6ZGV

Structure of human galactokinase 1 bound with 2-(4-chlorophenyl)-N-(pyrimidin-2-yl)acetamide

Summary for 6ZGV

• Entry DOI: 10.2210/pdb6zgv/pdb
• Descriptor: Galactokinase, beta-D-galactopyranose, 2-(1,3-benzoxazol-2-ylamino)spiro[1,6,7,8-tetrahydroquinazoline-4,1'-cyclohexane]-5-one, ... (5 entities in total)
• Functional Keywords: galk1, galactokinase 1, fragment screening, allosteric fragment, binding hotspt, transferase
• Biological source: Homo sapiens (Human)
• Total number of polymer chains: 4
• Total formula weight: 174251.30

Authors

Mackinnon, S.R.,Bezerra, G.A.,Zhang, M.,Foster, W.,Krojer, T.,Brandao-Neto, J.,Douangamath, A.,Arrowsmith, C.,Edwards, A.,Bountra, C.,Brennan, P.,Lai, K.,Yue, W.W. (deposition date: 2020-06-20, release date: 2020-07-29, Last modification date: 2024-01-24)

Primary citation

Mackinnon, S.R.,Krojer, T.,Foster, W.R.,Diaz-Saez, L.,Tang, M.,Huber, K.V.M.,von Delft, F.,Lai, K.,Brennan, P.E.,Arruda Bezerra, G.,Yue, W.W.
Fragment Screening Reveals Starting Points for Rational Design of Galactokinase 1 Inhibitors to Treat Classic Galactosemia.
Acs Chem.Biol., 16:586-595, 2021

PubMed 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.

PubMed: 33724769
DOI: 10.1021/acschembio.0c00498

Experimental method

X-RAY DIFFRACTION (2.3 Å)