shinorine

Abstract

Mycosporine-like amino acids (MAAs) are secondary metabolites, produced by a large variety of microorganisms including algae, cyanobacteria, lichen and fungi. MAAs act as UV-absorbers and photo-protectants. MAAs are suggested to exert pharmaceutical relevant bioactivities in the human system. We particularly focused on their effect on defence and regulatory pathways that are active in inflamed environments. The MAAs shinorine and porphyra-334 were isolated and purified from the red algae Porphyra sp. using chromatographic methods. The effect of MAAs on central signaling cascades, such as transcription factor nuclear factor kappa b (NF-κB) activation, as well as tryptophan metabolism, was investigated in human myelomonocytic THP-1 and THP-1-Blue cells. Cells were exposed to the MAAs in the presence or absence of lipopolysaccharide (LPS). NF-κB activity and the activity of tryptophan degrading enzyme indoleamine 2,3-dioxygenase (IDO-1) were used as readout. Compounds were tested in the concentration range from 12.5 to 200 µg/mL. Both MAAs were able to induce NF-κB activity in unstimulated THP-1-Blue cells, whereby the increase was dose-dependent and more pronounced with shinorine treatment. While shinorine also slightly superinduced NF-κB in LPS-stimulated cells, porphyra-334 reduced NF-κB activity in this inflammatory background. Modulation of tryptophan metabolism was moderate, suppressive in stimulated cells with the lower treatment concentration of both MAAs and with the unstimulated cells upon porphyra-334 treatment. Inflammatory pathways are affected by MAAs, but despite the structural similarity, diverse effects were observed.

2.1. Activation Degree of the Central Inflammation Marker NF-κB

Compared to control cells, treatment with shinorine in unstimulated cells activated NF-κB up to 33.63% at the highest treatment concentration (200 µg/mL). In addition, lower concentrations 12.5–100 µg/mL showed a dose-dependent and significant increase in NF-κB activity, starting from 4.78% upwards (p < 0.05). Treatment with shinorine in LPS-stimulated cells resulted in a slight but significant superinduction of NF-κB activity compared to LPS treatment alone, e.g., 100 and 200 µg/mL MAA treatment resulted in 6.01% and 10.16% activation compared to LPS-stimulated control cells (A).

2.3. Effects of Shinorine and Porphyra-334 on Tryptophan Metabolism

Treatment of LPS-stimulated cells with both MAAs at lower concentrations decreased Kyn/Trp levels slightly, but significantly. Shinorine decreased the Kyn/Trp at 12.5, 25 and 100 µg/mL treatment compared to LPS-stimulated control samples, with a maximum of 23.1% suppression. Shinorine treatment did not affect unstimulated cells (A).

ORAC antioxidant assay

The oxygen radical absorbance capacity for each MAA was carried out using the ORAC Antioxidant Assay Kit (Zenbio, North Carolina, USA) according to manufacturer's instructions. Trolox standards were prepared in the assay buffer (0–100 μM) along with serial dilutions of each MAA (500-0 μM) and an ascorbic acid positive control (0–100 μM). 150 μl of the fluorescein working solution was added to central wells of a 96 well plate, with 25 μl of each of the standards or MAA in duplicate, and the plate incubated at 37 °C for at least 15 min. The 2,2′-azobis(2-amidinopropane) dihydrochloride [AAPH; sold as 2,2′-azobis(2-methylpropanimidamine) dihydrochloride] working solution was added to each well (25 μl) to initiate the reaction. Fluorescence was measured in a preheated incubation chamber (37 °C) using a Spectra Max 384 Plus spectrophotometer with excitation/emission = 485/530 nm immediately (t = 0) and then every minute for 30 min. Standard curves were generated for each compound and the area under the curve calculated. Each MAA tested was expressed as a Trolox equivalent concentration.

MAAs Porphyra-334 and shinorine compete with Keap1-Nrf2 interaction in vitro

The MAAs porphyra-334 and shinorine together with eight selected antioxidants (ascorbic acid, tBHQ, caffeic acid, chlorogenic acid, curcumin, quercetin, EGCG, trans-resveratrol) plus the known electrophilic Nrf2 activator sulforaphane were evaluated for their interaction with the Kelch-repeat domain of the Keap1 protein using both in vitro FP and thermal shift assays. In the FP assay, the test compounds competed with a fluorescein labelled peptide (FITC-[β-Ala]-DEETGEF-OH) based on the high affinity ETGE motif that binds Nrf2 to the Kelch-repeat domain (). The protein-protein interaction of the FITC-[β-Ala]-DEETGEF-OH peptide had a KD of 96 nM in the presence of the Kelch-repeat domain protein and the unlabelled Ac-DEETGEF-OH peptide competed with this interaction with an IC50 of 5.4 μM []. Both porphyra-334 and shinorine gave nearly equivalent ligand-receptor estimated IC50 values of ∼100 μM, requiring a much greater concentration in the presence of excess Kelch-repeat domain protein than the binding of the native peptide sequence []. In contrast, no significant interactions were detected between the eight known antioxidants or sulforaphane and the Kelch-repeat domain protein.Fig. 3Measurement of specific, non-reactive binding of MAAs to the Kelch-repeat domain of Keap1. Ligand-receptor binding was measured using a fluorescence polarization assay in comparison to a high affinity, labelled synthetic peptide. The MAAs porphyra-334 and shinorine gave moderate IC50 values of ∼100 μM, whereas no significant interactions were detected between the Keap1 Kelch-repeat domain and any of the eight selected antioxidants or known electrophilic Nrf2 activator sulforaphane. The figure shows results for 100 μM concentration of MAAs and known antioxidants. Error bars represent one standard deviation of the mean, based on 3 experimental replicates, and statistically significant results (based on p-value of two sided t-test ≤ 0.05 adjusted for multiple testing based on Bonferroni correction) are marked with an asterisk.Fig. 3

MAAs Porphyra-334 and shinorine compete with Keap1-Nrf2 interaction in vitro

The MAAs and eight selected antioxidants were then tested as ligands of the Kelch-repeat domain protein using the thermal shift (differential scanning fluorimetry, DSF) assay. The assays involved treatment of the Kelch-repeat domain protein with the test compounds of interest, followed by heating of the samples to denature the protein. Ligands that bind and stabilise the Kelch-repeat domain protein result in higher denaturation (Tm) temperatures for the complex compared to the free protein. An unlabelled version of the peptide (Ac-DEETGEF-OH) used in the FP assay was used as the positive control in the thermal shift assays against which binding of the test compounds to the Kelch-repeat domain were compared. When used at a 50 μM concentration, the Ac-DEETGEF-OH peptide had a ΔTm relative to the unbound protein of 3.91 ± 0.04 °C. Both porphyra-334 and shinorine also demonstrated elevated ΔTm values to the Kelch-repeat domain protein, albeit when used at 100 μM (porphyra-334 ΔTm = 0.93 ± 0.02 °C, shinorine ΔTm = 0.64 ± 0.11 °C). However, no significant protein-ligand interactions were detectable when the eight antioxidants or the electrophilic Nrf2 activator sulforaphane were tested. The thermal shift assays reflected the results of the FP assays for binding of MAAs and test substrates to the Kelch-repeat domain protein of Keap1.

Porphya-334 and shinorine are in-vitro antioxidants

Scavenging of the DPPH free radical is a common assay used to assess the antioxidant activity of a compound. Ascorbic acid is a well-known DPPH free-radical scavenger and is commonly used as a positive control, which was used also for comparison in this study. The IC50 value for ascorbic acid was 24.5 ± 1.1 μM in the methanol reaction medium. The free radical scavenging activities of MAAs were markedly lower compared to that of ascorbic acid. The IC50 values were: 185.2 ± 3.2 μM for porphyra-334 (equivalent to 13.23% of the activity of ascorbic acid) and 399.0 ± 1.1 μM for shinorine (equivalent to 6.14% of the activity of ascorbic acid).