Zsofia Kutil

Natural compound cudraflavone B shows promising anti-inflammatory properties in vitro.

Cudraflavone B (1) is a prenylated flavonoid found in large amounts in the roots of Morus alba, a plant used as a herbal remedy for its reputed anti-inflammatory properties. The present study shows that this compound causes a significant inhibition of inflammatory mediators in selected in vitro models. Thus, 1 was identified as a potent inhibitor of tumor necrosis factor α (TNFα) gene expression and secretion by blocking the translocation of nuclear factor κB (NF-κB) from the cytoplasm to the nucleus in macrophages derived from a THP-1 human monocyte cell line. The NF-κB activity reduction resulted in the inhibition of cyclooxygenase 2 (COX-2) gene expression. Compound 1 acts as a COX-2 and COX-1 inhibitor with higher selectivity toward COX-2 than indomethacin. Pretreatment of cells by 1 shifted the peak in an regulatory gene zinc-finger protein 36 (ZFP36) expression assay. This natural product has noticeable anti-inflammatory properties, suggesting that 1 potentially could be used for development as a nonsteroidal anti-inflammatory drug lead.

Impact of Wines and Wine Constituents on Cyclooxygenase-1, Cyclooxygenase-2, and 5-Lipoxygenase Catalytic Activity

Cyclooxygenases and lipoxygenases are proinflammatory enzymes; the former affects platelet aggregation, vasoconstriction, vasodilatation and later the development of atherosclerosis. Red wines from Georgia and central and western Europe inhibited cyclooxygenase-1 (COX-1) activity in the range of 63–94%, cyclooxygenase-2 (COX-2) activity in the range of 20–44% (tested at a concentration of 5 mL/L), and 5-lipoxygenase (5-LOX) activity in the range of 72–84% (at a concentration of 18.87 mL/L). White wines inhibited 5-LOX in the range of 41–68% at a concentration of 18.87 mL/L and did not inhibit COX-1 and COX-2. Piceatannol (IC50 = 0.76 μM) was identified as a strong inhibitor of 5-LOX followed by luteolin (IC50 = 2.25 μM), quercetin (IC50 = 3.29 μM), and myricetin (IC50 = 4.02 μM). trans-Resveratrol was identified as an inhibitor of COX-1 (IC50 = 2.27 μM) and COX-2 (IC50 = 3.40 μM). Red wine as a complex mixture is a powerful inhibitor of COX-1, COX-2, and 5-LOX, the enzymes involved in eicosanoid biosynthetic pathway.

Redox and non-redox mechanism of in vitro cyclooxygenase inhibition by natural quinones.

In this study, ten anthra-, nine naphtho-, and five benzoquinone compounds of natural origin and five synthetic naphthoquinones were assessed, using an enzymatic in vitro assay, for their potential to inhibit cyclooxygenase-1 and -2 (COX-1 and COX-2), the key enzymes of the arachidonic acid cascade. IC₅₀ values comparable with COX reference inhibitor indomethacin were recorded for several quinones (primin, alkannin, diospyrin, juglone, 7-methyljuglone, and shikonin). For some of the compounds, we suggest the redox potential of quinones as the mechanism responsible for in vitro COX inhibition because of the quantitative correlation with their pro-oxidant effect. Structure-relationship activity studies revealed that the substitutions at positions 2 and 5 play the key roles in the COX inhibitory and pro-oxidant actions of naphthoquinones. In contrast, the redox mechanism alone could not explain the activity of primin, embelin, alkannin, and diospyrin. For these four quinones, molecular modeling suggested similar binding modes as for conventional nonsteroidal anti-inflammatory drugs (NSAIDs).

Prospective performance evaluation of selected common virtual screening tools. Case study: Cyclooxygenase (COX) 1 and 2.

Computational methods can be applied in drug development for the identification of novel lead candidates, but also for the prediction of pharmacokinetic properties and potential adverse effects, thereby aiding to prioritize and identify the most promising compounds. In principle, several techniques are available for this purpose, however, which one is the most suitable for a specific research objective still requires further investigation. Within this study, the performance of several programs, representing common virtual screening methods, was compared in a prospective manner. First, we selected top-ranked virtual screening hits from the three methods pharmacophore modeling, shape-based modeling, and docking. For comparison, these hits were then additionally predicted by external pharmacophore- and 2D similarity-based bioactivity profiling tools. Subsequently, the biological activities of the selected hits were assessed in vitro, which allowed for evaluating and comparing the prospective performance of the applied tools. Although all methods performed well, considerable differences were observed concerning hit rates, true positive and true negative hits, and hitlist composition. Our results suggest that a rational selection of the applied method represents a powerful strategy to maximize the success of a research project, tightly linked to its aims. We employed cyclooxygenase as application example, however, the focus of this study lied on highlighting the differences in the virtual screening tool performances and not in the identification of novel COX-inhibitors.

Pharmacophore modeling for COX-1 and -2 inhibitors with LigandScout in comparison to Discovery Studio.

BACKGROUND Pharmacophore modeling has become an integrated tool in drug discovery. However, no prospective study compares the performance of the available software. METHODS The two widely used pharmacophore modeling and screening software programs Discovery Studio and LigandScout were used to generate, validate, and prospectively apply COX-1 and -2 models. Selected virtual hits were tested in cell-free enzymatic assays. The correct retrieval of active compounds was compared. RESULTS In the enzymatic testing, 10.5% of the tested hits for COX-2 and 6.6% of the predicted compounds for COX-1 were active. To directly compare the two models, both based on the same PDB entry, were selected for virtual screening. The two programs yielded vastly different hit lists, but both predicted active compounds. CONCLUSION To obtain a comprehensive selection of active compounds, more than one program should be used for modeling.

Histone Deacetylase 11 Is a Fatty-Acid Deacylase

Histone deacetylase 11 (HDAC11) is a sole member of the class IV HDAC subfamily with negligible intrinsic deacetylation activity. Here, we report in vitro profiling of HDAC11 deacylase activities, and our data unequivocally show that the enzyme efficiently removes acyl moieties spanning 8-18 carbons from the side chain nitrogen of the lysine residue of a peptidic substrate. Additionally, N-linked lipoic acid and biotin are removed by the enzyme, although with lower efficacy. Catalytic efficiencies toward dodecanoylated and myristoylated peptides were 77 700 and 149 000 M-1 s-1, respectively, making HDAC11 the most proficient fatty-acid deacylase of the HDAC family. Interestingly, HDAC11 is strongly inhibited by free myristic, palmitic, and stearic acids with inhibition constants of 6.5, 0.9, and 1.6 μM, respectively. At the same time, its deacylase activity is stimulated more than 2.5-fold by both palmitoyl-coenzyme A and myristoyl-coenzyme A, pointing toward metabolic control of the enzymatic activity by fatty-acid metabolites. Our data reveal novel enzymatic activity of HDAC11 that can, in turn, facilitate the uncovering of additional biological functions of the enzyme as well as the design of isoform-specific HDAC inhibitors.

Discovery of the first dual inhibitor of the 5-lipoxygenase-activating protein and soluble epoxide hydrolase using pharmacophore-based virtual screening

Leukotrienes (LTs) are pro-inflammatory lipid mediators derived from arachidonic acid (AA) with roles in inflammatory and allergic diseases. The biosynthesis of LTs is initiated by transfer of AA via the 5-lipoxygenase-activating protein (FLAP) to 5-lipoxygenase (5-LO). FLAP inhibition abolishes LT formation exerting anti-inflammatory effects. The soluble epoxide hydrolase (sEH) converts AA-derived anti-inflammatory epoxyeicosatrienoic acids (EETs) to dihydroxyeicosatetraenoic acids (di-HETEs). Its inhibition consequently also counteracts inflammation. Targeting both LT biosynthesis and the conversion of EETs with a dual inhibitor of FLAP and sEH may represent a novel, powerful anti-inflammatory strategy. We present a pharmacophore-based virtual screening campaign that led to 20 hit compounds of which 4 targeted FLAP and 4 were sEH inhibitors. Among them, the first dual inhibitor for sEH and FLAP was identified, N-[4-(benzothiazol-2-ylmethoxy)-2-methylphenyl]-N’-(3,4-dichlorophenyl)urea with IC50 values of 200 nM in a cell-based FLAP test system and 20 nM for sEH activity in a cell-free assay.

Human histone deacetylase 6 shows strong preference for tubulin dimers over assembled microtubules

Human histone deacetylase 6 (HDAC6) is the major deacetylase responsible for removing the acetyl group from Lys40 of α-tubulin (αK40), which is located lumenally in polymerized microtubules. Here, we provide a detailed kinetic analysis of tubulin deacetylation and HDAC6/microtubule interactions using individual purified components. Our data unequivocally show that free tubulin dimers represent the preferred HDAC6 substrate, with a KM value of 0.23 µM and a deacetylation rate over 1,500-fold higher than that of assembled microtubules. We attribute the lower deacetylation rate of microtubules to both longitudinal and lateral lattice interactions within tubulin polymers. Using TIRF microscopy, we directly visualized stochastic binding of HDAC6 to assembled microtubules without any detectable preferential binding to microtubule tips. Likewise, indirect immunofluorescence microscopy revealed that microtubule deacetylation by HDAC6 is carried out stochastically along the whole microtubule length, rather than from the open extremities. Our data thus complement prior studies on tubulin acetylation and further strengthen the rationale for the correlation between tubulin acetylation and microtubule age.

Myrica rubra leaves as a potential source of a dual 5-LOX/COX inhibitor

ABSTRACT Myrica rubra Sieb. et Zucc. is a valuable fruit tree that is used in Chinese, Japanese and Taiwanese traditional medicine. We investigated the anti-inflammatory activity of M. rubra leaves extracted with four different solvents. Total phenolics were determined using the Folin–Ciocalteu method. Extracts were investigated for their inhibitory activity toward the pro-inflammatory enzymes cyclooxygenase-1 and -2 (COX-1, COX-2) and 5-lipoxygenase (5-LOX). The ethanol extract of M. rubra leaves demonstrated a strong inhibition of prostaglandin E2 (PGE2) biosynthesis catalyzed by both COX-1 (93.42%) and COX-2 (75.71%) and leukotriene B4 (LTB4) formation catalyzed by 5-LOX (82.72%). Further we identified selective COX-1 inhibition by the n-butanol and aqueous fractions of the ethanol extract (with an IC50 for COX-1 inhibition of 1.07 and 0.71 µg mL−1 , respectively) and dual 5-LOX/COX inhibition by the ethyl acetate fraction (with an IC50 of 3.29 for COX-1, 2.54 for COX-2 and 8.30 µg mL−1 for 5-LOX).

Effect of Dietary Stilbenes on 5-Lipoxygenase and Cyclooxygenases Activities In Vitro

Stilbenes contained in various foods are associated with health beneficial effects. In this study six natural and one synthetic stilbene were tested for their potential to regulate the activity of lipoxygenase and cyclooxygenase in vitro. The most potent inhibitor of 5-lipoxygenase was pterostilbene (IC50 = 9.32 μM), whereas the strongest inhibitor of cyclooxygenase-1 and cyclooxygenase-2 was pinostilbene (IC50s = 1.90 and 0.35 μM, respectively). Pterostilbene (IC50s = 11.70 and 27.04 μM for cyclooxygenase-1 and cyclooxygenase-2, respectively) and oxyresveratrol (IC50s = 18.49; 2.79 and 14.71 μM for 5-lipoxygenase, cyclooxygenase-1, and cyclooxygenase-2, respectively) were capable to inhibit catalytic activity of all three tested enzymes. Isorhapontigenin (IC50s = 8.81 and 24.00 μM for cyclooxygenase-1 and cyclooxygenase-2, respectively) and rhapontigenin (IC50s = 24.55 and 36.12 μM for cyclooxygenase-1 and cyclooxygenase-2, respectively) were only moderate or weak inhibitors of both cyclooxygenase forms. In summary, these results indicated that besides the known cyclooxygenase inhibitor resveratrol also other natural stilbenes could be potent inhibitors of the arachidonic acid pathway and deserve further attention as compounds with promising health benefits.