Antiproliferative Illudalane Sesquiterpenes from the Marine Sediment Ascomycete Aspergillus Oryzae

marine drugs

Article Antiproliferative Illudalane Sesquiterpenes from the Marine Sediment Ascomycete Aspergillus oryzae

Raha Orfali 1,*, Shagufta Perveen 1,* , Muhammad Farooq Khan 2 , Atallah F. Ahmed 1,3 , Mohammad A. Wadaan 2, Areej Mohammad Al-Taweel 1, Ali S. Alqahtani 1,4 , Fahd A. Nasr 4 , Sobia Tabassum 5, Paolo Luciano 6, Giuseppina Chianese 6 , Jyh-Horng Sheu 7,8 and Orazio Taglialatela-Scafati 6,*

1 Department of Pharmacognosy, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia; [email protected] (A.F.A.); [email protected] (A.M.A.-T.); [email protected] (A.S.A.) 2 Bio-Products Research Chair, Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; [email protected] (M.F.K.); [email protected] (M.A.W.) 3 Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt 4 Medicinal, Aromatic and Poisonous Plants Research Center, College of Pharmacy, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia; [email protected] 5 Interdisciplinary Research Centre in Biomedical Materials (IRCBM), Lahore Campus, COMSATS University Islamabad, Islamabad 44000, Pakistan; [email protected] 6 Department of Pharmacy, School of Medicine and Surgery, University of Naples Federico II, Via Montesano 49, 80131 Naples, Italy; [email protected] (P.L.); [email protected] (G.C.) 7 Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Citation: Orfali, R.; Perveen, S.; Kaohsiung 80424, Taiwan; [email protected] 8 Khan, M.F.; Ahmed, A.F.; Wadaan, Department of Medical Research, China Medical University Hospital, China Medical University, M.A.; Al-Taweel, A.M.; Alqahtani, Taichung 40447, Taiwan A.S.; Nasr, F.A.; Tabassum, S.; * Correspondence: [email protected] (R.O.); [email protected] (S.P.); [email protected] (O.T.-S.) Luciano, P.; et al. Antiproliferative Illudalane Sesquiterpenes from the Abstract: The new asperorlactone (1), along with the known illudalane sesquiterpene echinolactone Marine Sediment Ascomycete D(2), two known pyrones, 4-(hydroxymethyl)-5-hydroxy-2H-pyran-2-one (3) and its acetate 4, and Aspergillus oryzae. Mar. Drugs 2021, 19, 4-hydroxybenzaldehyde (5), were isolated from a culture of Aspergillus oryzae, collected from Red 333. https://doi.org/10.3390/ Sea marine sediments. The structure of asperorlactone (1) was elucidated by HR-ESIMS, 1D, and md19060333 2D NMR, and a comparison between experimental and DFT calculated electronic circular dichroism (ECD) spectra. This is the ﬁrst report of illudalane sesquiterpenoids from Aspergillus fungi and, more Academic Editors: in general, from ascomycetes. Asperorlactone (1) exhibited antiproliferative activity against human RuAngelie Edrada-Ebel, lung, liver, and breast carcinoma cell lines, with IC50 values < 100 µM. All the isolated compounds Chang-Yun Wang and Bin-Gui Wang were also evaluated for their toxicity using the zebraﬁsh embryo model.

Received: 20 May 2021 Keywords: Aspergillus oryzae Accepted: 7 June 2021 ; marine fungus; illudalane sesquiterpenes; antiproliferative activity; Published: 10 June 2021 zebraﬁsh toxicity

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional afﬁl- 1. Introduction iations. The marine environment is an unsurpassed casket of chemodiversity and a proliﬁc source of biologically active compounds with potential medicinal applications [1]. In the last 50 years, scientists all over the world have dedicated their efforts to uncover the potential of marine metabolites, succeeding in the isolation of thousands of natural products Copyright: © 2021 by the authors. with peculiar architectures and interesting bioactivities [2]. As per April 2021, the marine Licensee MDPI, Basel, Switzerland. pharmacology arsenal [3] includes 15 approved drugs (mainly for cancer treatment), seven This article is an open access article compounds in phase I, 12 compounds in phase II, and 5 compounds in phase III clinical distributed under the terms and trials, the latter including plitidepsin, recently proposed for COVID-19 symptomatic treat- conditions of the Creative Commons ment [4,5]. Sponges and tunicates are undoubtedly the most intensely studied marine Attribution (CC BY) license (https:// organisms, but it is now clear that marine microbes, isolated from sediments or from creativecommons.org/licenses/by/ symbiotic plants or invertebrates, constitute a rich source for secondary metabolites. In this 4.0/).

Mar. Drugs 2021, 19, 333. https://doi.org/10.3390/md19060333 https://www.mdpi.com/journal/marinedrugs Mar. Drugs 2021, 19, x FOR PEER REVIEW 2 of 9 Mar. Drugs 2021, 19, 333 2 of 9

In this context, although terrestrial fungi are more explored, in comparison to their marine counterparts, a surprisingcontext, although number of terrestrial structurally fungi unique are more compounds explored, have in comparison been isolated to their marine coun- from fungi livingterparts, in marine asurprising habitats [6]. number The peculiar of structurally properties unique of the compounds marine environ- have been isolated from ment with regardfungi to nutrients, living in temperature marine habitats, and [6 competition,]. The peculiar are properties likely crucial of the factors marine in environment with improving the abilityregard of tomarine nutrients, fungi temperature, to elaborate andcompounds competition, with arepromising likely crucial bioactivi- factors in improving ties, especially suitedthe ability for antibiotic of marine and fungi anticancer to elaborate applications compounds [7]. with promising bioactivities, especially The genus Aspergillussuited for is antibiotic one of the and most anticancer abundant applications among marine [7]. ascomycetes, characterized by high salt tolerance,The genus fastAspergillus growth rateis one, and of the capacity most abundant to adapt among to diverse marine hab- ascomycetes, char- itats. Marine Aspergillusacterized fungi by produce high salt a tolerance, wide range fast of growthsecondary rate, metabolites and the capacity belonging to adapt to diverse to different classeshabitats. and are MarineendowedAspergillus with a broadfungi array produce of biological a wide rangeactivities of secondaryof indus- metabolites be- trial and pharmaceuticallonging interest to different [8,9]. classesAs a part and of areour endowed ongoing research with a broad activity array aimed of biologicalat activities of industrial and pharmaceutical interest [8,9]. As a part of our ongoing research activity the isolation of bioactive compounds from terrestrial and marine fungi [10–13], A. oryzae aimed at the isolation of bioactive compounds from terrestrial and marine fungi [10–13], samples obtained from the sediments of the Red Sea, along the coasts of Saudi Arabia, A. oryzae samples obtained from the sediments of the Red Sea, along the coasts of Saudi were chemically investigated. This study resulted in the isolation of two illudalane ses- Arabia, were chemically investigated. This study resulted in the isolation of two illudalane quiterpenes, the new asperorlactone (1) and the known echinolactone D (2), along with sesquiterpenes, the new asperorlactone (1) and the known echinolactone D (2), along with two rare pyrone derivatives (3–4) and 4-hydroxybenzaldehyde (5). These compounds two rare pyrone derivatives (3–4) and 4-hydroxybenzaldehyde (5). These compounds were were evaluated for their antiproliferative activity against three human carcinomas (lung, evaluated for their antiproliferative activity against three human carcinomas (lung, liver, liver, and breast) cell lines. Chemotherapeutics used in cancer treatment are often charac- and breast) cell lines. Chemotherapeutics used in cancer treatment are often characterized terized by marked toxic effects on normal cells. Less than 2% of compounds emerging by marked toxic effects on normal cells. Less than 2% of compounds emerging from in vitro from in vitro drug screening could enter clinical trials since the majority of the newly de- drug screening could enter clinical trials since the majority of the newly developed leads veloped leads failfail in preclinical in preclinical testing testing due due to their to their toxicity toxicity in experimental in experimental animal animal models models [14]. In vitro [14]. In vitro drugdrug screening screening methods methods are areprevalently prevalently used used in inorder order to tocharacterize characterize the the bioactivity and bioactivity and thethe toxicity toxicity of of new new compounds, compounds, while while testing theirtheir safetysafety in in suitable suitable animal ani- models prior to mal models priorclinical to clinical trials trials would would save save time time and and money. money. Since Since a high a high throughput throughput screening approach is screening approachnot is feasible not feasible in higher in higher animals, animals, zebrafish zebrafish constitute constitute a valid optiona valid for option preclinical testing and for preclinical testinghave and shown have quite show reproduciblen quite reproducible results. It results. is predicted It is predicted that, following that, fol- further development lowing further developmentof technologies, of technologies, zebrafish will zebrafish play a will key roleplay ina key speeding role in up speeding the emergence up of precision the emergence of medicineprecision [medicine15,16]. On [15,16]. these bases,On these the bases, isolated the compounds isolated compounds1–5 have 1 been–5 tested for their have been tested forzebrafish their zebrafish animal toxicity, animal andtoxicity results, and are results reported are reported herein. herein.

2. Results and Discussion2. Results and Discussion 2.1. Extraction and2.1. Structural Extraction Identification and Structural Identiﬁcation A. oryzae samplesA. were oryzae isolatedsamples from were the isolatedRed Sea fromsediment the Red collected Sea sediment at a depth collected of— at a depth of— 50 m off Jeddah, Saudi50 m offArabia. Jeddah, Fermentation Saudi Arabia. of the Fermentation fungus on solid of therice fungus medium on and solid ex- rice medium and traction with EtOAcextraction afforded with a brownish EtOAc afforded residue a, brownishwhich was residue, chromatographed which waschromatographed by using by using silica gel and RP-silica18 column gel and chromatography, RP-18 column chromatography, affording one new affording (1) andone four new known (1) and (2– four known (2–5) 5) compounds (Figurecompounds 1). (Figure1).

Figure 1. Chemical structures of metabolites isolated from A. oryzae.

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Mar. Drugs 2021, 19, 333 3 of 9 Figure 1. Chemical structures of metabolites isolated from A. oryzae.

TheThe knownknown compounds compounds were were identified identified as echinolactoneas echinolactone D ( 2D)[ 17(2)], [17], 4-(hydroxymethyl)- 4-(hydroxyme- 5-hydroxy-2thyl)-5-hydroxyH-pyran-2-one-2H-pyran- (23-)[one18], (3 (5-hydroxy-2-oxo-2H-pyran-4-yl)methyl) [18], (5-hydroxy-2-oxo-2H-pyran-4-yl)methyl acetate ( 4acetate)[16], and(4) [16] 4-hydroxybenzaldehyde, and 4-hydroxybenzaldehyde (5), by a ( comparison5), by a comparison of their of spectroscopic their spectroscopic data with data those with reportedthose reported in the in literature. the literature. Theilludalane The illudalane echinolactone echinolactone D (2 )D had (2) had been been isolated isolated before be- fromfore from mycelia mycelia of the of fungus the fungusEchinodontium Echinodontium japonicum japonicum[17] and [17] from and the from wood the decomposingwood decom- fungusposing Granulobasidiumfungus Granulobasidium vellereum vellereum[19], and [19], therefore, and therefore this is its, this first is reportits first from report a marine from a source.marine Compoundsource. Compound3 is a rare 3 is isomeric a rare isomeric analog of analog kojic acidof kojic for whichacid for only which three only reports three werereports present were inpresent the literature, in the literature, all from allAspergillus from Aspergillusfungi (marine fungi (marineA. flavus A.[ 18flavus], terrestrial [18], ter- A.restrial niger [A.20 niger], and [20], freshwater and freshwaterA. austroafricanus A. austroafricanus[21]). [21]). AsperorlactoneAsperorlactone ((11)) waswas isolatedisolated as as a a colorless colorless oil oil with with molecular molecular formula formula C 15C15HH1818OO33,, determineddeterminedby by HR-ESIMS.HR-ESIMS.The The13 13C NMR and DEPT spectra of 1 (Table1 1)) showed showed the the pres- pres- enceence ofof oneone lactonelactone carbonylcarbonyl( δ(δCC 166.5),166.5), oneone aromaticaromatic methinemethine( δ(δCC 123.1),123.1), oneone oxymethineoxymethine 33 22 ((δδCC 60.8),60.8), oneone spsp ((δδC 39.3) and fivefive spsp ((δδC 122.7, 132.2, 136.2, 144.3,144.3, 150.1)150.1) nonprotonatednonprotonated 33 carbons,carbons, threethree spsp methylenes ((δδCC 46.5, 47.1, 72.7, thethe latterlatter O-bearing),O-bearing), andand threethree methylmethyl groupsgroups ((δδCC 13.1,13.1, 27.527.5 ×× 2). On the basis of thesethese data,data, thethe sevenseven unsaturationunsaturation equivalentsequivalents requiredrequired by the molecular molecular formula formula of of 1 1couldcould bebe accommodated accommodated by bythe thepresence presence of a ofben- a benzenezene ring ring and and of two of two additional additional rings, rings, including including a lactone. a lactone. 1 TheThe 1H NMR NMR spectrum spectrum of of 11 (Table(Table 1)1) showed showed only only one one aromatic aromatic methine methine signal signal (δH (7.75),δH 7.75 two), two methyl methyl singlets singlets (δH ( δ1.19H 1.19 × 2),× an2), arylmethyl an arylmethyl at δ atH δ2.37H 2.37 (s, (s,3H), 3H), and and two two pairs pairs of ofmethylenes methylenes around around δHδ H2.802.80–2.83.–2.83. The The single single-spin-spin system system of of 11 includedincluded a diastereoptopicdiastereoptopic hydroxymethylenehydroxymethylene ((δδHH 4.52 and 4.63) 4.63),, and and an an oxymethine oxymethine signal signal (δ (δHH 4.95).4.95). Having Having associ- asso- ciatedated all all these these proton proton signals signals to to those those of of the directly linked carbonscarbons withwith thethe 2D2D NMRNMR HSQCHSQC experiment,experiment, the the illudalane illudalane type type sesquiterpenes sesquiterpenes skeleton skeleton of compoundof compound1 could 1 could be es-be tablishedestablished by by following following the the correlation correlation network network of theof the 2D 2D NMR NMR HMBC HMBC spectrum spectrum(Figure (Figure2 ) (see2) (see Supplementary Supplementary Materials). Materials).

FigureFigure 2.2. COSYCOSY (blue)(blue) andand keykey HMBCHMBC (red(red arrows)arrows) correlationscorrelations forfor compoundcompound1 1..

Table 1. 1H (700 MHz) and 13C (175 MHz) NMR data for asperorlactone (1) in CD3OD. 1 13 Table 1. H (700 MHz) and C (175 MHz) NMR data for asperorlactone (1) in CD3OD. Positions δH (Mult., J in Hz) δC, Type

1aPositions 2.83 (overlapped)δH (Mult., J in Hz) 46.5,δC, Type CH2

1b 1a2.81 2.83(d, 17.5) (overlapped) 46.5, CH2 2 1b- 2.81 (d, 17.5) 150.1, C 3 2- -132.2, 150.1, C 3 - 132.2, C 4 - 136.2, C 4 - 136.2, C 5 54.95 (dd, 4.95 1.0, (dd, 2.1) 1.0, 2.1)60.8, 60.8, CH 6 64.63 (dd, 4.63 1.0, (dd, 12.0) 1.0, 12.0)72.7, 72.7, CH22 4.52 (dd,4.52 2.1, (dd, 12.0) 2.1, 12.0) 7 7- -166.5, 166.5, C 8 - 122.7, C 8 9- 7.75 (s) 123.1,122.7, CH C 9 107.75 (s) -123.1, 144.3, CH C 10 11 2.82- (overlapped) 47.1,144.3, CH C2 12 - 39.3, C 13 2.37 (s) 13.1, CH3

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11 2.82 (overlapped) 47.1, CH2 12 - 39.3, C Table 1. Cont. 13 2.37 (s) 13.1, CH3

3 14 Positions 1.19δH (s)(Mult., J in Hz) 27.5,δC, CH Type 15 1.19 (s) 27.5, CH3 14 1.19 (s) 27.5, CH3 In particular,15 correlations of H-9 with 1.19 C-8 (s) and the lactone C-7, of 27.5, H-5 CHwith3 C-4 and C-8, and of H2-6 with C-7 defined the structure of the hydroxyisochroman-1-one moiety of asperorlactone.In particular, The correlations structure of of H-9 the withcondensed C-8 and dimethylated the lactone five C-7,-membered of H-5 with ring C-4 was and deducedC-8, and from of H 2the-6 withcorrelation C-7 defined of H3- the14/H structure3-15 with of C- the1, C hydroxyisochroman-1-one-11, and C-12 and of H2-1 and moiety H2- 11of with asperorlactone. C-2 and C-10. The Finally, structure the remaining of the condensed methyl group dimethylated was attached five-membered at C-3, following ring itswas correlations deduced fromwith theC-2, correlation C-3, and C of-4. H3-14/H3-15 with C-1, C-11, and C-12 and of H2-1 andAsperorlactoneH2-11 with C-2 ( and1) is C-10.an optically Finally, active the remaining compound methyl ([α]23− group19.8) with was attacheda single stereo- at C-3, following its correlations with C-2, C-3, and C-4. D center (C-5). We first tried to define the absolute configuration of 1 by employing the mod- [ ]23 − ified MosherAsperorlactone’s method (1 [22]) is; an however optically, all active the attempts compound to obtain ( α D the 19.8)formation with aof single the MTPA stere- estersocenter from (C-5). the corresponding We first triedto chloride define failed. the absolute Most likely, configuration the reaction of 1ofby the employing bulky MTPA the groupmodified with Mosher’s the hydroxyl method group [22 ];at however, C-5 was hindered all the attempts by the tomethyl obtain group the formation present on of the the condensedMTPA esters aromatic from the ring. corresponding Therefore, we chloride decided failed. to rely Most on likely, computational the reaction calculations, of the bulky MTPA group with the hydroxyl group at C-5 was hindered by the methyl group present on reasoning that comparison between experimental and quantum mechanically calculated the condensed aromatic ring. Therefore, we decided to rely on computational calculations, ECD spectrum could provide an unambiguous indication on the absolute configuration reasoning that comparison between experimental and quantum mechanically calculated of 1. ECD spectrum could provide an unambiguous indication on the absolute configuration The structure of 1 was subjected to a geometry and energy optimization using DFT of 1. with the mPW1PW91/6-311+G(d,p) functional and basis set combination using the Gauss- The structure of 1 was subjected to a geometry and energy optimization using DFT ian 09 software. The reasonably populated conformations, their relative energy, and the with the mPW1PW91/6-311+G (d,p) functional and basis set combination using the Gaus- equilibrium room-temperature Boltzmann populations are reported in Figure 3. The two sian 09 software. The reasonably populated conformations, their relative energy, and the major conformers 1a and 1b, accounting for 99.5% of the total population, differ almost equilibrium room-temperature Boltzmann populations are reported in Figure3. The two exclusively for the pseudorotation of the five-membered ring. major conformers 1a and 1b, accounting for 99.5% of the total population, differ almost exclusively for the pseudorotation of the five-membered ring.

Conformer 1c Conformer 1b ΔG = 11.30 Kcal/mol Conformer 1a ΔG = 0.40 Kcal/mol Population 52.8% Population 0.5 % Population 46.7% FigureFigure 3. 3. TheThe reasonably reasonably populated conformersconformers1a 1a––1c1cof of1 1and and their their calculated calculated Boltzmann Boltzmann population. population. TDDFT calculations were run using the functional CAM-B3LYP and the basis sets 6-31GTDDFT (d,p) includingcalculations at leastwere30 run excited using statesthe functional in all cases, CAM and-B3LYP using IEF-PCMand the basis for MeOH.sets 6- 31GThe (d,p) rotatory including strength at valuesleast 30 were excited summed states afterin all acases Boltzmann, and using statistical IEF-PCM weighting, for MeOH. and The∆ε valuesrotatory were strength calculated values by were forming summed sums after of Gaussian a Boltzmann functions statistical centered weighting at the wave-, and Δεlengths values of were the respective calculated electronic by forming transitions sums of and Gaussian multiplied functions by the correspondingcentered at the rotatory wave- lengthsstrengths. of the Thus, respective the ECD electronic spectra transitions for R-1 and andS-1 multipliedwere obtained by the (Figure corresponding4). The exten- rota- torysive strengths. similarity Thus, of the the first ECD with spectra the experimental for R-1 and S ECD-1 were spectrum obtained allowed (Figure us4). aThe confident exten- siveassignment similarity of theof the absolute first with configuration the experimental of asperorlactone ECD spectrum as 5R .allowed us a confident assignmentThe isolation of the absolute of asperorlactone configuration (1), of as asperorlactone well as of the relatedas 5R. echinolactone D (2), is of great relevance because, to our knowledge, this is the first report of illudalane-type sesquiterpenes from an ascomycete (A. oryzae), since this class of metabolites has, until now, been found exclusively in basidiomycetes.

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Figure 4. Experimental ECD curve of asperorlactone (red) and calculated ECD curves for R-1 (black) and S-1 (green).

The isolation of asperorlactone (1), as well as of the related echinolactone D (2), is of FigureFiguregreat 4.relevance 4.Experimental Experimental because, ECD ECD curve to our ofcurve asperorlactoneknowledge, of asperorlactone (red)this andis the calculated (red) first andreport ECD calculated curvesof illudalane for RECD-1 (black)- typecurves ses- for R-1 quiterpenes from an ascomycete (A. oryzae), since this class of metabolites has, until now, and(black)S-1 (green). and S-1 (green). been found exclusively in basidiomycetes. ItIt has has been been reported reported that that illudalanes illudalanes derive derive biosynthetically biosynthetically from afrom humulene a humulene precur- pre- sorcursor that,The that, upon isolation upon cyclization, cyclization, of asperorlactone would would generate generate a ( protoilludane1), aas protoilludane well as that of ﬁnally the that related finally would would rearrangeechinolactone rearrange to D (2), is of formgreatto form the relevance the illudalane illudalane because, derivative derivative [to23 ].our [23]. In theknowledge, In light the light of this of this hypothesis, this is hypothesis, the afirst possible reporta possible biosynthesis of biosynthe-illudalane-type ses- ofquiterpenessis asperorlactone of asperorlactone from is reported an is reportedascomycete in Figure in 5Figure, where(A. oryzae 5, illudol where), [since 24illudol] is athis key [24] class intermediate is a keyof metabolites intermediate that could has, that until now, affordbeencould 1 foundaffordby dehydration, 1exclusively by dehydration, oxidation, in basidiomycetes. oxidation and four-membered, and four -membered ring opening. ring opening. It has been reported that illudalanes derive biosynthetically from a humulene pre- cursor that, upon cyclization, would generate a protoilludane that finally would rearrange to form the illudalane derivative [23]. In the light of this hypothesis, a possible biosynthesis of asperorlactone is reported in Figure 5, where illudol [24] is a key intermediate that could afford 1 by dehydration, oxidation, and four-membered ring opening.

FigureFigure 5. 5.Postulated Postulated biosynthesis biosynthesis of of asperorlactone asperorlactone (1). (1).

2.2. Pharmacological and Toxicological Evaluation of the Isolated Compounds 2.2. Pharmacological and Toxicological Evaluation of the Isolated Compounds Illudalane sesquiterpenes, obtained from different sources, have been reported to Illudalane sesquiterpenes, obtained from different sources, have been reported to possess several biological properties, with a special focus on anticancer activities [25,26]. possess several biological properties, with a special focus on anticancer activities [25,26]. This prompted us to evaluate the antiproliferative activity of compounds 1–5 against three humanThis prompted cancer cell us lines,to evaluate namely, the lung antiproliferative carcinoma (A549), activity liver of carcinomacompounds (HepG2), 1–5 against and three human cancer cell lines, namely, lung carcinoma (A549), liver carcinoma (HepG2), and breast carcinoma (MCF7). The obtained IC50 values are presented in Table2. Compounds breast carcinoma (MCF7). The obtained IC50 values are presented in Table 2. Compounds 1Figure–5 generally 5. Postulated showed moderate biosynthesis antiproliferative of asperorlactone activity against(1). all tested cell lines, invari- ably1–5 withgenerally higher showed potency moderate against lung antiproliferative carcinoma, as compared activity toagainst liver orall breast tested carcinoma. cell lines, in- Asvariably shown with in Table higher1, asperorlactone potency against ( 1) and lung compound carcinoma2 were, as compared the most potent to liver compounds or breast car- against2.2.cinoma. Pharmacological three As shown cancer in cell andTable lines, Toxicological 1, with asperorlactone the single Evaluation exception (1) and of compound of the the Isolated activity 2 were Compounds of 5 theagainst most the potent MCF-7compoundsIlludalane cell line. against Additionally, sesquiterpenes, three cancer interesting cell obtained lines, is the with comparison from the singledifferent between exception compoundssources, of the have 3activityand been4, of 5reported to evidencingpossessagainst the several that MCF acetylation -biological7 cell line. improves Additionally,properties, the activity with interesting against a special A549 is the andfocus comparison HepG2. on anticancer between activities com- [25,26]. pounds 3 and 4, evidencing that acetylation improves the activity against A549 and This prompted us to evaluate the antiproliferative activity of compounds 1–5 against three HepG2. human cancer cell lines, namely, lung carcinoma (A549), liver carcinoma (HepG2), and

breast carcinoma (MCF7). The obtained IC50 values are presented in Table 2. Compounds 1–5 generally showed moderate antiproliferative activity against all tested cell lines, in- variably with higher potency against lung carcinoma, as compared to liver or breast carcinoma. As shown in Table 1, asperorlactone (1) and compound 2 were the most potent compounds against three cancer cell lines, with the single exception of the activity of 5 against the MCF-7 cell line. Additionally, interesting is the comparison between compounds 3 and 4, evidencing that acetylation improves the activity against A549 and HepG2.

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TableTable 2. 2.Antiproliferative Antiproliferative activity activity of of compounds compounds1 1––55against against three three human human cancer cancer cell cell lines. lines.

Compound IC50 (µM) for the Different Carcinoma Cell Lines Compound IC50 (µM) for the Different Carcinoma Cell Lines A549 (Lung) HepG2 (Liver) MCF-7 (Breast) A549 (Lung) HepG2 (Liver) MCF-7 (Breast) 1 72.7 ± 1.1 86.6 ± 3.2 106.5 ± 4.2 1 72.7 ± 1.1 86.6 ± 3.2 106.5 ± 4.2 2 55.7 ± 2.5 148.4 ± 5.6 128.0 ± 2.8 2 55.7 ± 2.5 148.4 ± 5.6 128.0 ± 2.8 33 208.5208.5 ± ±6.86.8 220.4 ±± 3.6 225.4225.4± ±5.1 5.1 44 89.489.4 ± ±2.32.3 126.8 ±± 6.4 170.7170.7± ±4.5 4.5 55 97.597.5 ± ±2.62.6 242.6 ±± 6.4 158.2158.2± ±5.5 5.5 ± ± ± DoxorubicinDoxorubicin 2.1 2.1 ± 0.080.08 2.2 ± 0.15 1.91.9 ±0.05 0.05

AsAs anticipated,anticipated, screening in zebrafish zebrafish embryos embryos provides provides an an excellent excellent environment environment in inwhich which the the toxicity toxicity of of a acompound compound on on noncancer noncancer cells cells and and systems could bebe predicted.predicted. Thus,Thus, the the zebrafish zebrafish embryos embryos model model was was used used to evaluate to evaluate the animal the animal toxicity toxicity of compounds of com- 1pounds–5 (Figure 1–56 ).(Figure The LC 6). The50 values LC50 values (the concentration (the concentration required required to kill to50% kill 50% of embryos) of embryos) of allof theall the tested tested compounds compounds were were higher higher than than the the 1 mg/mL1 mg/mL range, range, indicating indicating their theirsafety safety onon noncancernoncancer cellscells andandselectivity selectivityindices indices>50. >50. TheThe zebrafishzebrafish embryosembryos thatthat wereweretreated treated withwith compoundscompounds 22,,3 3,, andand5 5did didnot notexhibit exhibitany anyobservable observabletoxicity, toxicity, and and they they developed developed normallynormally up up to to 3 days3 days post post treatment. treatment. On On the otherthe other hand, hand, compound compound4, for which4, for which the initial the developmentinitial development and growth and growth of zebrafish of zebrafish embryos embryos was normal, was normal, induced induced the death the ofdeath 100% of of100% treated of treated embryos embryos after 24 after h post 24 treatment.h post treatment. The zebrafish The zebrafish embryos embryos that were that treated were µ 1 withtreated more with than more 200 thanM compound200 µM compound(higher 1 than (higher IC50 than) developed IC50) developed normally; normally however,; how- the embryosever, the exhibited embryos cardiac exhibited toxicity cardiac (cardiac toxicity edema (cardiac and cardiac edema hypertrophy, and cardiac black hypertrophy, arrow in Figureblack 6arrow) after in 2 Figure days post 6) after treatment. 2 days post treatment.

FigureFigure 6.6. In vivo screening of compounds compounds 11––55 inin zebrafish zebrafish embryos. embryos. Representative Representative micrograph micrograph of ofembryos embryos at at3 days 3 days poster poster fertilization fertilization,, which which were were treated treated with with compounds compounds 1–5.1 The–5. Thezebrafish zebrafish embryos treated with compounds 2, 3, and 5 developed normally, and there were no obvious dif- embryos treated with compounds 2, 3, and 5 developed normally, and there were no obvious ferences in morphology and growth between control and treated embryos. The zebrafish embryos differences in morphology and growth between control and treated embryos. The zebrafish embryos treated with > 200 µM of 1, however, had cardiac edema and cardiac hypertrophy (black arrow). treatedThe zebrafish with >200 embryosµM of treated1, however, with hadcompound cardiac edema4 developed and cardiac normally hypertrophy but were found (black dead arrow). on The day zebrafish2. All theembryos images are treated in same with magnification, compound 4 developed scale is 200 normally µm. but were found dead on day 2. All the images are in same magnification, scale is 200 µm. 3. Materials and Methods 3. Materials and Methods 3.1.3.1. FungalFungal MaterialMaterial AspergillusAspergillus oryzae oryzaewas was isolated isolated from from the the marine marine sediment sediment collected collected at − at50 − m50 off m Jeddah,off Jed- Reddah, Sea, Red Saudi Sea, Saudi Arabia, Arabia in October, in October 2018. The2018. fungal The fungal identification identification was conducted was conducted by DNA by amplificationDNA amplification and sequencing and sequencing of the internal of the transcribedinternal transcribed spacer region spacer (GenBank region accession(GenBank accession No. MH608347) followed by a subsequent BLAST search in NCBI according to

Mar. Drugs 2021, 19, 333 7 of 9

No. MH608347) followed by a subsequent BLAST search in NCBI according to the protocol described before [27]. The specimen of the fungal strain was deep-frozen and deposited at the authors’ lab (R.O., S.P.).

3.2. Fermentation, Extraction, and Isolation A. oryzae was cultivated in 20 Erlenmeyer flasks on solid rice medium containing (100 g rice, 3.5 g sea salt, and 110 mL of demineralized water). After autoclaving at 121 ◦C for 20 min and then cooling to room temperature, each flask was inoculated and then incubated at 20 ◦C under static conditions. After four weeks, 500 mL EtOAc was added to each flask to stop the fermentation and extract. The total extract was collected after the flasks had been shaken at 150 rpm for 8 h on a laboratory shaker. The obtained crude extract after evaporation of the EtOAc (7 g) was then partitioned between n-hexane and MeOH. The polar phase was then subjected to Sephadex LH-20 column (100 × 2.5 cm) using 100% methanol as an eluting solvent. Similar fractions were combined with each other according to TLC readings and further purified by semipreparative HPLC using gradient system MeOH-H2O from 40:60 to 70:30 in 30 min to afford 1 (3.5 mg), 2 (5.0 mg), 3 (7.0 mg), 4 (5.0 mg), and 5 (2.6 mg). 23 Asperorlactone (1). Colorless oil; [ α]D −19.8 (c 0.6, MeOH); UV λmax (MeOH) nm (log ε): 1 230 (4.6); ECD λmax (MeOH) nm (∆ε): 238 (+ 9.6), 265 (−4.8); H NMR (700 MHz, CD3OD), 13 + and C NMR (175 MHz, CD3OD); see Table1; ESIMS m/z 269.1150 [M + Na] (calc. for C15H18 O3 Na m/z 269.1154).

3.3. Computational Calculations A preliminary conformational search on each stereoisomer was performed by Simu- lated Annealing in the INSIGHT II package. The MeOH solution phases were mimicked through the value of the corresponding dielectric constant. Using the steepest descent followed by quasi-Newton–Raphson method (VA09A) the conformational energy was min- imized. Restrained simulations were carried out for 500 ps using the CVFF force field as implemented in Discovery software (Version 4.0 Accelrys, San Diego, USA). The simulation started at 1000 K, and then the temperature was decreased stepwise to 300 K. The final step was again the energy minimization, performed in order to refine the conformers obtained, using the steepest descent and the quasi-Newton–Raphson (VA09A) algorithms succes- sively. Both dynamic and mechanic calculations were carried out by using 1 (kcal/mol)/A° 2 flat well distance restraints. In total, 100 structures were generated. TDDFT calculations were run using the functional CAM-B3LYP and the basis sets 6-31G (d,p) including at least 30 excited states in all cases, and using IEF-PCM for MeOH. The rotatory strength values were summed after a Boltzmann statistical weighting, and ∆ε values were calculated by forming sums of Gaussian functions centered at the wavelengths of the respective electronic transitions and multiplied by the corresponding rotatory strengths. The ECD spectra that were obtained were UV-corrected and compared with the experimental ones.

3.4. In Vitro Antiproliferative Activity The antiproliferative activity of compounds was measured using MTT (3-(4, 5-dime- thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. A549 (lung), HepG2 (liver), and MCF-7 (breast) cancer cells were purchased from the American Type Cell Collection (ATCC, Manassas, VA, USA). The cells were seeded at 5 × 104 cells/well (in 100 µL of DMEM) in 96-well microplates. After 24 h incubation at 37 ◦C, serial dilution (15–250 µM) of each compound was added and incubated for 48 h. Thereafter, 10 µL of the MTT solution (5 mg/mL) was added to each well. After 4 h incubation with the MTT solution, a volume of 100 µL of acidiﬁed isopropanol was added to solubilize the formazan product and incubated on a shaker for a further 10 min. Reduced MTT was assayed at 570 nm using a microplate reader (BioTek, Winoosky, VT, USA). Control groups received the same amount of DMSO (0.1%), untreated cells were used as a negative control, whereas cells treated with doxorubicin were used as a positive control. The IC50 (concentration that caused more Mar. Drugs 2021, 19, 333 8 of 9

than 50% inhibition of proliferation) was calculated from a dose-dependent curve. The cell viability percent was calculated = mean absorbance of treated sample/mean absorbance of control ×100.

3.5. Zebrafish Toxicity Screening Wild-type zebrafish strain AB/Tuebingen TAB-14(AB/TuebingenTAB-14) (Catalog ID: ZL1438) were obtained from zebrafish international resource center and grown in an animal facility at the Department of Zoology, King Saud University, Riyadh, Kingdom of Saudi Arabia. The fish were maintained and bred following guidelines of the Institutional Animal Care and Use Committee (ICUAC) and zebrafish book. The fertilized embryos were obtained by natural pairwise breeding of adult fish. The fertilized embryos were sorted, dead embryos were removed, and synchronous stage embryos were used for screening. A stock solution of 25 mM was made by dissolving the compounds in molecular biology grade DMSO (Sigma Aldrich, St. Louis, MI, USA). Zebrafish embryos were exposed to serial dilution (1, 5, 15, 45, 150, and 300 µM) of each compound. The embryos were remained exposed to the compounds for 3 days, and the embryos medium containing the compounds were changed every day. The response of the embryos toward mortality, and embryonic toxicity (teratogenicity) was monitored once after 12 h and then after every 24 h until the end of the experiment. The experiment was repeated at least three times (triplicate biological repeats) by using a new batch of embryos every time. LC50 for zebrafish embryonic toxicity was calculated by using an updated Probit analysis by Finney method [28].

4. Conclusions Samples of A. oryzae, obtained from the Red Sea sediments collected off Jeddah, Saudi Arabia, afforded the first illudalane sesquiterpenoids isolated from an ascomycete, including the new asperorlactone (1), characterized by hydroxylation of the lactone ring. Elucidation of the structure and absolute configuration of this compound needed application of ESIMS and NMR spectral analyses and computational calculations of ECD spectra. The isolated compounds showed moderate antiproliferative activity against three human carcinoma cell lines (lung, liver, and breast). The zebrafish embryo model was used to evaluate the animal toxicity of compounds and selected echinolactone D (2) and the rare pyrone 3 as targets worthy of further investigation.

Supplementary Materials: The following are available online at https://www.mdpi.com/article/ 10.3390/md19060333/s1, Figures S1–S5: 1D and 2D NMR spectra of asperorlactone (1). Author Contributions: Conceptualization, R.O., S.P., and O.T.-S.; methodology, M.F.K., F.A.N., S.P., and A.F.A.; software, A.M.A.-T. and P.L.; validation, S.T.; formal analysis, M.F.K., F.A.N., R.O., and G.C.; investigation, M.F.K., F.A.N., P.L., and G.C.; resources A.S.A. and M.A.W.; writing—original draft preparation M.F.K., F.A.N., and O.T.-S.; writing—review and editing A.S.A., M.A.W., and O.T.-S.; funding acquisition M.F.K., M.A.W., and J.-H.S. All authors have read and agreed to the published version of the manuscript. Funding: This study was funded by the Deanship of Scientific Research, King Saud University, through the Vice Deanship of Scientific Research Chairs. Institutional Review Board Statement: In this study, only zebrafish embryos were used which aged less than 5 days post fertilization, and hence, as reported in Reprod. Toxicol. 2012, 33, 128– 132, doi:10.1016/j.reprotox.2011.06.121, they are exempted to take the ethical approval from ethical committee for the use and care of laboratory animals. Informed Consent Statement: Not applicable. Data Availability Statement: The data presented in this study are available in the article and Supplementary Material. Conflicts of Interest: The authors declare no conflict of interest. Mar. Drugs 2021, 19, 333 9 of 9

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