Structure-based inhibitor design for substrate reduction therapy towards primary hyperoxaluria and galactosaemia

Inborn errors of metabolism are a group of more than 1000 inherited single gene disorders for which few effective treatments are available. In almost all of these disorders, mutations cause a loss of function in the encoded protein that is essential to normal metabolism, resulting in accumulation of metabolites upstream of the variant and/or deficiency of downstream metabolites. Where the disease pathogenesis results from toxicity of accumulated metabolites, inhibition of proteins preceding the metabolic block has been shown to be an effective therapeutic strategy. For example, proof-of-concept, genetic knockdown studies support the inhibition of galactokinase 1 (hGALK1), which precedes the defective variant in galactose metabolism, to treat classic galactosaemia; and inhibition of hydroxyacid oxidase 1 (hHAO1), which precedes the defect in glycolate metabolism, to treat primary hyperoxaluria. However, existing inhibitors for these two targets have limited scaffold diversity and their poor pharmacokinetic and toxicity profiles preclude therapeutic use. In this work, fragment screening by X-ray crystallography was combined with solution binding and activity assays to generate novel chemical starting points for the development of hGALK1 and hHAO1 inhibitors. Two fragments weakly inhibited hGALK1 upon binding to novel allosteric pockets away from the active site. Merging of fragments in one such pocket yielded two inhibitor hits of good potency and efficacy with potential for future drug development. For hHAO1, analogues of fragments binding to the active site, a nearby novel pocket and at the subunit-subunit interface yielded a total of 9 high efficacy inhibitors, spanning all three sites and diverse scaffolds. Altogether, the compounds described in this work hold promise for addressing the unmet therapeutic need in classic galactosaemia and primary hyperoxaluria, and the methodology developed suggests a broader application to the field of inborn errors of metabolism.