Phage-assisted, active site-directed ligand evolution of a potent and selective histone deacetylase 8 inhibitor.
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Phage-assisted, active site-directed ligand evolution (PADLE) is a recently developed technique that uses an amber codon-encoded noncanonical amino acid (ncAA) as an anchor to direct phage-displayed peptides to a target for an enhanced ligand identification process. 2-Amino-8-oxodecanoic acid (Aoda) is a ketone-containing ncAA residue in the macrocyclic peptide natural product apicidin that is a pan-inhibitor of Zn2+ -dependent histone deacetylases (HDACs). Its ketone serves as an anchoring point to coordinate the catalytic zinc ion in HDACs. Using a previously evolved N -acetyl-lysyl-tRNA synthetase in combination with tRNAPyl , we showed that Aoda was efficiently incorporated into proteins in Escherichia coli by amber suppression. By propagating an amber codon-obligate phagemid library in E. coli encoding Aoda, we generated an Aoda-containing phage-displayed peptide library. Using this library to conduct PADLE against HDAC8 revealed a 7-mer peptide GH8P01F1 with Aoda-flanking amino acid residues that matched existing peptide sequences in identified HDAC8 substrates. Switching Aoda in GH8P01F1 to a more Zn2+ -chelating ncAA S-2-amino-8-hydroxyamino-8-oxooctanoic acid (Asuha) led to an extremely potent compound GH8HA01, which has an HDAC8-inhibition Ki value of 0.67nM. GH8HA01 and its 5-mer truncation analogue Ac-GH8HA0117 that has an HDAC8-inhibition Ki value of 0.31nM are two of the most potent HDAC8 inhibitors that have been developed. Furthermore, both are highly selective against HDAC8 compared with other HDACs tested, demonstrating the great potential of using PADLE to identify highly potent and selective ligands for targets with conserved active sites among homologues.
Morse, J. S., Sheng, Y. J., Hampton, J. T., Sylvain, L. D., Das, S., Alugubelli, Y. R., ... Liu, W. R.
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Morse, Jared S||Sheng, Yan J||Hampton, Joshua Trae||Sylvain, Lauralee D||Das, Sukant||Alugubelli, Yugendar R||Chen, Peng-Hsun Chase||Yang, Kai S||Xu, Shiqing||Fierke, Carol A||Liu, Wenshe Ray
