br Conflict of interest br Acknowledgments br Introduction
Conflict of interest
Introduction Inflammation is a protective response of the microcirculation to harmful stimuli. Yet, excessive inflammation is potentially harmful and a characteristic of many chronic diseases . 5-Lipoxygenase (5-LO, EC188.8.131.52) contributes to the inflammatory reaction by catalyzing the conversion of arachidonic MK-1775 receptor (AA) to potent lipid mediators, namely leukotriene (LT)B4 and LTC4. These mediators drive chemotaxis and leukocyte activation, or in case of LTC4 lead to increased vasopermeability and bronchoconstriction . Possible 5-LO linked indications therefore range from asthma  and allergic rhinitis , to atherosclerosis , Alzheimer’s disease , leukemia ,  and certain types of cancer . In order to widen the therapeutic armamentarium in these diseases many efforts have been made to develop anti-leukotriene drugs, which climaxed in the approval of the direct 5-LO inhibitor Zileuton ,  (FDA approval for the therapy of asthma ). Unfortunately the use of Zileuton is limited, partly due to hepatotoxic adverse effects . Therefore many novel compounds were designed, resulting in various promising strategies. Given the catalytic cycle of 5-LO, direct 5-LO inhibitors can act via chelating the active site iron (iron ligand inhibitors; e.g. Zileuton) or by keeping the catalytic iron in the inactive ferrous state by reduction (redox-active inhibitors). Further on there are active site directed, non-redox type inhibitors which compete with AA for binding to 5-LO and allosteric inhibitors, which e.g. act at the regulatory C2-like domain (e.g. Hyperforin , EP6 ). Despite these rational approaches, besides Zileuton, a great number of investigated compounds subsequently failed in preclinical or clinical trials . This was partly due to insufficient in vitro screening. Apart from toxicities, several compounds suffer from a lack of specificity or display only a poor efficacy in physiologically relevant conditions. The potencies of ZM 230487 or L-739,010 for example depend on the phosphorylation status of 5-LO . Thus there is still a pressing need to develop novel well-characterized 5-LO inhibitors. In a previous study we presented comprehensive structure-activity relationships (SAR) of a class of potent aminothiazole-comprising 5-LO inhibitors . Based on the parent compound ST-1083 we developed two lead compounds (ST-1853 and ST-1906) out of this SAR with high 5-LO inhibitory potency in purified enzyme and intact cell assays (IC50=50nM in intact human polymorphonuclear leukocytes (PMNL)) (Fig. 1) . Yet, comprehensive evaluation of the lead likeness of the compounds, as well as the detailed mechanism of action of this novel 5-LO inhibitor type is still due. Therefore, we elaborated various in vitro assays to clarify these unmet questions and present a characterization of the 5-LO inhibition by N-phenyl-4-aryl-1,3-thiazole-2-amines with focus on the two lead compounds ST-1853 and ST-1906.
Materials & methods
Discussion Based on the results of our previous SAR study on 2-aminothiazole 5-LO inhibitors , we encountered a potency loss between r5-LO and intact PMNL for unsubstituted compounds and a positive shift towards a higher potency in PMNL for compounds bearing a methylated substitution motif. In the present study we demonstrated that the high potency of N-phenyl-4-aryl-1,3-thiazole-2-amine derivatives in the purified r5-LO assay might be due to covalent binding of the unsubstituted aminophenolic moiety to C159 and C418. We further give an explanation for the reduced activity in intact cells, as the compounds are capable of binding GSH. Corresponding previous studies have shown that compounds with tampered aminophenolic moiety show only a diminished potency . Further on, compound ST-1905, bearing the quinone imine moiety, and its reduced equivalent ST-1083 presented similar potencies at the r5-LO wt (high potency) and 4C mutant assay (impaired potency) (cfFig. 5). In line, ST-1905 showed a more pronounced binding of GSH in comparison with the hydroquinone amine and a lower potency in intact cells. Together with the previously deduced SAR this suggests that redox activity is one aspect of the inhibitory mode of action. While the DPPH assay attests antiradical activity of the compounds, the Ferene-S assay also approved the capability of reducing Fe3+, therewith providing the basis of a partly redox active mechanism of inhibition. Both lead compounds ST-1853 and ST-1906 showed a higher potency in intact cells than with the purified recombinant enzyme. This is rather unusual, since intact cells have to be permeated and present metabolizing enzymes as well as off targets. Inhibition of 5-LO upstream enzymes (cPLA2, FLAP) would be a plausible explanation for this behavior. Yet in addition to a FLAP inhibitor assay there is no impairment of potency, when analyzing 5-LO product formation with varying AA concentrations or 5-LO stimuli. Furthermore, ST-1853 and ST-1906 could, like the untypical 5-LO inhibitor Hyperforin, interfere with the membrane binding C2-like domain . However, unlike Hyperforin, the potency of ST-1853 is not impaired with the presence of 20μg/ml phosphatidylcholine, indicating a different mechanism of action. Both lead compounds bear the 2,6-dimethyl substitution pattern which may add inductive effects to enhance reductive properties of the compound. As shown for acetaminophen derivatives this substitution pattern does not impair redox cycling . Yet potentially toxic protein modification by the oxidized form or certain phase II metabolism (in this study shown for glucuronidation) can be prevented by this substitution pattern. Nonetheless modification of the reducing milieu of the intact cell via alteration of the thiol level did not alter the inhibitory potency. An alternative explanation for the higher potency in intact cells could be an enrichment of compound in intact cells leading to higher concentrations at the target area. In contrast to the unsubstituted compounds, ST-1853 and ST-1906 had a higher potency using the purified 4C and C416S enzyme compared to wt 5-LO. C416 of the 5-LO wt may therefore feature negative effects on binding of the lead compounds.