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.