The antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl) urea (LY181984) inhibits NADH oxidase activity of HeLa plasma membranes

Abstract

Plasma membrane vesicles from HeLa S cells grown in culture bound with high affinity the antitumor sulfonylurea N-(4-methylphenylsulfonyl)-N'-(4-chlorophenyl)urea (LY181984). Based on binding site protection experiments with the radiolabeled thiol reagent N-[14C]ethylmaleimide, a ca. 34 kDa binding protein was identified. By analogy with a 36 kDa NADH oxidase from plant plasma membranes where activity was blocked by a growth-inhibitory herbicidal sulfonylurea, the sulfonylurea-binding protein of the HeLa plasma membranes has now been identified as a comparable sulfonylurea-inhibited NADH oxidase activity. The drug inhibited half maximally at about 50 nM which corresponded closely to the Kd for binding of [3H]LY181984 of 25 nM. A closely related but growth-inactive sulfonylurea N-(4-methylphenylsulfonyl)-N'-(phenyl)urea (LY181985) inhibited the activity only weakly. The inhibition by LY181984 was analyzed kinetically and shown to be noncompetitive or uncompetitive depending on the concentration of NADH. With sealed right-side out plasma membrane vesicles, the NADH oxidase activity was about 90% inhibited by 1 microM LY181984. With frozen and thawed plasma membrane vesicles or with vesicles first solubilized with 1% Triton X-100, activity also was inhibited by LY181984 and not by LY181985 but the maximum inhibition at 10 microM LY181984 was about 50%. With plasma membranes from rat liver, neither LY181984 nor LY181985 affected the NADH oxidase even in the presence of detergent. Thus, selective inhibition or stimulation of the oxidation of NADH of tumor plasma membranes by the antitumor sulfonylurea LY181984 may be related to its antitumor activity.