Department of Metabolism and Pharmacokinetics, Bristol-Myers Squibb Company, Princeton, NJ 08648, USA.
Int J Mol Sci. 2022 Apr 12;23(8):4269. doi: 10.3390/ijms23084269.
Cyclic peptides are an attractive option as therapeutics due to their ability to disrupt crucial protein-protein interactions and their flexibility in display type screening strategies, but they come with their own bioanalytical challenges in metabolite identification. Initial amide hydrolysis of a cyclic peptide results in a ring opening event in which the sequence is linearized. Unfortunately, the mass of the singly hydrolyzed sequence is the same (M + 18.0106 Da) irrespective of the initial site of hydrolysis, or soft spot. Soft spot identification at this point typically requires time-consuming manual interpretation of the tandem mass spectra, resulting in a substantial bottleneck in the hit to lead process. To overcome this, derivatization using 2-pyridine carboxaldehyde, which shows high selectivity for the alpha amine on the N-terminus, was employed. This strategy results in moderate- to high-efficiency derivatization with a unique mass tag and diagnostic ions that serve to highlight the first amino acid in the newly linearized peptide. The derivatization method and analytical strategy are demonstrated on a whole cell lysate digest, and the soft spot identification strategy is shown with two commercially available cyclic peptides: JB1 and somatostatin. Effective utilization of the automated sample preparation and interpretation of the resulting spectra shown here will serve to reduce the hit-to-lead time for generating promising proteolytically stable peptide candidates.
由于环状肽能够破坏关键的蛋白质-蛋白质相互作用，并且在展示类型筛选策略方面具有灵活性，因此它们是一种有吸引力的治疗方法选择。但是，在代谢产物鉴定方面，它们也存在自身的生物分析挑战。环状肽的初始酰胺水解会导致环打开事件，其中序列线性化。不幸的是，无论水解的初始位置（软点）如何，单水解序列的质量都是相同的（M + 18.0106 Da）。此时，软点的鉴定通常需要耗时的串联质谱手动解释，从而导致命中先导过程中的实质性瓶颈。为了克服这一问题，使用 2-吡啶甲醛进行了衍生化，该试剂对 N 末端的α 胺具有高选择性。该策略导致具有独特质量标签和诊断离子的中等到高效衍生化，这些标签和诊断离子用于突出新线性化肽中的第一个氨基酸。该衍生化方法和分析策略在全细胞裂解物消化物上进行了演示，并使用两种市售的环状肽：JB1 和生长抑素展示了软点鉴定策略。有效利用这里显示的自动样品制备和对所得光谱的解释，将有助于减少生成有前途的蛋白水解稳定肽候选物的命中到先导时间。
