New Anti-Inflammatory Metabolites by Microbial Transformation of Medrysone

Microbial transformation of the anti-inflammatory steroid medrysone (1) was carried out for the first time with the filamentous fungi Cunninghamella blakesleeana (ATCC 8688a), Neurospora crassa (ATCC 18419), and Rhizopus stolonifer (TSY 0471). The objective was to evaluate the anti-inflammatory potential of the substrate (1) and its metabolites. This yielded seven new metabolites, 14α-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (2), 6β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 15β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (4), 6β,17α-dihydroxy-6α-methylpregn-4-ene-3,11,20-trione (5), 6β,20S-dihydroxy-6α-methylpregn-4-ene-3,11-dione (6), 11β,16β-dihydroxy-6α-methylpregn-4-ene-3,11-dione (7), and 15β,20R-dihydroxy-6α-methylpregn-4-ene-3,11-dione (8). Single-crystal X-ray diffraction technique unambiguously established the structures of the metabolites 2, 4, 6, and 8. Fungal transformation of 1 yielded oxidation at the C-6β, -11β, -14α, -15β, -16β positions. Various cellular anti-inflammatory assays, including inhibition of phagocyte oxidative burst, T-cell proliferation, and cytokine were performed. Among all the tested compounds, metabolite 6 (IC50 = 30.3 μg/mL) moderately inhibited the reactive oxygen species (ROS) produced from zymosan-induced human whole blood cells. Compounds 1, 4, 5, 7, and 8 strongly inhibited the proliferation of T-cells with IC50 values between <0.2–10.4 μg/mL. Compound 7 was found to be the most potent inhibitor (IC50 < 0.2 μg/mL), whereas compounds 2, 3, and 6 showed moderate levels of inhibition (IC50 = 14.6–20.0 μg/mL). Compounds 1, and 7 also inhibited the production of pro-inflammatory cytokine TNF-α. All these compounds were found to be non-toxic to 3T3 cells (mouse fibroblast), and also showed no activity when tested against HeLa (human epithelial carcinoma), or against PC3 (prostate cancer) cancer cell lines.


Introduction
Microbial transformation is an effective tool for structural derivatizations that are difficult to achieve by conventional chemical methods.Microbial systems are also extensively employed in the study of drug metabolism and bioremediation [1,2].
Steroids are among the most widely marketed pharmaceutical products.Several steroids are used as anabolic, contraceptive, anti-androgenic, anti-inflammatory, and anti-cancer agents.Microbial hydroxylation of steroids is an efficient method for the synthesis of new hydroxysteroids with high stereo-and regio-selectivity, and for study of the steroidal metabolism [1,[3][4][5].The bioconversion of steroids was initiated in 1950.In 1952, progesterone was converted into 11α-hydroxyprogesterone, which was later used as an intermediate in the synthesis of cortisone [6].
SAINT program was used to analyze data, solved with the aid of direct methods [24], and refined with the help of SHELXTL-PC package [25].The figures were plotted by ORTEP program [26].

General fermentation and extraction protocol
Biotransformation studies were carried out by using stage II fermentation protocol [27].Stage I cultured flasks were prepared by transferring the spores from 3 day old slants, which were then incubated for 4 days on a rotary shaker (128 rpm) at 25-28°C.Aliquots (5 mL) from the stage I cultured flask were then transferred to the remaining flasks, and incubated on a rotary shaker (128 rpm) at 25-28°C.After 2 days, compound 1 was dissolved in acetone, and evenly distributed among all the flasks.Fermentation was continued and time course studies were performed after different time intervals to assess the degree of transformation.After completion of 12-14 days, the broth was filtered to separate mycelia and washed with dichloromethane.The filtrate was then extracted with the same solvent i.e. dichloromethane.The solvent was dried with anhydrous sodium sulfate (Na 2 SO 4 ), and evaporated under reduced pressure to obtain the crude extract.Two parallel control experiments were also performed as positive (media with compound only), and negative (media with fungus only) controls.

Biological activity
Oxidative burst inhibition assay.Oxidative burst assay was carried out by chemiluminescence technique using luminol as a probe to determine the effect of test compounds on the  96-well round bottom plates.Each compound was added in triplicate using four concentrations (0.2, 1, 5, and 25 μg/mL).Positive control wells contained cells and PHA, whereas cells alone served as a negative control.The plate was incubated in 5% CO 2 at 37°C for 72 h, and cells were pulsed with 25 μL of tritiated thymidine (0.5 μci/well).The incubation was continued for further 18 h.The cells were finally harvested on a glass fiber filter, and the plate was read as count per minute (CPM) using β-scintillation counter.
Cytotoxicity assay.The cytotoxicity of the test compounds against various cell lines was evaluated by MTT (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyl-tetrazolium bromide] colorimetric assay in 96-well flat-bottomed microplates [30].The prostate cancer PC3 (ATCC CRL-1435), and mouse fibroblast 3T3 (ATCC CRL-1658) cell lines were purchased from the American Type Culture Collection (ATCC, Virginia, USA).The human epithelial adenocarcinoma HeLa cells were kindly provided by Prof. Dr. Anwar Ali Siddiqui from Aga Khan University, Karachi, Pakistan.Dulbecco's Modified Eagle Medium (DMEM), added with 5% of fetal bovine serum (FBS), 100 IU/mL of penicillin, and 100 μg/mL of streptomycin was used for culturing of the HeLa (human epithelial carcinoma), PC3 (prostate cancer), and 3T3 (mouse fibroblast) cell lines.The cells were grown in 75 cm 3 flask and incubated at 37°C in 5% CO 2 incubator.After incubation 100 μL/well cells were introduced in 96-well plate with the concentration of 5×10 4 cells/mL and incubated again on the same parameters mentioned above.After 24 h incubation, the old media was removed and various concentrations of test compounds (1-30 μM), diluted in 200 μL of fresh media, were added.The plates were further incubated for 48 h, followed by addition of 200 μL MTT (0.5 mg/mL).After 4 h of incubation, 100 μL of DMSO was introduced into each well.The absorbance was measured by microplate reader (Spectra Max plus, Molecular Devices, CA, USA) at 570 nm for the extent of MTT reduction to formazan within cells.The cytotoxicity was recorded as concentrations causing 50% growth inhibition (IC 50 ) for HeLa, PC3, and 3T3 cell lines.The results (% inhibition) were processed by using Soft-Max Pro software (Molecular Devices, CA, USA).% Inhibition ¼ 100ÀðOD of test compoundÀOD of negative controlÞ ðOD of positive controlÀOD of negative controlÞ Â 100
Effect on phagocytes oxidative burst.The production of reactive oxygen species (ROS) plays a key role in the development of many inflammatory disorders.This includes endothelial dysfunction and tissue injury by an enhanced ROS generation by polymorphonuclear neutrophils (PMNs) at the site of inflammation.In our study the luminol-enhanced chemiluminescence assay was used for the measurement of the production of ROS.Metabolites 2-8 were evaluated for the inhibition of the production of intracellular ROS (OH, O 2 ˗ , H 2 O 2 , HOCl) by using luminol as a probe and zymosan as an activator [34][35][36].The results indicated that all the tested compounds were found to be inactive, except metabolite 6 (IC 50 = 30.3± 8.8 μg/mL), which showed moderate activity against the zymosan-induced oxidative burst in PMNs (Table 3).
Effect on T-cell proliferation.T-Lymphocytes are the main cells of adaptive immune responses and are known to play a central role in pathogenesis of various autoimmune diseases.During chronic inflammation the cytokines secreted by activated T-cells are known to activate and proliferate the population of various other immune cells.They are also involved in graft rejection process during transplantation, where they destroy the graft directly through cell mediated lysis or indirectly by enhancing antibody production or by activating complement.Hence the inhibition of proliferation of T-cells provides strong immunosuppressive approach for the treatment of various autoimmune diseases and transplantation rejection [37].During this study, medrysone (1), and its metabolites 2-8 were evaluated for the inhibition of T-cell proliferation by using PBMCs, activated with PHA.The results indicated that compounds 1, 4,   4).

Effect on TNF-α
Cytokines are regulators of host responses to infection and inflammation, while the presence of pro-inflammatory cytokines worsens the disease condition.TNF-α is a pro-inflammatory cytokine, which promotes systemic inflammation.Blocking of TNF-α during overwhelming infection can improve the disease condition.During our studies, the substrate and transformed products 2-8 were evaluated against the production of TNF-α [38].Among them, compound 1 (IC 50 = 30.54± 1.69 μg/mL), and its metabolite 7 (IC 50 = 28.6 ± 11.5 μg/mL) were found to be moderate inhibitors of TNF-α production (Table 5).These results have helped us to identify compounds with potential anti-inflammatory activity.Compounds 1-8 were analyzed for their immunomodulatory effect on different parameters of both innate and adaptive immune responses, including their effect on generation of ROS,  were found to be the moderate inhibitors.Compound 7 was found to be the most potent inhibitor of T-cells proliferation from this group.These results indicate that all compounds have the potential to inhibit cellular immune responses and might be useful in suppressing various chronic inflammatory and autoimmune disorders as well as for treatment of transplantation rejection.Among all compounds, the compounds 1, and 7 were also found to inhibit proinflammatory cytokine TNF-α produced from LPS activated macrophages.The blockade of TNF-α proved to be beneficial in many pathological conditions including rheumatoid arthritis (RA), inflammatory bowel disease (IBD), and psoriasis.All compounds showed their suppressive effects on various parameters of innate and adaptive immune responses, and can provide valuable insight for the treatment of different chronic inflammatory and autoimmune illnesses.

Cytotoxicity activity
Substrate 1 and its metabolites 2-8 were evaluated for their cytotoxicity against HeLa (human epithelial carcinoma), PC3 (prostate cancer), and 3T3 (mouse fibroblast) cells.All the compounds were found to be non-cytotoxic against above mentioned cell lines.

Conclusion
In conclusion, this is the first report of the fungal transformation of steroidal anti-inflammatory drug medrysone (1) into several new derivatives 2-8 with C. blakesleeana (ATCC 8688a), N. crassa (ATCC 18419), and R. stolonifer (TSY 0471).Through this study we identified an efficient route towards the synthesis of C-6β, 11β, 14α, 15β, 16β, and 20β oxidation products.Single-crystal X-ray diffraction analyses were performed to unambiguously deduce the structures of metabolites 2, 4, 6, and 8.Among all the metabolites, compound 6 (IC 50 = 30.3μg/mL) showed moderate inhibitory activity against the zymosan-induced oxidative burst in human whole blood cells whereas rest of the compounds were found to be inactive.When tested for their effects on proliferation of T-cells, compounds 1, 4, 5, 7, and 8 showed a strong inhibitory activity against these cells.Compound 7 (IC 50 < 0.2 μg/mL) was the most potent inhibitor of T-cell proliferation.The compounds 2, 3, and 6 showed moderate levels of inhibition with an IC 50 values ranges between 14.6 to 20.0 μg/mL.When tested for their effect on production of pro-inflammatory cytokine, TNF-α, compounds 1, and 7 showed moderate levels of inhibition while remaining compounds showed no inhibitory activity.All the compounds were found to be non-toxic when tested on 3T3 (mouse fibroblast) cells and showed no activity when tested against HeLa (human epithelial carcinoma), and PC3 (prostate cancer) cell lines.The work presented here can be helpful for the study of in vivo metabolism of medrysone (1), as well as for the identification of new anti-inflammatory agents, based on bio-catalysed structural transformation of various steroids.

doi: 10 .
1371/journal.pone.0153951.t005proliferation of T-cells, and production of pro-inflammatory cytokine TNF-α.During an innate immune response, the phagocytes release several chemical mediators like reactive oxygen species (ROS) and cytokines, which perpetuate the inflammatory process and activate the adaptive immune responses.During the current study, compound 6 exhibited moderate inhibitory activity on ROS produced from professional phagocytes, activated with the serum opsonized zymosan (as an antigen) by myeloperoxidase dependent pathway.It also showed moderate inhibition of proliferation of PHA activated T-cells.Compounds 1, 4, 5, 7, and 8 strongly inhibited the PHA activated proliferation of T-cells, whereas compounds 2, 3, and 6

Table 3 .
Effect of compounds 1-8 on luminol enhanced oxidative burst using zymosan activated PMNs.and 8 possess strong inhibitory activity with an IC 50 values between < 0.2 to 10.4 μg/mL.Among them compound 7 was the most potent inhibitor (IC 50 < 0.2 μg/mL) of T-cell proliferation.Compounds 2, 3, and 6 were found to be moderately active with an IC 50 ranges between 14.6-20.0μg/mL as compared with the standard drug prednisolone (IC 50 < 3.1 μg/mL) (Table aThe IC 50 values were obtained using various concentrations of test compounds, and readings are presented as mean ± SD of triplicates.doi:10.1371/journal.pone.0153951.t0035, 7,

Table 4 .
Effect of compounds 1-8 on PHA activated T-cells proliferation.
a Results are presented as mean ± SD of triplicates.The level of TNF-α was monitored using ELISA kits.vesign = indicates increase in the cytokine level as compared to activated cells without test compound.