Mackinnon Sabrina R, Bezerra Gustavo A, Krojer Tobias, Szommer Tamas, von Delft Frank, Brennan Paul E, Yue Wyatt W
Centre for Medicines Discovery, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.
Target Discovery Institute, University of Oxford, Oxford, United Kingdom.
Front Chem. 2022 May 4;10:844598. doi: 10.3389/fchem.2022.844598. eCollection 2022.
Primary hyperoxaluria type I (PH1) is caused by AGXT gene mutations that decrease the functional activity of alanine:glyoxylate aminotransferase. A build-up of the enzyme's substrate, glyoxylate, results in excessive deposition of calcium oxalate crystals in the renal tract, leading to debilitating renal failure. Oxidation of glycolate by glycolate oxidase (or hydroxy acid oxidase 1, HAO1) is a major cellular source of glyoxylate, and siRNA studies have shown phenotypic rescue of PH1 by the knockdown of HAO1, representing a promising inhibitor target. Here, we report the discovery and optimization of six low-molecular-weight fragments, identified by crystallography-based fragment screening, that bind to two different sites on the HAO1 structure: at the active site and an allosteric pocket above the active site. The active site fragments expand known scaffolds for substrate-mimetic inhibitors to include more chemically attractive molecules. The allosteric fragments represent the first report of non-orthosteric inhibition of any hydroxy acid oxidase and hold significant promise for improving inhibitor selectivity. The fragment hits were verified to bind and inhibit HAO1 in solution by fluorescence-based activity assay and surface plasmon resonance. Further optimization cycle by crystallography and biophysical assays have generated two hit compounds of micromolar (44 and 158 µM) potency that do not compete with the substrate and provide attractive starting points for the development of potent and selective HAO1 inhibitors.
I型原发性高草酸尿症（PH1）由AGXT基因突变引起，该突变会降低丙氨酸：乙醛酸转氨酶的功能活性。该酶的底物乙醛酸积累，导致草酸钙晶体在尿路中过度沉积，进而引发使人衰弱的肾衰竭。乙醇酸氧化酶（或羟基酸氧化酶1，HAO1）将乙醇酸氧化是乙醛酸的主要细胞来源，并且RNA干扰研究表明，通过敲低HAO1可实现PH1的表型拯救，这表明HAO1是一个有前景的抑制剂靶点。在此，我们报告了通过基于晶体学的片段筛选鉴定出的六个低分子量片段的发现和优化，这些片段与HAO1结构上的两个不同位点结合：活性位点和活性位点上方的变构口袋。活性位点片段扩展了用于底物模拟抑制剂的已知支架，使其包括更具化学吸引力的分子。变构片段代表了对任何羟基酸氧化酶的非正构抑制的首次报道，在提高抑制剂选择性方面具有重大前景。通过基于荧光的活性测定和表面等离子体共振验证了片段命中物在溶液中与HAO1结合并抑制HAO1。通过晶体学和生物物理测定进行的进一步优化循环产生了两种微摩尔级（44和158 μM）效力的命中化合物，它们不与底物竞争，并为开发强效和选择性的HAO1抑制剂提供了有吸引力的起点。
