Activity of either HO-1 or HO-2 as shown in Figure 1. Thus
Activity of either HO-1 or HO-2 as shown in Figure 1. Thus, the HO-1 and HO-2 activity curves for vitamin K1 (panel a) were flat from 10 nM PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27735993 to10 M with a small upward trend at 100 M for brain microsomal HO-2, and the curves for vitamin K2 (panel b) were flat throughout the full concentration range. In comparison, menadione activated HO-2 about 7-fold (Figure 2). Structurally menadione differs from vitamins K1 and K2 in that it possesses a methyl moiety at the 2-position whereas vitamins K1 and K2 each possess an additional, adjacent long unsaturated aliphatic side chain (Figure 1). Because this side chain could possibly interfere with binding of vitamin K1 or K2 to HO-2 through steric hindrance, we decided to explore the activity of other menadione analogs that possessed large non-aliphatic BX795 site substituents at positions-2 and -3. For these experiments, we tested menadione analogs that possessed various substituents at the 2- and 3-positions including- naphthoquinones containing furan-carboxylic acid substituents (Table 1), naphthoquinones containing furan-benzoxazine (Table 2), naphthoquinones containing aminophenyl and piperidinyl moieties (Table 3), naphthoquinones containing 2-phenyl (Table 4) and other naphthoquinones containing various substituents at positions-2 and -3 (Table 5). Tables 1, 2, 3 and 4 show activation of rat brain HO-2 by menadioneanalogs possessing a wide variety of substituents at the 2- and 3-position. For example, compound 71830417 (a furan-containing naphthoquinone) activated HO-2 to 693 of control, SI-859 (a 2-phenylnaphthoquinone) activated HO-2 to 691 , and 7010100237 (a 2-(aminophenyl)3-piperidin-1-yl)naphthoquinone) activated HO-2 to 249 of control. Clearly, a variety of moieties could be present at the 2- and 3-positions of naphthoquinone, with maintenance of the ability to activate HO-2. Thus, unlike vitamins K1 and K2, the mere presence of a large substituent at the 2- and 3-positions in these compounds did not render them unable to activate HO-2.Mechanism of actionAccording to classical receptor theory, an agonist drug such as menadione possesses affinity for its receptor on HO-2 and efficacy once it has bound. The inactivity of vitamin K1 might be explained by an inability to bind to HO-2. To explore this, we tested the effects of menadione in the presence of vitamin K1. This experiment was done in two ways- by adding vitamin K1 (10 ?100 M) to the reaction mixture before menadione (25 M) or after menadione. Both iterations yielded no effect on menadione activation of HO-2; vitamin K1 did not alter the menadione-induced activation of HO-2 (data not shown). An obvious characteristic of menadione and its analogs studied herein is their naphthoquinone core, which is known to participate in redox reactions [16]. Consequently,OCHC o n t r o l A c t iv it y600O CHCHDMDOCHO CH400 300 200 100 0 -9 -8 -OFFMDF F O5F-MDF—-lo g [ D r u g ]M D /H O – 2 D M D /H O -2 M D /H O – 1 5 F -M D /H O -Figure 2 Activation of rat brain HO-2 by menadione, and lack of activation by dimethoxymenadione (DMD) and pentafluromenadione (5 F-MD). HO-1 and HO-2 activity were measured as described in Methods using spleen and brain microsomes, respectively. The abscissa shows drug concentration and the ordinate shows HO activity as a percent of control in the absence of added drug. Open circles show menadione activation of HO-2, and solid triangles show lack of activation of rat spleen HO-1. Solid circles show lack of activation of HO-2.