Nonclinical Drug Safety, MRL, Merck & Co., Inc., 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States.
Pharmacokinetics, Dynamics, Metabolism, and Bioanalytics, MRL, Merck & Co., Inc., 770 Sumneytown Pike, West Point, Pennsylvania 19486, United States.
Anal Chem. 2024 May 14;96(19):7756-7762. doi: 10.1021/acs.analchem.4c01372. Epub 2024 May 1.
Cyclic peptides are an emerging therapeutic modality over the past few decades. To identify drug candidates with sufficient proteolytic stability for oral administration, it is critical to pinpoint the amide bond hydrolysis sites, or soft spots, to better understand their metabolism and provide guidance on further structure optimization. However, the unambiguous characterization of cyclic peptide soft spots remains a significant challenge during early stage discovery studies, as amide bond hydrolysis forms a linearized isobaric sequence with the addition of a water molecule, regardless of the amide hydrolysis location. In this study, an innovative strategy was developed to enable the rapid and definitive identification of cyclic peptide soft spots by isotope-labeled reductive dimethylation and mass spectrometry fragmentation. The dimethylated immonium ion with enhanced MS signal at a distinctive / in MS/MS fragmentation spectra reveals the N-terminal amino acid on a linearized peptide sequence definitively and, thus, significantly simplifies the soft spot identification workflow. This approach has been evaluated to demonstrate the potential of isotope-labeled dimethylation to be a powerful analytical tool in cyclic peptide drug discovery and development.
在过去的几十年中，环状肽是一种新兴的治疗模式。为了确定具有足够的口服给药蛋白水解稳定性的药物候选物，确定酰胺键水解部位（即软点）至关重要，这有助于更好地了解它们的代谢，并为进一步的结构优化提供指导。然而，在早期发现研究中，明确表征环状肽软点仍然是一个重大挑战，因为酰胺键水解会形成一个线性等摩尔序列，其中添加了一个水分子，而与酰胺水解的位置无关。在这项研究中，开发了一种创新策略，通过同位素标记的还原二甲化和质谱碎裂来实现环状肽软点的快速和明确鉴定。在 MS/MS 碎裂谱中，具有增强的 MS 信号的二甲化亚氨鎓离子揭示了线性化肽序列上的 N 末端氨基酸，从而大大简化了软点鉴定工作流程。已经评估了该方法，以证明同位素标记二甲化有可能成为环状肽药物发现和开发的强大分析工具。
