Dissection, Optimization, and Structural Analysis of a Covalent Irreversible DDAH1 Inhibitor
2018
Burstein-Teitelbaum, Gayle | Er, Joyce A.V. | Monzingo, Arthur F. | Tuley, Alfred | Fast, Walter
Inhibitors of the human enzyme dimethylarginine dimethylaminohydrolase-1 (DDAH1) can control endogenous nitric oxide production. A time-dependent covalent inactivator of DDAH1, N⁵-(1-imino-2-chloroethyl)-l-ornithine (KI = 1.3 μM, kᵢₙₐcₜ = 0.34 min–¹), was conceptually dissected into two fragments and each characterized separately: l-norvaline (Kᵢ = 470 μM) and 2-chloroacetamidine (KI = 310 μM, kᵢₙₐcₜ = 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, N⁵-(1-imino-2-chloroisopropyl)-l-ornithine (kᵢₙₐcₜ/KI = 208 M–¹ s–¹) and N⁵-(1-imino-2-chlorisopropyl)-l-lysine (kᵢₙₐcₜ/KI = 440 M–¹ s–¹), and one that lengthens the linker beyond that found in the substrate, N⁵-(1-imino-2-chloroethyl)-l-lysine (Cl-NIL, KI = 0.19 μM, kᵢₙₐcₜ = 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.
显示更多 [+] 显示较少 [-]