Inhibition of 5-lipoxygenase by U73122 is due to covalent binding to cysteine 416.

U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.

Thiazole derivatives as dual inhibitors of deoxyribonuclease I and 5-lipoxygenase: A promising scaffold for the development of neuroprotective drugs.

A library of 43 thiazole derivatives, including 31 previously and 12 newly synthesized in the present study, was evaluated in vitro for their inhibitory properties against bovine pancreatic DNase I. Nine compounds (including three newly synthesized) inhibited the enzyme showing improved inhibitory properties compared to that of the reference crystal violet (IC50 = 346.39 µM). Two compounds (5 and 29) stood out as the most potent DNase I inhibitors, with IC50 values below 100 µM. The 5-LO inhibitory properties of the investigated derivatives were also analyzed due to the importance of this enzyme in the development of neurodegenerative diseases. Compounds (12 and 29) proved to be the most prominent new 5-LO inhibitors, with IC50 values of 60 nM and 56 nM, respectively, in cell-free assay. Four compounds, including one previously (41) and three newly (12, 29 and 30) synthesized, have the ability to inhibit DNase I with IC50 values below 200 µM and 5-LO with IC50 values below 150 nM in cell-free assay. Molecular docking and molecular dynamics simulations were used to clarify DNase I and 5-LO inhibitory properties of the most potent representatives at the molecular level. The newly synthesized compound 29 (4-((4-(3-bromo-4-morpholinophenyl)thiazol-2-yl)amino)phenol) represents the most promising dual DNase I and 5-LO inhibitor, as it inhibited 5-LO in the nanomolar and DNase I in the double-digit micromolar concentration ranges. The results obtained in the present study, together with our recently published results for 4-(4-chlorophenyl)thiazol-2-amines, represent a good basis for the development of new neuroprotective therapeutics based on dual inhibition of DNase I and 5-LO.

SAR-study on a new class of imidazo[1,2-a]pyridine-based inhibitors of 5-lipoxygenase.

A novel class of 5-lipoxygenase (5-LO) inhibitors characterized by a central imidazo[1,2-a]pyridine scaffold, a cyclohexyl moiety and an aromatic system, is presented. This scaffold was identified in a virtual screening study and exhibits promising inhibitory potential on the 5-LO. Here, we investigate the structure-activity relationships of this compound class. With N-cyclohexyl-6-methyl-2-(4-morpholinophenyl)imidazo[1,2-a]pyridine-3-amine (14), we identified a potent 5-LO inhibitor (IC(50)=0.16µM (intact cells) and 0.1µM (cell-free)), which may possess potential as an effective lead compound intervening with inflammatory diseases and certain types of cancer.

Synthesis and biological evaluation of a class of 5-benzylidene-2-phenyl-thiazolinones as potent 5-lipoxygenase inhibitors.

A class of 5-lipoxygenase (5-LO) inhibitors characterized by a central 5-benzylidene-2-phenyl-thiazolinone scaffold was synthesized as a new series of molecular modifications and extensions of a previously reported series. Compounds were tested in a cell-based and a cell-free assay and furthermore evaluated for their influence on cell viability. The presented substituted thiazolinone scaffold turned out to be essential for both the 5-LO inhibitory activity and the non-cytotoxic profile. With (Z)-5-(4-methoxybenzylidene)-2-(naphthalen-2-yl)-5H-thiazol-4-one (2k, ST1237), a potent, direct, non-cytotoxic 5-LO inhibitor with IC(50) of 0.08 µM and 0.12 µM (cell-free assay and intact cells), we present a promising lead optimization and development for further investigations as novel anti-inflammatory drug.

Development of novel aminothiazole-comprising 5-LO inhibitors.

BACKGROUND: Leukotrienes are pivotal lipid mediators in various immune and inflammatory reactions. Herein, 5-LO is a validated target. 2-Aminothiazoles, as a privileged structure, implicate known 5-LO inhibitors like ST-1083 (IC50 [polymorphonuclear leukocytes (PMNL)] = 0.68 µM), yet deep structure-activity relationships (SAR) have not been established. MATERIALS & METHODS: Compounds were synthesized via Hantzsch thiazole synthesis. Inhibitory activities were evaluated using intact PMNL and purified 5-LO together with cytotoxicity measurements in U937 cells. RESULTS: We introduced novel functionalities at 2-, 3-, 4- and 5-position of the 2-aminothiazole scaffold and conducted bioisosteric replacement to optimize the parent scaffold. SARs of the 2-aminothiazole scaffold were deduced and extended primarily for inhibition of the 5-LO enzyme. CONCLUSION: SAR studies provided at least two optimized leads (ST-1853, ST-1906) with high potency (IC50 [polymorphonuclear leukocytes] = 0.05 µM), specificity and noncytotoxic behavior.

Development and evaluation of ST-1829 based on 5-benzylidene-2-phenylthiazolones as promising agent for anti-leukotriene therapy.

Different inflammatory diseases and allergic reactions are mediated by leukotrienes, which arise from the oxygenation of arachidonic acid catalyzed by 5-lipoxygenase (5-LO). One promising approach for an effective anti-leukotriene therapy is the inhibition of this key enzyme. This study presents the synthesis and development of a potent and direct 5-LO inhibitor based on the well characterized 5-benzylidene-2-phenylthiazolone C06, whose further pharmacological investigation was precluded due to its low solubility. Through optimization of C06, evaluation of structure-activity relationships including profound assessment of the thiazolone core and consideration of the solubility, the 5-benzyl-2-phenyl-4-hydroxythiazoles represented by 46 (ST-1829, 5-(4-chlorobenzyl)-2-p-tolylthiazol-4-ol) were developed. Compound 46 showed an improved 5-LO inhibitory activity in cell-based (IC50 values 0.14 µM) and cell-free assays (IC50 values 0.03 µM) as well as a prominent enhanced solubility. Furthermore, it kept its promising inhibitory potency in the presence of blood serum, excluding excessive binding to serum proteins. These facts combined with the non-cytotoxic profile mark a major step towards an effective anti-inflammatory therapy.

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.

Structure-activity relationship and in vitro pharmacological evaluation of imidazo[1,2-a]pyridine-based inhibitors of 5-LO.

BACKGROUND: 5-LO is an important enzyme involved in the biosynthesis of leukotrienes, which are lipid mediators of immune and inflammation responses, with important roles in respiratory disease, cardiovascular disease, immune responses and certain types of cancer. Therefore, this enzyme has been investigated as a potential target for the treatment of these pathophysiological conditions. RESULTS: 5-LO inhibitory potential was investigated in intact polymorphonuclear leukocytes, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and in vitro pharmacological evaluation. Chemical modifications for lead optimization via straight forward synthesis was used to combine small polar groups, which led to a suitable candidate (IC50 [polymorphonuclear leukocytes] = 1.15 µM, IC50 [S100] = 0.29 µM) with desired in vitro biopharmaceutical profiles in terms of solubility (451.9 µg/ml) and intrinsic clearance without demonstrating any cytotoxicity. CONCLUSION: Compound 9l is a novel, potent and selective 5-LO inhibitor with favorable preclinical drug-like properties.

Exploring the chemical space of multitarget ligands using aligned self-organizing maps.

Design of multitarget drugs and polypharmacological compounds has become popular during the past decade. However, the main approach to design such compounds is to link two selective ligands via a flexible linker. Although such chimeric ligands often have reasonable potency in vitro, the in vivo efficacy is low due to high molecular weight, low ligand efficiency, and poor pharmacokinetic profile. We developed an unprecedented in silico approach for fragment-based design of multitarget ligands. It relies on superposition of the chemical spaces related to the affinity on single targets represented by self-organizing maps. We used this approach for screening of molecular fragments, which bind to the enzymes 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH). Using STD-NMR and activity-based assays, we were able to identify fragments binding to both targets. Furthermore, we were able to expand one of the fragments to a potent dual inhibitor bearing a reasonable molecular weight (MW = 446) and high affinity to both targets (IC50 of 0.03 µM toward 5-LO and 0.17 µM toward sEH).

Synthesis and structure-activity relationship studies of novel dual inhibitors of soluble epoxide hydrolase and 5-lipoxygenase.

Current research leads to the assumption that drugs affecting more than one target could result in a more efficient treatment of diseases and fewer safety concerns. Administration of drugs inhibiting only one branch of the arachidonic acid cascade is usually accompanied by side effects. We therefore designed and synthesized a library of hybrid molecules incorporating an imidazo[1,2-a]pyridine and an urea moiety as novel soluble epoxide hydrolase (sEH)/5-lipoxygenase (5-LO) dual inhibitors. Evaluation of the compounds was accomplished by in vitro testing using recombinant enzyme assays.

Molecular characterization of EP6--a novel imidazo[1,2-a]pyridine based direct 5-lipoxygenase inhibitor.

5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic acid (AA) cascade and catalyzes the formation of bioactive leukotrienes (LTs) which are involved in inflammatory diseases and allergic reactions. The pathophysiological effects of LTs are considered to be prevented by 5-LO inhibitors. In this study we present cyclohexyl-[6-methyl-2-(4-morpholin-4-yl-phenyl)-imidazo[1,2-a]pyridin-3-yl]-amine (EP6), a novel imidazo[1,2-a]pyridine based compound and its characterization in several in vitro assays. EP6 suppresses 5-LO activity in intact polymorphonuclear leukocytes with an IC(50) value of 0.16µM and exhibits full inhibitory potency in cell free assays (IC(50) value of 0.05µM for purified 5-LO). The efficacy of EP6 was not affected by the redox tone or the concentration of exogenous AA, characteristic drawbacks known for the class of nonredox-type 5-LO inhibitors. Furthermore, EP6 suppressed 5-LO activity independently of the cell stimulus or the activation pathway of 5-LO contrary to what is known for some nonredox-type inhibitors. Using molecular modeling and site-directed mutagenesis studies, we were able to derive a feasible binding region within the C2-like domain of 5-LO that can serve as a new starting point for optimization and development of new 5-LO inhibitors targeting this site. EP6 has promising effects on cell viability of tumor cells without mutagenic activity. Hence the drug may possess potential for intervention with inflammatory and allergic diseases and certain types of cancer including leukemia.

A class of 5-benzylidene-2-phenylthiazolinones with high potency as direct 5-lipoxygenase inhibitors.

A novel class of potent direct 5-lipoxygenase (5-LO) inhibitors bearing a thiazolinone-scaffold identified by virtual screening is presented. A range of substitutions and the importance of the 2-phenyl moiety were evaluated. This series is characterized by high potency in intact polymorphonuclear leukocytes and a cell-free system, exemplified by (Z)-2-(4-chlorophenyl)-5-(4-methoxybenzylidene)-5H-thiazol-4-one (18, IC(50) = 0.28 and 0.09 µM). These disubstituted thiazolinones may possess potential for intervention with inflammatory and allergic diseases and certain cancer types.