和力

生长抑素类似物通过与癌细胞表面的生长抑素受体结合，在神经内分泌肿瘤的治疗中发挥重要作用。在这项工作中，我们分析了一种新型超短生长抑素类似物 Thz-Phe-D-Trp-Lys-Thr-DOTA (DOTA-P4)的受体结合亲和力和体外稳定性。该偶联物成功地用⁴⁴ Sc、⁹⁰ Y、¹⁵² Eu 和²⁰⁷ Bi 进行了放射性标记，并通过薄层和高效液相色谱进行了表征和验证。找到了 M-DOTA-P4 标记的最佳条件。体外稳定性研究是在盐水中、在血清蛋白存在下以及与生物学相关的金属阳离子下进行的。所有复合物在体外均无阳离子释放在 4-24 小时内。通过圆二色光谱研究 DOTA 缀合物的构象。DOTA-P4 偶联物的圆二色光谱在 225 nm 处显示负峰，这可能对应于所需的 β-折叠构象。IMR-32 细胞在 4°C 下与 2 型和 5 型生长抑素受体结合，非特异性结合占总结合的 26%。Scatchard 图的两线近似值导致表观解离常数为 0.10 和 2.25 nM。结果表明，相对于氨基酸序列的螯合剂位置显着影响不同离子半径的阳离子的标记条件。首次证明了线性型超短肽缀合物与 DOTA 与生长抑素受体的结合。在接种了肿瘤的小鼠体内。

更新日期：2021-07-21

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