Hsiao M.H.Miao Y.Liu Z.Schütze K.Limjunyawong N.Chien D.C.C.Monteiro W.D.Chu L.S.Morgenlander W.Jayaraman S.Jang S.E.Gray J.J.Zhu H.Dong X.Steinegger M.Larman H.B.Mahidol University2025-02-242025-02-242025-01-01Molecular and Cellular Proteomics Vol.24 No.2 (2025)15359476https://repository.li.mahidol.ac.th/handle/123456789/105409Animal venoms, distinguished by their unique structural features and potent bioactivities, represent a vast and relatively untapped reservoir of therapeutic molecules. However, limitations associated with comprehensively constructing and expressing highly complex venom and venom-like molecule libraries have precluded their therapeutic evaluation via high-throughput screening. Here, we developed an innovative computational approach to design a highly diverse library of animal venoms and “metavenoms”. We used programmable M13 hyperphage display to preserve critical disulfide-bonded structures for highly parallelized single-round biopanning with quantitation via high-throughput DNA sequencing. Our approach led to the discovery of Kunitz-type domain containing proteins that target the human itch receptor Mas-related G-protein coupled receptor member X4, which plays a crucial role in itch perception. Deep learning-based structural homology mining identified two endogenous human homologs, tissue factor pathway inhibitor (TFPI), and serine peptidase inhibitor, Kunitz type 2 (SPINT2), which exhibit agonist-dependent potentiation of Mas-related G-protein coupled receptor member X4. Highly multiplexed screening of animal venoms and metavenoms is therefore a promising approach to uncover new drug candidates.ChemistryBiochemistry, Genetics and Molecular BiologyArticleSCOPUS10.1016/j.mcpro.2024.1009012-s2.0-852179049381535948439746545