Ann-Kathrin Häfner

Lipoxin and resolvin biosynthesis is dependent on 5-lipoxygenase activating protein

Resolution of acute inflammation is an active process coordinated by proresolving lipid mediators (SPMs) such as lipoxins (LXs) and resolvins (Rvs), which are formed by the concerted action of 2 lipoxygenases (LOs). Because the exact molecular mechanisms of SPM biosynthesis are not completely understood, we aimed to investigate LX and D‐type Rv formation in human leukocytes and HEK293T cells overexpressing leukotriene (LT) pathway enzymes. Activity assays in precursor (15‐hydroxyeicosatetraenoic acids, 17‐HDoHE)‐treated granulocytes [polymorphonuclear leukocytes (PMNLs)] showed a strict dependence of LXA4/RvD1 biosynthesis on cell integrity, and incubation with recombinant human 5‐LO did not lead to LX or Rv formation. Pharmacologic inhibition of 5‐LO activating protein (FLAP) by MK‐886 inhibited LXA4/RvD1 biosynthesis in precursor‐treated PMNLs (drug concentration causing 50% inhibition ∼0.3/0.2 μM), as did knockdown of the enzyme in MM6 cells, and precursor‐treated HEK293T overexpressing 5‐LO produced high amounts of LXA4 only in the presence of FLAP. In addition, inhibition of cytosolic phospholipase A2α (cPLA2α) interfered with LXA4/RvD1 formation from exogenous precursors in PMNLs. Furthermore, inhibition of the LT synthases LTA4 hydrolase and LTC4 synthase in PMNL/platelet coincubations augmented LXA4 levels. These findings show that several enzymes known to be involved in the biosynthesis of proinflammatory LTs, such as FLAP and cPLA2α, also contribute to LX and Rv formation.—Lehmann, C., Homann, J., Ball, A.‐K., Blöcher, R., Kleinschmidt, T. K., Basavarajappa, D., Angioni, C., Ferreirós, N., Häfner, A.‐K., Rådmark, O., Proschak, E., Haeggström, J. Z., Geisslinger, G., Parnham, M. J., Steinhilber, D., Kahnt, A. S. Lipoxin and resolvin biosynthesis is dependent on 5‐lipoxygenase activating protein. FASEB J. 29, 5029–5043 (2015). www.fasebj.org

Dimerization of human 5-lipoxygenase

Abstract Human 5-lipoxygenase (5-LO) can form dimers as shown here via native gel electrophoresis, gel filtration chromatography and LILBID (laser induced liquid bead ion desorption) mass spectrometry. After glutathionylation of 5-LO by diamide/glutathione treatment, dimeric 5-LO was no longer detectable and 5-LO almost exclusively exists in the monomeric form which showed full catalytic activity. Incubation of 5-LO with diamide alone led to a disulfide-bridged dimer and to oligomer formation which displays a strongly reduced catalytic activity. The bioinformatic analysis of the 5-LO surface for putative protein-protein interaction domains and molecular modeling of the dimer interface suggests a head to tail orientation of the dimer which also explains the localization of previously reported ATP binding sites. This interface domain was confirmed by the observation that 5-LO dimer formation and inhibition of activity by diamide was largely prevented when four cysteines (C159S, C300S, C416S, C418S) in this domain were mutated to serines.

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.

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).

Electrophilic Fatty Acid Species Inhibit 5-Lipoxygenase and Attenuate Sepsis-Induced Pulmonary Inflammation

AIMS The reaction of nitric oxide and nitrite-derived species with polyunsaturated fatty acids yields electrophilic fatty acid nitroalkene derivatives (NO2-FA), which display anti-inflammatory properties. Given that the 5-lipoxygenase (5-LO, ALOX5) possesses critical nucleophilic amino acids, which are potentially sensitive to electrophilic modifications, we determined the consequences of NO2-FA on 5-LO activity in vitro and on 5-LO-mediated inflammation in vivo. RESULTS Stimulation of human polymorphonuclear leukocytes (PMNL) with nitro-oleic (NO2-OA) or nitro-linoleic acid (NO2-LA) (but not the parent lipids) resulted in the concentration-dependent and irreversible inhibition of 5-LO activity. Similar effects were observed in cell lysates and using the recombinant human protein, indicating a direct reaction with 5-LO. NO2-FAs did not affect the activity of the platelet-type 12-LO (ALOX12) or 15-LO-1 (ALOX15) in intact cells or the recombinant protein. The NO2-FA-induced inhibition of 5-LO was attributed to the alkylation of Cys418, and the exchange of Cys418 to serine rendered 5-LO insensitive to NO2-FA. In vivo, the systemic administration of NO2-OA to mice decreased neutrophil and monocyte mobilization in response to lipopolysaccharide (LPS), attenuated the formation of the 5-LO product 5-hydroxyeicosatetraenoic acid (5-HETE), and inhibited lung injury. The administration of NO2-OA to 5-LO knockout mice had no effect on LPS-induced neutrophil or monocyte mobilization as well as on lung injury. INNOVATION Prophylactic administration of NO2-OA to septic mice inhibits inflammation and promotes its resolution by interfering in 5-LO-mediated inflammatory processes. CONCLUSION NO2-FAs directly and irreversibly inhibit 5-LO and attenuate downstream acute inflammatory responses.

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.

Stabilisation and characterisation of the isolated regulatory domain of human 5-lipoxygenase.

5-Lipoxygenase (5-LOX) is the key player of pro-inflammatory leukotriene biosynthesis. Its regulatory or so-called PLAT (polycystin-1, lipoxygenase, α-toxin) domain binds allosteric modulators like calcium, membranes, coactosin-like protein and Dicer, thereby influencing 5-LOX activity at the nuclear membrane by mediating translocation. The PLAT domain may also regulate cytosolic 5-LOX activity and possibly influence microRNA metabolism. Hence, it has also evolved as a promising target for anti-inflammatory therapy. Research focusing on this substructure of 5-LOX requires an assay system based on the isolated domain. However, we found that the isolated PLAT domain was highly prone to aggregation and therefore unsuitable for interaction studies. Substitution of the single, membrane-binding tryptophan 75 with glycine reduced aggregation and substantially increased its thermal stability. Calcium interaction of the single mutant was confirmed by differential scanning fluorimetry. Moreover, crosslinking experiments demonstrated the ability of the isolated PLAT domain to bind Dicer C-terminus whereas the interaction with coactosin-like protein required the interplay of the catalytic and the PLAT domain.

Characterization of the interaction of human 5-lipoxygenase with its activating protein FLAP

Human 5-lipoxygenase (5-LO) is the key enzyme in the formation of leukotrienes (LTs), important mediators of inflammation. Cellular 5-LO activity is regulated in a complex manner, e.g. by calcium influx, the cellular redox status or 5-LO phosphorylation. Being a mobile enzyme, 5-LO migrates from the cytosol to the nuclear envelope where it is believed to interact with 5-lipoxygenase-activating protein (FLAP) and receives the substrate arachidonic acid (AA). 5-LO contains four cysteine residues located close to the AA entry site. In the present study, we show that in vitro glutathionylation of recombinant purified 5-LO wildtype (WT) as well as 5-LO 4C, a mutant where the four surface cysteines are replaced by serines (Cys159/300/416/418Ser), does not alter the product synthesis. However, in 5-LO/FLAP-transfected HeLa cells, treatment with the thiol-oxidizing agent diamide which promotes glutathionylation at surface Cys residues led to a decreased LT synthesis by 5-LO WT. In contrast to the WT enzyme, LT formation of the 4C mutant was stimulated by addition of diamide. Immunofluorescence studies in human monocytes and HEK293 cells, expressing 5-LO and FLAP, revealed that diamide prevented the translocation of 5-LO WT whereas it enhanced the translocation of the fourfold cysteine mutant. Therefore, we could demonstrate that the interface, involving the four cysteines 159, 300, 416 and 418, is important for the translocation to the nuclear membrane and the colocalization with FLAP.

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.

Identification and Characterization of a New Protein Isoform of Human 5-Lipoxygenase.

Leukotrienes (LTs) are inflammatory mediators that play a pivotal role in many diseases like asthma bronchiale, atherosclerosis and in various types of cancer. The key enzyme for generation of LTs is the 5-lipoxygenase (5-LO). Here, we present a novel putative protein isoform of human 5-LO that lacks exon 4, termed 5-LOΔ4, identified in cells of lymphoid origin, namely the Burkitt lymphoma cell lines Raji and BL41 as well as primary B and T cells. Deletion of exon 4 does not shift the reading frame and therefore the mRNA is not subjected to non-mediated mRNA decay (NMD). By eliminating exon 4, the amino acids Trp144 until Ala184 are omitted in the corresponding protein. Transfection of HEK293T cells with a 5-LOΔ4 expression plasmid led to expression of the corresponding protein which suggests that the 5-LOΔ4 isoform is a stable protein in eukaryotic cells. We were also able to obtain soluble protein after expression in E. coli and purification. The isoform itself lacks canonical enzymatic activity as it misses the non-heme iron but it still retains ATP-binding affinity. Differential scanning fluorimetric analysis shows two transitions, corresponding to the two domains of 5-LO. Whilst the catalytic domain of 5-LO WT is destabilized by calcium, addition of calcium has no influence on the catalytic domain of 5-LOΔ4. Furthermore, we investigated the influence of 5-LOΔ4 on the activity of 5-LO WT and proved that it stimulates 5-LO product formation at low protein concentrations. Therefore regulation of 5-LO by its isoform 5-LOΔ4 might represent a novel mechanism of controlling the biosynthesis of lipid mediators.