Advancing ASMS with LC‐MS/MS for the discovery of novel PDCL2 ligands from DNA‐encoded chemical library selections
Year: 2022
Type: article
Abstract: BACKGROUND: 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. OBJECTIVE: 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. MATERIALS AND METHODS: 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). RESULTS: 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. DISCUSSION AND CONCLUSION AND RELEVANCE: 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.
Source: Andrology
DOI: 10.1111/andr.13309
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: hybrid
Estimated APC paid: $3,000
Funders: Bill and Melinda Gates Foundation, Welch Foundation, Cancer Prevention and Research Institute of Texas, National Institutes of Health, Japan Society for the Promotion of Science, National Cancer Institute, National Institute of Child Health and Human Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development
Awards: G1043300375, Elucidating the molecular mechanisms of fertilization common to vertebrate animal models, G1301834326, Development of new diagnostic markers and reversible fertility inhibitors for mammalian spermatozoa, G2805889465, G2901508018, Preclinical Candidate Discovery Core, Recruitment of First-Time, Tenure-Track Faculty Members, G4030898082, Development of a Precision Drug to target Y537S Mutant Estrogen Receptor in Metastatic Breast Cancer, Development of Next-Generation Estrogen Receptor Mutant Inhibitors for Endocrine Resistant Metastatic Breast Cancer, G6670048745, Functional genomics and DEC-Tec to identify germ cell-specific contraceptives, Baylor College of Medicine - to apply additional target validation and prioritization filters to a portfolio of potential male contraceptive drug targets that could accelerate ..., Elucidating the universality and specificity of reproductive mechanisms between species using genetically modified animals, Functional analysis of novel testis-expressed secreted and transmembrane proteins
OpenAlex ID: W4303696576