Fragment Screening Reveals Starting Points for Rational Design of Galactokinase 1 Inhibitors to Treat Classic Galactosemia

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.

Multi‐omics in classical galactosemia: Evidence for the involvement of multiple metabolic pathways

“…New therapeutic options should focus on intracellular phosphate homeostasis, for example by preventing the accumulation of Gal‐1P though GALK inhibition. Already in 2002, we suggested galactokinase inhibition as an effective treatment for CG, 22 and much effort is made worldwide to develop this therapeutic option 23,24 . Clinical trials would benefit from a clear predictive biomarker that parallels the disease burden and ideally indicates the phenotypic course.…”

“…Already in 2002, we suggested galactokinase inhibition as an effective treatment for CG, 22 and much effort is made worldwide to develop this therapeutic option. 23,24 Clinical trials would benefit from a clear predictive biomarker that parallels the disease burden and ideally indicates the phenotypic course. However, the above-described complex pathophysiological process of CG makes it difficult to find this prognostic biomarker, as implicated by our findings that none of the detected metabolites were predictive for intellectual-and neurological outcome and our previous finding that the most recent Gal-1P also was not predictive for clinical outcome.…”

The Importance of Neonatal Screening for Galactosemia

“…However, this type of test lacks sensitivity and specificity, leading to false positive results in the case of fructosuria, lactosuria (caused by intestinal lactase deficiency), or conditions that affect the clearance of blood galactose, such as severe liver disease or antimicrobial therapy. On the other hand, if the infant is receiving intravenous nutrition, galactosuria may be absent, thus leading to false negative results [ 25 , 26 ].…”

Characterization of novel mevalonate kinases from the tardigrade Ramazzottius varieornatus and the psychrophilic archaeon Methanococcoides burtonii

“…The following references are cited in the supporting information for this article: Hartley et al (2004) and Mackinnon et al (2021).…”