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

ABSTRACT: 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 ?M, kinact = 0.34 min-1), was conceptually dissected into two fragments and each characterized separately: l-norvaline ( Ki = 470 ?M) and 2-chloroacetamidine ( KI = 310 ?M, 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 ?M, 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 ?M) with cytotoxicity appearing only at higher concentrations (ED50 = 118 ?M). 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.

SUBMITTER: Burstein-Teitelbaum G

PROVIDER: S-EPMC6074031 | biostudies-literature | 2018 Jul

REPOSITORIES: biostudies-literature

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

Burstein-Teitelbaum Gayle G Er Joyce A V JAV Monzingo Arthur F AF Tuley Alfred A Fast Walter W

Biochemistry 20180720 30

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 ...[more]

PMID: 29983043

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Dataset Information

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

Publications

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

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