Advancing ASMS with LC‐MS/MS for the discovery of novel PDCL2 ligands from DNA‐encoded chemical library selections
| Autor: | Qiuji Ye, Hassane Belabed, Yong Wang, Zhifeng Yu, Murugesan Palaniappan, Jian‐Yuan Li, Stacey A. Kalovidouris, Kevin R. MacKenzie, Mingxing Teng, Damian W. Young, Yoshitaka Fujihara, Martin M. Matzuk |
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| Rok vydání: | 2022 |
| Předmět: | |
| Zdroj: | Andrology. |
| ISSN: | 2047-2927 2047-2919 |
| DOI: | 10.1111/andr.13309 |
| Popis: | A safe, effective, and reversible nonhormonal male contraceptive drug is greatly needed for male contraception as well as for circumventing the side effects of female hormonal contraceptives. Phosducin-like 2 (PDCL2) is a testis-specific phosphoprotein in mice and humans. We recently found that male PDCL2 knockout mice are sterile due to globozoospermia caused by impaired sperm head formation, indicating that PDCL2 is a potential target for male contraception. Herein, our study for the first time developed a biophysical assay for PDCL2 allowing us to screen a series of small molecules, to study structure-activity relationships, and to discover two PDCL2 binders with novel chemical structure.To identify a PDCL2 ligand for therapeutic male contraception, we performed DNA-encoded chemical library (DECL) screening and off-DNA hit validation using a unique affinity selection mass spectrometry (ASMS) biophysical profiling strategy.We employed the screening process of DECL, which contains billions of chemically unique DNA-barcoded compounds generated through individual sequences of reactions and different combinations of functionalized building blocks. The structures of the PDCL2 binders are proposed based on the sequencing analysis of the DNA barcode attached to each individual DECL compound. The proposed structure is synthesized through multistep reactions. To confirm and determine binding affinity between the DECL identified molecules and PDCL2, we developed an ASMS assay that incorporates liquid chromatography with tandem mass spectrometry (LC-MS/MS).After a screening process of PDCL2 with DECLs containing 440 billion compounds, we identified a series of hits. The selected compounds were synthesized as off-DNA small molecules, characterized by spectroscopy data, and subjected to our ASMS/LC-MS/MS binding assay. By this assay, we discovered two novel compounds, which showed good binding affinity for PDCL2 in comparison to other molecules generated in our laboratory and which were further confirmed by a thermal shift assay.With the ASMS/LC-MS/MS assay developed in this paper, we successfully discovered a PDCL2 ligand that warrants further development as a male contraceptive. |
| Databáze: | OpenAIRE |
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