Design, Synthesis, and Structure-Activity Relationship Studies of Dual Inhibitors of Soluble Epoxide Hydrolase and 5-Lipoxygenase.

Inhibition of multiple enzymes of the arachidonic acid cascade leads to synergistic anti-inflammatory effects. Merging of 5-lipoxygenase (5-LOX) and soluble epoxide hydrolase (sEH) pharmacophores led to the discovery of a dual 5-LOX/sEH inhibitor, which was subsequently optimized in terms of potency toward both targets and metabolic stability. The optimized lead structure displayed cellular activity in human polymorphonuclear leukocytes, oral bioavailability, and target engagement in vivo and demonstrated profound anti-inflammatory and anti-fibrotic efficiency in a kidney injury model caused by unilateral ureteral obstruction in mice. These results pave the way for investigating the therapeutic potential of dual 5-LOX/sEH inhibitors in other inflammation- and fibrosis-related disease models.

Characterization of the molecular mechanism of 5-lipoxygenase inhibition by 2-aminothiazoles.

5-Lipoxygenase (5-LO, EC1.13.11.34) has been implicated in the pathogenesis of inflammatory and immune diseases. Recently, aminothiazole comprising inhibitors have been discovered for this valuable target. Yet, the molecular mode of action of this class of substances is only poorly understood. Here, we present the detailed molecular mechanism of action of the compound class and the in vitro pharmacological profile of two lead compounds ST-1853 and ST-1906. Mechanistic studies with recombinant proteins as well as intact cell assays enabled us to define this class as a novel type of 5-LO inhibitors with unique characteristics. The parent compounds herein presented a certain reactivity concerning oxidation and thiol binding: Unsubstituted aminophenols bound covalently to C159 and C418 of human 5-LO. Yet, dimethyl substitution of the aminophenol prevented this reactivity and slowed down phase II metabolism. Both ST-1853 and ST-1906 confirmed their lead likeness by retaining their high potency in physiologically relevant 5-LO activity assays, high metabolic stability, high specificity and non-cytotoxicity.

Michael acceptor containing drugs are a novel class of 5-lipoxygenase inhibitor targeting the surface cysteines C416 and C418.

Recently, we published that nitro-fatty acids (NFA) are potent electrophilic molecules which inhibit 5-lipoxygenase (5-LO) by interacting catalytically with cysteine residues next to a substrate entry channel. The electrophilicity is derived from an intramolecular Michael acceptor moiety consisting of an electron-withdrawing group in close proximity to a double bond. The potential of the Michael acceptor moiety to interact with functionally relevant cysteines of proteins potentially renders them effective and sustained enzyme activity modulators. We screened a large library of naturally derived and synthetic electrophilic compounds to investigate whether other types of Michael acceptor containing drugs suppress 5-LO enzyme activity. The activity was measured by assessing the effect on the 5-LO product formation of intact human polymorphonuclear leukocytes. We demonstrated that a number of structurally different compounds were suppressive in the activity assays and showed that Michael acceptors of the quinone and nitro-alkene group produced the strongest inhibition of 5-LO product formation. Reactivity with the catalytically relevant cysteines 416 and 418 was confirmed using mutated recombinant 5-LO and mass spectrometric analysis (MALDI-MS). In the present study, we show for the first time that a number of well-recognized naturally occurring or synthetic anti-inflammatory compounds carrying a Michael acceptor, such as thymoquinone (TQ), the paracetamol metabolite NAPQI, the 5-LO inhibitor AA-861, and bardoxolone methyl (also known as RTA 402 or CDDO-methyl ester) are direct covalent 5-LO enzyme inhibitors that target the catalytically relevant cysteines 416 and 418.

Multi-dimensional target profiling of N,4-diaryl-1,3-thiazole-2-amines as potent inhibitors of eicosanoid metabolism.

Eicosanoids like leukotrienes and prostaglandins play a considerable role in inflammation. Produced within the arachidonic acid (AA) cascade, these lipid mediators are involved in the pathogenesis of pain as well as acute and chronic inflammatory diseases like rheumatoid arthritis and asthma. With regard to the lipid cross-talk within the AA pathway, a promising approach for an effective anti-inflammatory therapy is the development of inhibitors targeting more than one enzyme of this cascade. Within this study, thirty N-4-diaryl-1,3-thiazole-2-amine based compounds with different substitution patterns were synthesized and tested in various cell-based assays to investigate their activity and selectivity profile concerning five key enzymes involved in eicosanoid metabolism (5-, 12-, 15-lipoxygenase (LO), cyclooxygenase-1 and -2 (COX-1/-2)). With compound 7, 2-(4-phenyl)thiazol-2-ylamino)phenol (ST-1355), a multi-target ligand targeting all tested enzymes is presented, whereas compound 9, 2-(4-(4-chlorophenyl)thiazol-2-ylamino)phenol (ST-1705), represents a potent and selective 5-LO and COX-2 inhibitor with an IC50 value of 0.9 ± 0.2 µM (5-LO) and a residual activity of 9.1 ± 1.1% at 10 µM (COX-2 product formation). The promising characteristics and the additional non-cytotoxic profile of both compounds reveal new lead structures for the treatment of eicosanoid-mediated diseases.