Dissection, Optimization, and Structural Analysis of a Covalent Irreversible DDAH1 Inhibitor

Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1) can control endogenous nitric oxide production. A time-dependent covalent inactivator of DDAH1, N 5-(1-imino-2-chloroethyl)-l-ornithine (K I = 1.3 μM, k inact = 0.34 min–1), was conceptually dissected into two fragments and each characterized separately: l-norvaline (K i = 470 μM) and 2-chloroacetamidine (K I = 310 μM, k inact = 4.0 min–1). This analysis suggested that the two fragments were not linked in a manner that allows either to reach full affinity or reactivity, prompting the synthesis and characterization of three analogues: two that mimic the dimethylation status of the substrate, N 5-(1-imino-2-chloroisopropyl)-l-ornithine (k inact /K I = 208 M–1 s–1) and N 5-(1-imino-2-chlorisopropyl)-l-lysine (k inact /K I = 440 M–1 s–1), and one that lengthens the linker beyond that found in the substrate, N 5-(1-imino-2-chloroethyl)-l-lysine (Cl-NIL, K I = 0.19 μM, k inact = 0.22 min–1). Cl-NIL is one of the most potent inhibitors reported for DDAH1, inactivates with a second order rate constant (1.9 × 104 M–1 s–1) larger than the catalytic efficiency of DDAH1 for its endogenous substrate (1.6 × 102 M–1 s–1), and has a partition ratio of 1 with a >100 000-fold selectivity for DDAH1 over arginase. An activity-based protein-profiling probe is used to show inhibition of DDAH1 within cultured HEK293T cells (IC50 = 10 μM) with cytotoxicity appearing only at higher concentrations (ED50 = 118 μM). A 1.91 Å resolution X-ray crystal structure reveals specific interactions made with DDAH1 upon covalent inactivation by Cl-NIL. Dissecting a covalent inactivator and analysis of its constituent fragments proved useful for the design and optimization of this potent and effective DDAH1 inhibitor.