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Cutting Edge Peptide Derivatization Approaches
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Part 1: 2-Pyridine Carboxaldehyde for Semi-Automated Soft Spot Identification in Cyclic Peptides: 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.
Part 2: Rapid and Definitive Identification of Cyclic Peptide Soft Spots by Isotope-Labeled Reductive Dimethylation and Mass Spectrometry Fragmentation: Cyclic peptides have been 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 addition of a water molecule regardless of the amide hydrolysis location. In this study, an innovative strategy was developed to enable 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 m/z 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.
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Yu Feng
Merck
Dr. Yu Feng is an associate principal scientist at Merck in the Nonclinical Drug Safety (NDS) department. Dr. Feng received his doctoral degree in pharmaceutical sciences from the University of Wisconsin-Madison under the supervision of Dr. Lingjun Li. Yu also holds Bachelor's degree in both organic chemistry and economics from Peking University, and completes MBA program at University of Illinois Urbana-Champaign. His research in Merck focuses on utilizing mass spectrometry techniques to advance the understanding mechanisms of toxicity and evaluate drug candidates. Dr. Feng has contributed significantly to the field, with more than 20 peer-reviewed publications and patents in biomolecule characterization, quantitative proteomics/glycomics and natural product synthesis.
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Joe Cannon
Bristol Myers Squibb
Joe Cannon received his PhD in Biochemistry at the University of Maryland under Catherine Fenselau in 2012, and proceeded to consecutive post doctoral appointments at the University of Texas at Austin with Jennifer Brodbelt and Harvard Medical School with Wade Harper and Steve Gygi. He then joined Merck in 2018 and put his mass spectrometry skills to work with the biotransformation group and later as an ADME PI. He is currently an Associate Director of Biotransformation at Bristol Myers Squibb and supports company wide efforts in biotransformation, DMPK, and ADME across modalities.
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Komal Kedia
Merck
Dr. Kedia received her Bachelors in Pharmaceutical Sciences from the Delhi University in New Delhi, India and her Ph.D. in Analytical Chemistry from Brigham Young University (BYU) under Dr. Steven Graves. Her work in Dr. Graves’s lab focused on identifying biomarkers (including proteins, peptides, lipids and other small molecules) of Preeclampsia, from placental tissue and blood to better understand the pathogenesis of this disorder. Following graduation, Dr. Kedia joined Pacific Northwest National Lab (PNNL) in Dr. Richard Smith’s group where she participated in multiple projects starting from biomarker discovery for several diseases, to method development for xenobiotics, specifically using LCMS and IMMS based approaches. She is currently an Associate Principal Scientist at Merck within PDMB group, where her responsibilities include supporting PK/PD studies and biomarker analysis across various internal programs. Additionally, Dr. Kedia is the project lead for the development and evaluation of different applications on a state-of-the-art, high-resolution ion mobility instrument based on SLIM technology, with the goal of providing program support across different modalities, especially for challenging analytes such as cyclic peptides and lipids.
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