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bioRxiv · 10.1101/2023.01.08.523168

Phage-Assisted, Active Site-Directed Ligand Evolution with a Genetically Encoded Nε-Butyryl-ʟ-Lysine to Identify a Cellularly Potent and Selective Inhibitor for the ENL YEATS Domain as an Anti-Leukemia Agent

Chen, P.-H. C.·Guo, X. S.·Zhang, H. E.·Geng, Z. Z.·Dubey, G. K.·Fierke, C. A.·Xu, S.·Liu, W. R.
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Abstract

Eleven-nineteen leukemia protein (ENL) plays pivotal roles in the leukemogenesis. As a YEATS domain protein, ENL reads histone acylation marks and recruits key transcription factors to leukemic drivers such as HOXA9, MEIS1, and MYB and therefore promotes leukemia development. The histone-reading function of ENL has been proven essential in the onset and progression of several acute leukemias, suggesting a putative therapeutic window for ENL inhibition. In this study, we developed a phage-assisted, active site-directed ligand evolution (PADLE) approach for the identification of potent and selective ENL inhibitors, where N{varepsilon}-butyryl-O_SCPLOWLC_SCPLOW-lysine (BuK) that possesses known target-protein interactions with the ENL YEATS domain was genetically incorporated into a phage display library to serve as a warhead to direct displayed peptides to the active site of ENL YEATS for enrichment. Using this novel strategy in combination with structure-activity relationship that replaced BuK with other ncAAs for de novo {pi}-{pi}-{pi} stacking interactions with two aromatic residues in ENL YEATS, selective and potent ENL inhibitors with a Kd value as low as 2.0 nM were identified. One pentapeptide inhibitor tENL-S1f displayed selective inhibition of ENL over other YEATS domains as well as strong cellular target engagement and on-target effects in inhibiting leukemia cell growth and suppressing the expression of ENL target genes. As the first of its kind study, the current work opens a large avenue of research of using PADLE to develop selective and potent peptidyl inhibitors for a large variety of epigenetic reader proteins.

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Chen, P.-H. C., Guo, X. S., Zhang, H. E., Geng, Z. Z., Dubey, G. K., Fierke, C. A., Xu, S., Liu, W. R.. 2023-01-09. Phage-Assisted, Active Site-Directed Ligand Evolution with a Genetically Encoded Nε-Butyryl-ʟ-Lysine to Identify a Cellularly Potent and Selective Inhibitor for the ENL YEATS Domain as an Anti-Leukemia Agent. https://doi.org/10.1101/2023.01.08.523168

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