Structural Basis of ALDH1A2 Inhibition by Irreversible and Reversible Small Molecule Inhibitors

Enzymes of the ALDH1A subfamily of aldehyde dehydrogenases are crucial in regulating retinoic acid (RA) signaling and have received attention as potential drug targets. ALDH1A2 is the primary RA-synthesizing enzyme in mammalian spermatogenesis and is therefore considered a viable drug target for male contraceptive development. However, only a small number of ALDH1A2 inhibitors have been reported, and information on the structure of ALDH1A2 was limited to the NAD-liganded enzyme void of substrate or inhibitors. Herein, we describe the mechanism of action of structurally unrelated reversible and irreversible inhibitors of human ALDH1A2 using direct binding studies and X-ray crystallography. All inhibitors bind to the active sites of tetrameric ALDH1A2. Compound WIN18,446 covalently reacts with the side chain of the catalytic residue Cys320, resulting in a chiral adduct in (R) configuration. The covalent adduct directly affects the neighboring NAD molecule, which assumes a contracted conformation suboptimal for the dehydrogenase reaction. The reversible inhibitors interact predominantly through direct hydrogen bonding interactions with residues in the vicinity of Cys320 without affecting NAD. Upon interaction with inhibitors, a large flexible loop assumes regular structure, thereby shielding the active site from solvent. The precise knowledge of the binding modes provides a new framework for the rational design of novel inhibitors of ALDH1A2 with improved potency and selectivity profiles.醛脱氢酶ALDH1A亚家族（ALDH1A subfamily）的酶在调控视黄酸（retinoic acid, RA）信号通路中发挥关键作用，作为潜在药物靶点受到广泛关注。ALDH1A2是哺乳动物精子发生过程中合成视黄酸的主要酶，因此被视为开发男性避孕药的可行药物靶点。然而，目前仅报道了少量ALDH1A2抑制剂，且此前关于ALDH1A2的结构信息仅局限于结合烟酰胺腺嘌呤二核苷酸（nicotinamide adenine dinucleotide, NAD）但不含底物或抑制剂的酶结构。本文中，我们通过直接结合实验与X射线晶体学（X-ray crystallography）方法，阐明了人类ALDH1A2的两类结构无关的可逆与不可逆抑制剂的作用机制。所有抑制剂均结合至四聚体ALDH1A2的活性位点。化合物WIN18,446可与催化残基Cys320的侧链发生共价反应，形成构型为(R)的手性加合物。该共价加合物会直接影响邻近的NAD分子，使其呈现出不利于脱氢酶反应的收缩构象。可逆抑制剂则主要通过与Cys320附近残基形成直接氢键相互作用发挥作用，且不会对NAD造成影响。在与抑制剂结合后，一段较大的柔性环会形成规整结构，从而将活性位点与溶剂环境隔绝开来。对结合模式的精准认知，为合理设计具有更优活性与选择性的新型ALDH1A2抑制剂提供了全新的研究框架。