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

Abstract

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.

Design, synthesis, and ex vivo evaluation of a selective inhibitor for retinaldehyde dehydrogenase enzymes

“…Interactions between the γ-sulfur of the catalytic cysteine of RALDH2 and the carbonyl carbon of DAR were ruled out as modeling predicted insufficient space in the substrate binding pocket to accommodate the additional methylene group of DAR. We predict that, similar to what has been observed with WIN 18446, 75 upon nucleophilic attack by the catalytic cysteine, a chloride atom is displaced from DAR, resulting in an irreversible interaction (Figure 7C). Modeling of DAR in the binding pocket of RALDH2 using the hydride transfer configuration of NAD + 87 indicated significant atomic collision between the nicotinamide ring of NAD + with the dichloromethyl end group of compound 2, suggesting steric inhibition of DAR binding in the active site of RALDH2 when NAD + is present (data not shown).…”

“…67, 68, 74 Based on an x-ray crystal structure of WIN 18446 bound to human RALDH2, the ALDH active cysteine sulfhydryl group forms a covalent bond with the terminal carbon of WIN 18446, causing displacement of a chloride atom from WIN 18446. 75 We postulated that the relatively large size of the substrate entrance tunnel for the RALDH enzymes, as compared with the other ALDH enzymes, would allow for the specificity of retinaldehyde (RAL) (Figure 1B) to the RALDH enzymes. 20, 76 Using this knowledge, we suspected that a RALDH selective inhibitor could be generated by attaching a dichloro-methane moiety to the retinyl group (beta-ionone ring with an isoprenoid chain) of RAL, thereby forming dichloro-all- trans -retinone (DAR) (Figure 1C).…”

“…Modeling of DAR into the active site of RALDH2 was based on the crystal structure of human RALDH2 with bound WIN 18,446 retaining one chloride atom (residue # DAR 700-Cl1) as seen in the crystal structure. 75 The retinyl group (beta-ionone ring with isoprenoid chain) was positioned and energy minimization was carried out using AMBER version 17. Binding poses were visualized and figures generated with PyMOL v. 1.8.6 (Schrodinger, Cambridge MA).…”

Computer-Aided Modeling of Interaction between Aldehyde Dehydrogenase and Garcinia Biflavonoids

“…Herein, Table 1 summarizes the binding energy values estimated for each of the Garcinia-derived biflavonoids against ALDH and their molecular interaction revealing the essential amino acid residues that contributed to the ALDHbiflavonoids complex formation and stability. The binding energy ranges from -8.7 kcal/mol to -9.0 kcal/mol compared to the reference ligand (-11.6 kcal/mol) which correlates with its IC 50 value (0.54 µM) [4], thus indicating the relatively moderate potential of the biflavonoids in inhibiting the functions of ALDH. The binding energy can provide insights into the affinity of the biflavonoids to the active site of ALDH.…”

“…This well-established function of ALDH in alcohol metabolism became a bedrock that drove research efforts towards identification of ALDH inhibitors. Moreover, ALDH isozymes have been reported for their importance in oxidizing lipid peroxidation-derive aldehydes which are reactive towards cellular components [4]. Hence, the enzymes contribute to maintenance of cellular homeostasis [5].…”