Enzyme Kinetics and Binding Studies on Inhibitors of MEK Protein Kinase

• Published in: Biochemistry (Easton) 2008-04, Vol.47 (17), p.5017-5027
• Main Authors: VanScyoc, Wendy S, Holdgate, Geoffrey A, Sullivan, Jane E, Ward, Walter H. J
• Format: Article
• Language: English
• Subjects: Adenosine Triphosphate - metabolism; Benzamides - chemistry; Benzamides - metabolism; Benzamides - pharmacology; Butadienes - metabolism; Butadienes - pharmacology; Catalysis - drug effects; Enzyme Activation - drug effects; Enzyme Inhibitors - metabolism; Enzyme Inhibitors - pharmacology; Inhibitory Concentration 50; Kinetics; MAP Kinase Kinase 1 - antagonists & inhibitors; MAP Kinase Kinase 1 - metabolism; Nitriles - metabolism; Nitriles - pharmacology; Protein Binding; Quinolines - chemistry; Quinolines - metabolism; Quinolines - pharmacology; Thermodynamics
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi701811x

Inhibition of the protein kinase, MEK1, is a potential approach for the treatment of cancer. Inhibitors may act by prevention of activation (PoA), which involves interfering with phosphorylation of nonactivated MEK1 by the upstream kinase, B-RAF. Modulation also may occur by inhibition of catalysis (IoC) during phosphorylation of the downstream substrate, ERK2, by activated MEK1. Here, five MEK inhibitors are characterized in terms of binding affinity, PoA, and IoC. The compounds are a butadiene (U-0126), an N-alkoxy amide (CI-1040), two CI-1040 analogues (an anthranilic acid and an N-alkyl amide), and a cyanoquinoline. Some compounds give different mechanisms of inhibition (ATP-competitive, noncompetitive, or uncompetitive) in PoA compared to IoC or show a change in potency between the assays. The inhibitors also exhibit different shifts in potency when either PoA or IoC is compared with binding to nonactivated MEK. The inhibitor potency ranking, therefore, is dependent upon the assay format. When the ATP concentration equals K m, IoC IC50 increases in the order CI-1040 ≈ cyanoquinoline < anthranilic acid ≈ U-0126 < alkyl amide. Conversely, the K d from nonactivated MEK1 for four of the compounds varies between more than 6-fold lower and over 18-fold higher than this IC50, with U-0126 having the lowest K d and CI-1040 having the highest. In PoA when the ATP concentration equals K m, U-0126 has the lowest IC50, becoming more potent than CI-1040, the cyanoquinoline, and the anthranilic acid. These observations have implications for understanding structure–activity relationships of MEK inhibitors and illustrate how assays can be designed to favor different compounds.