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Lookup NU author(s): Dr Sabrina MackinnonORCiD, Professor Wyatt YueORCiD
This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).
Classic galactosemia is caused by loss-of-function mutations in galactose-1-phosphate uridylyltransferase (GALT) that lead to toxic accumulation of its substrate, galactose-1-phosphate. One proposed therapy is to inhibit the biosynthesis of galactose-1-phosphate, catalyzed by galactokinase 1 (GALK1). Existing inhibitors of human GALK1 (hGALK1) are primarily ATP-competitive with limited clinical utility to date. Here, we determined crystal structures of hGALK1 bound with reported ATP-competitive inhibitors of the spiro-benzoxazole series, to reveal their binding mode in the active site. Spurred by the need for additional chemotypes of hGALK1 inhibitors, desirably targeting a nonorthosteric site, we also performed crystallography-based screening by soaking hundreds of hGALK1 crystals, already containing active site ligands, with fragments from a custom library. Two fragments were found to bind close to the ATP binding site, and a further eight were found in a hotspot distal from the active site, highlighting the strength of this method in identifying previously uncharacterized allosteric sites. To generate inhibitors of improved potency and selectivity targeting the newly identified binding hotspot, new compounds were designed by merging overlapping fragments. This yielded two micromolar inhibitors of hGALK1 that were not competitive with respect to either substrate (ATP or galactose) and demonstrated good selectivity over hGALK1 homologues, galactokinase 2 and mevalonate kinase. Our findings are therefore the first to demonstrate inhibition of hGALK1 from an allosteric site, with potential for further development of potent and selective inhibitors to provide novel therapeutics for classic galactosemia.
Author(s): Mackinnon SR, Krojer T, Foster WR, Diaz-Saez L, Tang M, Huber KVM, von Delft F, Lai K, Brennan PE, Arruda Bezerra G, Yue WW
Publication type: Article
Publication status: Published
Journal: ACS Chemical Biology
Year: 2021
Volume: 16
Issue: 4
Pages: 586-595
Print publication date: 16/04/2021
Online publication date: 16/03/2021
Acceptance date: 18/02/2021
Date deposited: 06/10/2023
ISSN (print): 1554-8929
ISSN (electronic): 1554-8937
Publisher: American Chemical Society
URL: https://doi.org/10.1021/acschembio.0c00498
DOI: 10.1021/acschembio.0c00498
PubMed id: 33724769
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