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Fragment screening reveals starting points for rational design of galactokinase 1 inhibitors to treat classic galactosemia

DOI: 10.1021/acschembio.0c00498 DOI Help

Authors: Sabrina R. Mackinnon (Structural Genomic Consortium, University of Oxford) , Tobias Krojer (Structural Genomics Consortium, University of Oxford) , William R. Foster (Structural Genomic Consortium, University of Oxford) , Laura Diaz-Saez (Structural Genomic Consortium, University of Oxford) , Manshu Tang (University of Utah) , Kilian V. M. Huber (Structural Genomics Consortium and Target Discovery Institute, University of Oxford) , Frank Von Delft (Diamond Light Source) , Kent Lai (University of Utah) , Paul Brennan (Structural Genomic Consortium, University of Oxford) , Gustavo Arruda Bezerra (Structural Genomic Consortium, University of Oxford) , Wyatt W. Yue (Structural Genomics Consortium, University of Oxford)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Acs Chemical Biology , VOL 152

State: Published (Approved)
Published: March 2021
Diamond Proposal Number(s): 18145

Abstract: 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.

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography

Discipline Tags:

Life Sciences & Biotech Health & Wellbeing Drug Discovery Non-Communicable Diseases Structural biology

Technical Tags:

Diffraction Macromolecular Crystallography (MX)