Lead Compounds from Mangrove-Associated Microorganisms

marine drugs

Review Lead Compounds from Mangrove-Associated Microorganisms

Elena Ancheeva †, Georgios Daletos *,† and Peter Proksch *

Institute of Pharmaceutical Biology and Biotechnology, Heinrich-Heine-University, Universitaetsstrasse 1, 40225 Düsseldorf, Germany; [email protected] * Correspondence: [email protected] (G.D.); [email protected] (P.P.); Tel.: +49-211-81-14173 (G.D.); +49-211-81-14163 (P.P.) † These authors contributed equally to this work.

Received: 7 August 2018; Accepted: 29 August 2018; Published: 7 September 2018

Abstract: The mangrove ecosystem is considered as an attractive biodiversity hotspot that is intensively studied in the hope of discovering new useful chemical scaffolds, including those with potential medicinal application. In the past two decades, mangrove-derived microorganisms, along with mangrove plants, proved to be rich sources of bioactive secondary metabolites as exempliﬁed by the constant rise in the number of publications, which suggests the great potential of this important ecological niche. The present review summarizes selected examples of bioactive compounds either from mangrove endophytes or from soil-derived mangrove fungi and bacteria, covering the literature from 2014 to March 2018. Accordingly, 163 natural products are described in this review, possessing a wide range of potent bioactivities, such as cytotoxic, antibacterial, antifungal, α-glucosidase inhibitory, protein tyrosine phosphatase B inhibitory, and antiviral activities, among others.

Keywords: mangrove microorganisms; bioactive natural products; endophytes; drug leads

1. Introduction Mangrove (mangal) communities represent a coastal habitat located in tropical and subtropical intertidal estuarine zones, occurring in 112 countries, and mostly attributed to latitudes between 30◦ N and 30◦ S[1]. Special ecological conditions of mangroves include relatively high tidal range, high average temperature with little seasonal fluctuation, high salinity, strong winds, and muddy anaerobic or sandy soil [1–3]. The flora of these communities includes the so-called “exclusive” or true mangroves as well as “nonexclusive” mangrove species that inhabit other terrestrial or aquatic ecosystems (semi-mangrove or mangrove associates). Tomlinson defines true mangroves by several criteria, including exclusive occurrence in mangrove ecosystem, morphological adaptations (e.g., aerial roots and viviparous, water-dispersed propagules), physiological mechanisms for salt exclusion/excretion, as well as taxonomical distinctness from terrestrial species (at least at the generic level), albeit all these characteristics are not necessary to be present among one plant species [1]. One of the main extreme habitat factors that influences mangroves is high salinity, resulting in the following specific leaf traits and osmotic properties of true mangroves: lower specific leaf area, higher succulence, lower K+/Na+ ratio, higher Na+ and Cl− contents, and hence higher osmolality in contrast to semi-mangrove plants [4]. Since mangrove ecosystems can be viewed as an extreme environment, which demands various morphological and physiological adaptations of inhabiting species, the biosynthetic potential of the latter to produce a distinct array of novel chemical entities is apparent. Thus, the mangrove ecosystem is considered as an attractive biodiversity hotspot that is intensively investigated in the hope of discovering new structural scaffolds, including those with

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applications. Indeed, studies on secondary metabolites from mangrove plants in the past were found medicinalto be quite applications.fruitful, and gave Indeed, rise to studies new carbon on secondary frameworks metabolites accompanied from by mangrove pronounced plants biological in the pastactiviti werees [2]. found Notable to be quite recent fruitful, examples and of gave bioactive rise to derivatives new carbon from frameworks mangroves accompanied include the by pronouncedtetranortriterpenoids biological xylogranatins activities [2]. Notable A–D [5] recent and krischnadimerexamples of bioactive A [6], derivatives as well as fromthe triterpenoid mangroves includeparacaseolin the tetranortriterpenoids [7] possessing potent xylogranatins cytotoxic activitA–D [y5 ]toward and krischnadimer cancerous cellA [s,6], the as well limonoids as the triterpenoidkrishnolides paracaseolinA–D with anti [7]-HIV possessing activity potent [8], and cytotoxic thaixylomolin activity towardB with anti cancerous-inflammatory cells, the properties limonoids krishnolides[9]. Furthermore, A–D with secondary anti-HIV metabolites activity [8 ], from and thaixylomolin typical mangrove B with plants anti-inflammatory of the genera properties Xylocarpus [9]., Furthermore,Avicennia, Rhizophora secondary, and metabolites Bruguiera signific from typicalantly mangroveenriched our plants current of the knowledge genera Xylocarpus of the chemistry, Avicennia of, Rhizophoracomplex tetranorterpenoids, and Bruguiera significantly (limonoids), enriched di- and ourtriterpenoids, current knowledge and iridoids, of the among chemistry others of [2,10]. complex tetranorterpenoidsMangrove-associated (limonoids), microorganisms, di- and triterpenoids, including and fungal iridoids, and among bacterial others endophytes [2,10]. , as well as microbesMangrove-associated derived from soil microorganisms, samples, have including likewise fungal drawn and the bacterial attention endophytes, of natural as product well as microbesresearchers. derived Remarkable from soil discoveries samples, have of bioactivelikewise drawn xyloketals the attention with unprecedented of natural product structures researchers. from RemarkableXylaria sp. [11] discoveries and salinosporamide of bioactive xyloketals A from the with mangrove unprecedented bacterium structures Salinospora from Xylaria tropicasp. [12] [11 in] andthe salinosporamideearly 2000s indicated A from a great the mangrovepotential for bacterium drug discoverySalinospora and tropica inspired[12] further in the early studies 2000s on indicatedmicrobes aderived great potential from mangroves. for drug discovery Intensive andinvestigation inspired further of the studieslatter resulted on microbes in more derived than from350 publications mangroves. Intensivethat appeared investigation during of the the latter last ten resulted years in, morefocusing than on 350 naturalpublications product that appeared chemistry, during bioactivity, the last tenbiotechnology years, focusing, and on chemical natural product synthesis chemistry, of their bioactivity, bioactive biotechnology, metabolites, thus and demonstrating chemical synthesis the ofpotential their bioactive of mangrove metabolites,-associated thus microbes demonstrating as a prolific the potential source of of lead mangrove-associated compounds [13]. Particularly, microbes as aover prolific the sourcelast decade, of lead research compounds on these [13]. Particularly, microbes resulted over the in last characteri decade,z researchation of onalmost these 1 microbes000 new resultedmetabolites, in characterization among them, ~850 of almost derived 1000 from new fungi metabolites, (the majority among of them,them obtained ~850 derived as endophytes) from fungi, (theand majority~120 from of bacteria them obtained [14]. Thus, as endophytes),the overall trend and in ~120 the fromnumber bacteria of experimental [14]. Thus, articles the overall dedicated trend into the description number of of experimental new/bioactive articles mangrove dedicated-derived to description metabolites of over new/bioactive the past ten mangrove-derived years, remains metabolitespromising (Figure over the 1) past[13].ten years, remains promising (Figure1)[13].

Figure 1.1. NumberNumber ofof publicationspublications describingdescribing newnew and/orand/or bioactive mangrove-associatedmangrove-associated secondary metabolites covering the period 2007–2017.2007–2017. Source: MarinLit database and series of annual reviews by Blunt etet al.al. in Natural Product ReportsReports [[13,14].13,14]. ArticlesArticles onon mangrove-associatedmangrove-associated fungi and bacteria include bothboth ecologicalecological groups,groups, plant-plant- andand soil-derivedsoil-derived microorganisms.microorganisms.

Attempts to estimate estimate fungal fungal and and bacterial bacterial diversity diversity from from soil soil samples samples collected collected in inmangroves mangroves of ofSaudi Saudi Arabia, Arabia, China, China, Brazil Brazil,, and and India India showed showed a ahigh high diversity diversity of of associated associated microbes microbes [15 [15––1919].]. Interestingly, a a study study directed directed towards towards the the estimation estimation of bacterial diversity, utilizing 16S rRNA sequencing, demonstrated that the bacterial community community of pristine pristine mangrove mangrove sediments sediments containscontains species that are mostlymostly unrelatedunrelated toto knownknown bacteriabacteria [[19].19]. Moreover, analysis of culturableculturable fungal endophytes collected collected in in Brazil Brazil indicated indicated that that among among fungal fungal isolates isolates some species some speciescould not could be classiﬁed not be withinclassified any within known an generay known [20 genera]. These [20] investigations,. These investigations, together with together promising with promising results from results studies from on

Mar. Drugs 2018, 16, x 319 FOR PEER REVIEW 33 ofof 3132 studies on mangrove-associated secondary metabolites, demonstrate this unique ecosystem as a rich mangrove-associated secondary metabolites, demonstrate this unique ecosystem as a rich reservoir of reservoir of natural product diversity that could be utilized in the exploration of new drug leads. natural product diversity that could be utilized in the exploration of new drug leads. Advances in natural products derived from mangrove actinomycetes were discussed by Xu et Advances in natural products derived from mangrove actinomycetes were discussed by Xu et al. al. (up to 2013) [21]. Bioactive natural products from mangrove-associated microorganisms, their (up to 2013) [21]. Bioactive natural products from mangrove-associated microorganisms, their chemical chemical features and biosynthetic aspects, covering the time span of 2011–2013, were summarized features and biosynthetic aspects, covering the time span of 2011–2013, were summarized by by Jing Xu [22]. In this review, we highlight selected examples (studies in which at least one Jing Xu [22]. In this review, we highlight selected examples (studies in which at least one compound compound exhibits IC50 (/MIC) values less than 10 μM (/10 μg/mL) and/or comparable or higher exhibits IC (/MIC) values less than 10 µM (/10 µg/mL) and/or comparable or higher activity activity than50 that of the positive control in the respective bioassay) of recently described microbial than that of the positive control in the respective bioassay) of recently described microbial bioactive bioactive compounds of mangrove origin. The time frame covered in this paper extends from 2014 to compounds of mangrove origin. The time frame covered in this paper extends from 2014 to March 2018. March 2018. The articles selected for the review contain natural products with mechanism-of-action The articles selected for the review contain natural products with mechanism-of-action studies and/or studies and/or compounds with pronounced activity. The natural products described herein are compounds with pronounced activity. The natural products described herein are grouped according grouped according to the microorganism ecological source into endophytes and those derived from to the microorganism ecological source into endophytes and those derived from soil, the latter group soil, the latter group including studies on compounds of fungal or bacterial origin. including studies on compounds of fungal or bacterial origin.

2. Bioactive Compounds from Mangrove Mangrove-Associated-Associated Microorganisms

2.1. Bioactive Compounds from Endophytic Fungi

2.1.1. Cytotoxic Compounds Meng et al. successfully successfully isolated isolated twenty twenty-one-one new new compounds compounds from from a a single fungal strain Penicillium brocae MAMA-231,-231, obtained from fresh tissue of the marine mangrove plant Avicennia marina (Hainan Island, Island, China) China) using using different different types types of media of media for cultivation for cultivation of this fungus of this [23 fungus–26]. The [23 –first26]. fermentationThe ﬁrst fermentation on potato on- potato-dextrosedextrose broth broth (PDB) (PDB) medium medium afforded afforded six six new new disulﬁde-bridgeddisulfide-bridged diketopiperazines brocazinesbrocazines A–FA–F [[23].23]. AmongAmong them,them, brocazines brocazines A A ( 1(),1), B B (2 (),2), E E (3 (),3), and and F F (4 ()4 (Figure) (Figure2) 2displayed) displayed cytotoxicity cytotoxicity against against a a panel panel of of human human tumor tumor cell cell lines, lines, includingincluding Du145Du145 (human prostate cancer), HeLa (cervical cancer), HepG2 (liver cancer), MCF-7MCF-7 (breast adenocarcinoma), NCI-H460NCI-H460 (non(non-small-cell-small-cell lung cancer), SGC SGC-7901-7901 (gastric cancer), SW1990 SW1990 (pancreatic (pancreatic adenocarcinoma), adenocarcinoma), SW480 (colon cancer), cancer), and and U251 U251 (glioblastoma) (glioblastoma) with with IC50 ICvalues50 values in the in range the from range 0.89 from to 12.4 0.89 μ toM, 12.4whereasµM, brocazineswhereas brocazines C and D C did and not D show did not activity show, indicating activity, indicating that the presence that the ofpresence two double of two bonds double at positionsbonds at positionsC-6 and C C-6-6′ or and of C-6one0 doubleor of one bond double at C- bond6/6′ in at conjugation C-6/60 in conjugation with a keto with group a keto(at C group-5/5′), plays(at C-5/5 an0 ), important plays an important role in the role cytotoxicity in the cytotoxicity of these of these metabolites. metabolites. In a In further a further study, study, chemical chemical investigation of of the the extract from from the fungus grown in Czapek medium showed induction induction of of new new natural products, among them, the bisthiodiketopi bisthiodiketopiperazineperazine derivative brocazine G ( 5) (Figu (Figurere 22)) thatthat was activeactive againstagainst A2780 A2780 (human (human ovarian ovarian cancer) cancer) cells cells that that are eitherare either sensitive sensitive (sens) (sens) or resistant or resistant (CisR) (CisR)to the cytostaticto the cytostatic drug cisplatin drug cisplatin [24]. Interestingly, [24]. Interestingly, compound compound5 displayed 5 displayed potent activity potent toactivity both cell to botlinesh cell with lines IC50 withvalues IC50 of values 664 and of 664 661 and nM, 661 respectively. nM, respectively. In addition, In addition,5 showed 5 showed strong activity strong activity against againstStaphylococcus Staphylococcus aureus with aureus an with MIC an value MIC of value 0.62 µ ofM 0.62 (0.25 μµMg/mL), (0.25 μ strongerg/mL), stronger than that than of the that positive of the positivecontrol chloromycetincontrol chloromycetin (MIC = 1.55(MICµM/0.5 = 1.55 µμg/mL).M/0.5 μ Moreover,g/mL). Moreover, other studies other onstudies this funguson this yieldedfungus yieldeda series ofa series antibacterial of antibacterial compounds compounds (20–26) that(20– are26) describedthat are described in Section in 2.1.2Section. 2.1.2.

Figure 2. Chemical structures of 1–5. Figure 2. Chemical structures of 1–5.

The endophytic fungus Lasiodiplodia theobromae ZJ-HQ1, isolated from healthy leaves of the marine mangrove Acanthus ilicifolius (Guangdong Province, China), afforded two new chlorinated

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Mar. DrugsThe 2018 endophytic, 16, x FOR fungusPEER REVIEWLasiodiplodia theobromae ZJ-HQ1, isolated from healthy leaves of4 of the 32 marine mangrove Acanthus ilicifolius (Guangdong Province, China), afforded two new chlorinated preussomerinspreussomerins (6 andand 77)) along along with with nine nine known known analogs analogs [ [2727].]. Interestingly, 6,, 7,, andand the the known known compoundscompounds 88–14–14(Figure (Figur3e), exhibited3), exhibited cytotoxicity cytotoxicity against against a panel a of panel human of cancer human cell cancer lines, including cell lines, A549including (lung A549 adenocarcinoma), (lung adenocarcinoma), HepG2, MCF-7, HepG2, HeLa, MCF and-7, HEK HeLa 293T, and (embryonic HEK 293T kidney) (embryonic cells, with kidney) IC50 valuescells, with ranging IC50 values from 2.5 ranging to 83 µfromM. Structure–activity 2.5 to 83 μM. Structure relationship–activity analyses relationship revealed analyses that arevealed ketone groupthat a atketone C-1 and/orgroup at a chlorineC-1 and/or group a chlorine at C-2 ingroup ring Aat areC-2 favorable in ring A substituents are favorable for substituents cytotoxicity for of preussomerins.cytotoxicity of Furthermore, preussomerins. compounds Furthermore,6, 7, 10 compounds, 11, 13, and 614, 7showed, 10, 11, activity13, and toward 14 showedS. aureus activitywith MICtoward values S. aureus between with 4.4 MIC and values 35.9 µ betweenM (1.6 and 4.4 13 andµg/mL). 35.9 μM However, (1.6 and the 13 respectiveμg/mL). However, compounds the wererespective found compounds to be inactive were againstfound to a be panel inactive of Gram-negativeagainst a panel of bacteria, Gram-negative including bacteria,Escherichia including coli, PseudomonasEscherichia coli aeruginosa, Pseudomonas, and Salmonella aeruginosa enteritidis, and Salmonella, thus suggesting enteritidis, their thus selective suggesting inhibitory their selective activity againstinhibitory Gram-positive activity against bacteria, Gram which-positive is probably bacteria,connected which is probablywith the exerted connected cytotoxicity with the of exerted these metabolitescytotoxicity (Figureof these3 )[metabolites27]. (Figure 3) [27].

Figure 3. Chemical structures of 6–14. Figure 3. Chemical structures of 6–14. The endophyte Annulohypoxylon sp. CA-2013, isolated from the mangrove plant Rhizophora racemosaThe, endophyte collected in Annulohypoxylon Cameroon, led sp. to the CA characterization-2013, isolated from of new the benzo[ mangrovej]fluoranthene-based plant Rhizophora congeners,racemosa, collected daldinones in H–J Cameroon [28]. All, compoundsled to the characterization were tested for their of new cytotoxicity, benzo[j]fluoranthene and daldinone-based I (15) wascongeners, shown daldinones to be the most H–J active [28]. All derivative compounds (Figure were4). Interestingly,tested for their15 cytotoxicitywas shown, toand be daldinone an artefact I formed(15) was from shown daldinone to be the H throughmost active intramolecular derivative (Figure dehydration. 4). Interestingly, Compound 1515 wasexhibited shown strongto be an to moderateartefact formed cytotoxicity from against daldinone adult H lymphoblastic through intramolecular leukemia T cells dehydration. (Jurkat J16) Compound and Burkitt’s 15 lymphoma exhibited Bstrong lymphocytes to moderate (Ramos), cytotoxicity with IC 50againstvalues adult of 14.1 lymphoblastic and 6.6 µM, respectively.leukemia T ce Mechanism-of-actionlls (Jurkat J16) and Burkitt studies’s showedlymphoma that B 15lymphocytesactivates caspases, (Ramos) and, with subsequently IC50 values of induces 14.1 and apoptosis 6.6 μM, respectively. in Ramos cells Mechanism with a rapid-of- kineticaction studies profile, showed comparable that 15 to thatactivates of the caspases potent, known and subsequently apoptosis inducer induces staurosporine. apoptosis in Ramos Treatment cells ofwith caspase-9-deficient a rapid kinetic profile, and caspase-9-reconstituted comparable to that of the Jurkat potent cells known with 15apoptosisrevealed inducer that its staurosporine. pro-apoptotic Treatment of caspase-9-deficient and caspase-9-reconstituted Jurkat cells with 15 revealed that its pro-apoptotic effect is connected with caspase-9-dependent intrinsic (mitochondrial) apoptosis. Moreover, treatment of murine embryonic fibroblasts (MEF) cells possessing high expression level of mCitrine-hLC3B with 15, and subsequent analysis of the level of LC3 protein (component of the

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effectMar.membrane Drugs is connected 2018 struct, 16, xure FOR with of PEER caspase-9-dependentautophagosomes) REVIEW in intrinsiccells, indicated (mitochondrial) that daldinone apoptosis. I (15 Moreover,), alone or treatment together5 of 32 ofwith murine the panembryonic-caspase ﬁbroblasts inhibitor (MEF) QVD,cells shows possessing inhibition high of expression autophagy level in of a mCitrine-hLC3B caspase-independent with 15membranemanner, and subsequent(Figure struct 4)ure. analysis of autophagosomes) of the level of in LC3 cells protein, indicated (component that daldinone of the I membrane (15), alone structureor together of autophagosomes)with the pan-caspase in cells, inhibitor indicated QVD that, shows daldinone inhibition I (15 ), of alone autophagy or together in a with caspase the-independent pan-caspase inhibitormanner (Figure QVD, shows4). inhibition of autophagy in a caspase-independent manner (Figure4).

Figure 4. Chemical structure of 15. Figure 4. Chemical structure of 15. Investigation of the fungal strainFigure Rhytidhysteron 4. Chemical structure rufulum ofAS21B 15. , isolated from Azima sarmentosa, whichInvestigation was collected of from the fungal a mangrove strain Rhytidhysteron area in Samutsakhon rufulum Province,AS21B, isolated Thailand, from affordedAzima sarmentosaa series of, whichspirobisnaphtInvestigation was collectedhalene of fromtheanalogs, fungal a mangrove among strain Rhytidhysteronthem area,in compounds Samutsakhon rufulum 16– Province,1AS21B8 and , 4isolated0 Thailand,–41, exhibiting from afforded Azima cytotoxic sarmentosa a series and of, spirobisnaphthalenewhichnitric oxide was collected (NO) production analogs,from a mangrove among inhibitory them, area activities, compounds in Samutsakhon respectively16–18 andProvince, (see40– 41Section, Thailand, exhibiting 2.1.3 ).afforded cytotoxic Cultivation a and series of nitric the of oxidespirobisnaphtfungus (NO) under productionh slightlyalene analogs, acidic inhibitory conditionamong activities, thems (pH, respectivelycompounds = 5) led to(see 1 a6 – distinct1 Section8 and change42.1.30–4).1, Cultivation exhibiting in its metabolic cytotoxic of the profilefungus and, undernitricaffording oxide slightly two (NO) acidic new production spirobisnaphthalenes,conditions inhibitory (pH = 5) ledactivities, rhytidenones to a distinct respectively change G and (see in H its Section (17 metabolic), along 2.1.3 proﬁle, with). Cultivation eleven affording known of twothe newfungcompounds,us spirobisnaphthalenes, under among slightly them, acidic rhytidenones rhytidenones conditions EG(pH and and = F H 5)(16 ( 17 led and), along to 18 a, distinctrespectively) with eleven change known[2 9 in]. Theits compounds, metabolicproduction profile among of the, them,affordinglatter compound rhytidenones two new (18 E spirobisnaphthalenes,) andwas Fincreased (16 and 18 8-,fold respectively) compared rhytidenones [29 to]. the The G andculture production H (under17), of along normal the latter with conditions compound eleven known (pH (18 =) wascompounds,7), allowing increased itsamong 8-fold detection them, compared and rhytidenones isolation to the culture fromE and under the F ( 16culture normal and 18 extract. conditions, respectively) Compounds (pH [ =29 7),]. The allowing16 andproduction 18 its displayed detection of the andlatterpotent isolation compound activity from against (18 the) was culture Ramos increased extract. and drug 8 Compounds-fold resistant compared NSCLC16 and to the18 H1975 displayedculture (non under- potentsmall normal cell activity lung conditions against cancer) Ramos (pH cells =, 7), allowing its detection and isolation from the culture extract. Compounds 16 and 18 displayed andwith drugIC50 values resistant in the NSCLC range H1975 between (non-small 0.018 and cell 1.17 lung μM. cancer) Moreover, cells, compound with IC50 17values showed in the selective range betweenpotentactivity activity toward 0.018 andagainstthe Ramos 1.17 RamosµM. lymphoma Moreover, and drug cell compound resistant line with NSCLC an17 ICshowed50 valueH1975 selective of (non 0.461-small μ activityM. Thesecell towardlung findings cancer) the suggest Ramos cells, with IC50 values in the range between 0.018 and 1.17 μM. Moreover, compound 17 showed selective lymphomathat the C-4 cell α,β line-unsaturated with an IC ketone50 value moiety of 0.461 is essentialµM. These for ﬁndings the potent suggest cytotoxicity that the of C-4 theseα,β-unsaturated metabolites ketoneactivity(Figure moiety 5)toward [29]. is the essential Ramos for lymphoma the potent cell cytotoxicity line with an of IC these50 value metabolites of 0.461 μ (FigureM. These5)[29 findings]. suggest that the C-4 α,β-unsaturated ketone moiety is essential for the potent cytotoxicity of these metabolites (Figure 5) [29].

Figure 5. Chemical structures of 1616–18..

Phomoxanthone A (19) is a tetrahydroxanthone dimeric natural product that has attracted Phomoxanthone A (19) is Figure a tetrahydroxanthone 5. Chemical structures dimeric of 16 – natural18. product that has attracted significantsignificant attention attention due due to to its its cytotoxic, cytotoxic, antibacterial, antibacterial and, and antifungal antifungal properties. properties. In our In research our research group, thisgroup intriguingPhomoxanthone, this intriguing metabolite A metabolite ( was19) is isolated a tetrahydroxanthone was from isolated the endophyte from the dimeric Phomopsis endophyte natural longicolla Phomopsis productthat longicolla that was has derived that attracted from was thesignificantderived mangrove from attention plantthe mangrove Sonneratia due to its plant caesolariscytotoxic, Sonneratia, collected antibacterial caesolaris on Hainan, , and collected antifungal Island, on South Hainan properties. China Island, (Figure In Southour6 )[ research 30 Ch,31ina]. Initialgroup(Figure, cytotoxicity this 6) [30 intriguing,31]. screening Initial metabolite cytotoxicity of 19 showed was screening isolated pronounced from of 19 the growthshowed endophyte inhibition pronounced Phomopsis of cisplatin-sensitive growth longicolla inhibition that wasand of -resistant cell lines (IC50 in the range from 0.7 to 5.2 µM) in our bioassays, confirming the literature derivedcisplatin - fromsensitive the andmangrove -resistant plant cell Sonneratia lines (IC 50 caesolaris in the range, collected from on0.7 Hainanto 5.2 μ M) Island, in our South bioassays, China data(Figureconfirming [30 ]. 6) [ Preliminary30 the,31 ]. literature Initial mechanistic cytotoxicity data [30] investigations. screening Preliminary of revealed19mechanistic showed caspase pronounced investigations activation growth and revealed proapoptotic inhibition caspase of activities of 19. Moreover, potent activation of murine T cells, NK cells, and macrophages after cisplatinactivation-sensitive and proapoptotic and -resistant activities cell linesof 19 .(IC Moreover,50 in the rangepotent from activation 0.7 to of 5.2 murine μM) inT cells,our bioassays, NK cells, treatmentconfirmingand macrophages with the 19 literature afterwere treatment observed data [30]with assuming. Preliminary19 were cell observed immune mechanistic assuming stimulation investigations cell to immune be part revealedstimulation of the biological caspase to be 0 profileactivationpart of ofthe and phomoxanthone biol proapoptoticogical profile Aactivities of ( 19phomoxanthone). Semisyntheticof 19. Moreover, A ( studies19 potent). Semisynthetic revealed activation that studies of murine a 4–4 revealed linkageT cells, that NK between a cells, 4–4′ andlinkage macrophages between the after tetrahydroxanthone treatment with 19 monomers were observed is favorable assuming for mediating cell immune cytotoxicity stimulation [30 ].to The be part of the biological profile of phomoxanthone A (19). Semisynthetic studies revealed that a 4–4′ linkage between the tetrahydroxanthone monomers is favorable for mediating cytotoxicity [30]. The

Mar. Drugs 2018, 16, 319 6 of 31 the tetrahydroxanthone monomers is favorable for mediating cytotoxicity [30]. The pronounced cytotoxicity of phomoxanthone A (19) towards cancer cells encouraged further investigation of its mode of action, through which the apoptotic events in the cells are released. At first, the effect of 19 on cellular Ca2+ levels was examined [32]. As a result, the cytosolic Ca2+ concentration in Ramos cells treated with 19 was strongly increased, showing a similar pattern to that of the tyrosine phosphatase inhibitor pervanadate; however, 19 did not show any activity when tested against a broad panel of protein kinases. Furthermore, 19 was able to increase the cytosolic Ca2+ level in the absence of extracellular Ca2+, thus suggesting that the Ca2+ increase is caused by direct effects of the compound on the endoplasmic reticulum (ER) or on mitochondria. Co-treatment of the cells with thapsigargin, an inhibitor of the ER CaATPase, and 19 resulted in a higher increase of the Ca2+ concentration than that observed for cells treated solely with 19, thus indicating that the Ca2+ origin caused by 19 should be connected with other cell sources of these ions. Indeed, estimation of the effect of 19 on Ca2+ using HeLa cells expressing CEPIA (calcium-measuring organelle-entrapped protein indicators) Ca2+ probes targeted to the ER or mitochondria confirmed that 19 causes strong and rapid depletion of Ca2+ stored in mitochondria. Further comparison of the mitochondrial response after treatment with 19 with effects of the known mitochondrial permeability transition pore (mPTP) inducer ionomycin, and the mPTP inhibitor, cyclosporine A, allowed the conclusion that Ca2+ release caused by 19 is largely independent from the mPTP mechanism. Therefore, the mitochondrial Ca2+ release is likely to be connected with changes in other mitochondrial ion gradients. Further analysis of the effect of 19 revealed immediate depolarization of the membrane potential ∆Ψm, similar to carbonyl cyanide m-chlorophenyl hydrazone (CCCP), an inhibitor of oxidative phosphorylation. However, 19 produced only a slight decrease in cellular O2 consumption, in contrast to the rapid kinetics by CCCP, caused through the uncoupling of the proton gradient. Moreover, measurement of O2 consumption after respiration enhancement by CCCP showed a strong decrease in O2 utilization, pointing to the inhibition of cellular respiration and of electron transport chain (ETC) by 19. Detailed analysis of the effects of 19 on these mitochondrial processes based on comparison with known ETC inhibitors that target different complexes of the ETC and ATP synthase, such as rotenone, thenoyltrifluoroacetone, and antimycin A, suggested that 19 disturbs complex I and II of ETC or interferes with the shuttling of electrons between complex I/II and III. Moreover, 19, like CCCP, caused stress-induced OPA1 (enzyme essential for regulation of the equilibrium between mitochondrial fusion and mitochondrial fission) cleavage that was dependent on metalloendopeptidase OMA1, and led to cristae disruption, although it was proven that the disruption occurs independently of OMA1. Since mitochondrial fission is also regulated by the dynamin-related protein DRP1, which mediates outer mitochondrial membrane fission, the DRP1-deficient MEF cells were analyzed in the presence of 19, and the fragmentation was likewise observed. Experiments with dual staining of the mitochondrial matrix and the outer mitochondrial membrane, affected by 19 and CCCP (a positive control for fragmentation), showed that the outer membrane appeared to remain connected. This effect could also be observed in cells deficient for both DRP1 and OPA1, indicating induction of mitochondrial fission by 19 independently of canonical regulators. Finally, a close examination of the mitochondrial ultrastructure by transmission electron microscopy (TEM) revealed that 19 causes OMA1-independent disruption of mitochondrial matrix morphology, complete loss of cristae, and condensation of inner membrane structures at the outer membrane. Taken together, it was shown that the inner, but not the outer, mitochondrial membrane structure rapidly collapses into fragments, leading to cristae disruption, and eventually apoptosis upon treatment with 19, thus implying that phomoxanthone A (19) is a mitochondrial toxin with a novel mode of action, which is distinct from other known ETC inhibitors, OXPHOS uncouplers, and ionophores. Further studies on the identification of the molecular target of 19, through which mitochondrial Ca2+ release and inner mitochondrial membrane fission is induced, will undoubtedly uncover the mechanism of action of this interesting mycotoxin [32]. Mar. Drugs 2018, 16, 319 7 of 31 Mar. Drugs 2018, 16, x FOR PEER REVIEW 7 of 32

Figure 6. Chemical structure of 19. Figure 6. Chemical structure of 19. 2.1.2. Antimicrobial Compounds 2.1.2. Antimicrobial Compounds Subsequent work on the fungal extract of P. brocae MA-231 (see Section 2.1.1), cultured on PDB medium,Subsequent allowed work for the on characterizationthe fungal extract of of five P. brocae new sulfide MA-231 diketopiperazine (see Section 2.1.1) derivatives,, cultured on namely, PDB penicibrocazinesmedium, allowed A–E,for the and characterization one known analog, of five phomazine new sulfide B diketopiperazine (24)[25]. All compounds derivatives, were namely, tested forpenicibrocazines their antimicrobial A–E, and properties one known against analog, several phomazine human- B or(24 plant-pathogenic,) [25]. All compounds as wellwere as tested marine for microorganisms.their antimicrobial Penicibrocazines properties against B–D (20 several–22) and human24 (Figure- or7 ) plant showed-pathogenic activity against, as wellS. aureus as marinewith MICmicroorganisms. values of 82, 0.55,Penicibrocazines 17.6, and 0.55 Bµ–MD (32.0,(20–22 0.25,) and 8.0 24 and (Figure 0.25 µ 7)g/mL), showed respectively, activity against compared S. aureus to the positivewith MIC control values chloromycetin of 82, 0.55, 17.6 (MIC, and =0.55 12.4 μµMM/4.0 (32.0, µ0.25,g/mL). 8.0 and Compound 0.25 μg/mL),21 showed respectively, strong compared inhibiting activityto the positive against controlMicrococcus chloromycetin luteus with (MIC an MIC = 12.4 value μ ofM/ 0.554.0 μµg/mL).M (0.25 Compoundµg/mL), which 21 showed is higher strong than thatinhibiting of chloromycetin activity against (MIC Micrococcus = 6.2 µM/2.0 luteusµg/mL). with Moreover,an MIC value derivatives of 0.5520 μM, 22 (,0.25 penicibrocazine μg/mL), which E (23 is) andhigher24 were than active that of against chloromycetin the plant pathogen (MIC = 6.2Gaeumannomyces μM/2.0 μg/mL). graminis Moreover,with MICs derivatives 0.64, 17.6, 20,0.55, 22, andpenicibrocazine 159 µM (0.25, E 8.0,(23) 0.25, and and24 were 64.0 µactiveg/mL), against respectively, the plant whereas pathogen the positiveGaeumannomyces control amphotericin graminis with B displayedMICs 0.64, an17.6, MIC 0.55 value, and of 159 17.3 μMµ M(0.25, (16.0 8.0,µg/mL). 0.25, and Evaluation 64.0 μg/mL), of penicibrocazines respectively, whereas A–E against the positive eight tumorcontrol cell amphotericin lines showed B no displayed significant an cytotoxicity MIC value (IC of50 > 17.3 10 µμM).M Further(16.0 μg/mL cultivation). Evaluation of P. brocae of inpenicibrocazines liquid Czapek A medium–E against yielded eight tumor four new cell lines thiodiketopiperazine showed no significant alkaloids, cytotoxicity penicibrocazines (IC50 > 10 μ F–I,M). asFurther well ascultivation two new ofN -containingP. brocae inp liquid-hydroxyphenopyrrozin Czapek medium yielded derivatives four brocapyrrozins new thiodiketopiperazine A (25) and B[alkaloids,26]. Subjected penicibrocazines to the same F– bioactivityI, as well assays as two as in new the previousN-containing report p- [hydroxyphenopyrrozin25], 25 and the known compoundderivatives 4-hydroxy-3-phenyl-1 brocapyrrozins A (25H)-pyrrol-2(5 and B [26H].)-one Subjected (26) (Figure to the7 ) same showed bioactivity strong inhibitory assays as activity in the againstprevious the report bacterium [25], 25S. aureusand theand known the fungus compoundFusarium 4-hydroxy oxysporum-3-phenylwith- MIC1H-pyrrol values-2(5 rangingH)-one from (26) 0.41(Figure to 2.857) showedµM (0.125 strong to inhibitory 0.5 µg/mL). activity These against values the were bacterium equipotent S. aureus or stronger and the than fungus those Fusarium of the positiveoxysporum antibacterial with MIC andvalues antifungal ranging controls,from 0.41 chloromycetin to 2.85 μM (0.125 (MIC =to 1.550.5 μµM/0.5g/mL). µTheseg/mL) values and zeocin were (MICequipotent = 0.35 orµ M/0.5strongerµg/mL), than those respectively. of the positive The aforementioned antibacterial and results antifungal indicated controls that, thechloro presencemycetin of an(MIC acetonyl = 1.55 μ groupM/0.5 at μg/mL) C-2 is favorableand zeocin for (MIC the antibiotic= 0.35 μM/ activity0.5 μg/mL), of brocapyrrozins. respectively. The aforementioned results indicated that the presence of an acetonyl group at C-2 is favorable for the antibiotic activity of brocapyrrozins.

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Figure 7. Chemical structures of 20–26. Figure 7. Chemical structures of 20–26. A fungal strain Stemphylium sp. 33231 isolated from the mangrove Bruguiera sexangula var. Arhynchopetala fungal straincollected Stemphylium from thesp. South33231 isolatedChina Sea from yielded the mangrove eight new Bruguiera and seventeen sexangula known var. metabolitesrhynchopetala [33 collected]. Among from them, the twoSouth new China anthraquinone Sea yielded derivatives eight new and (27 andseventeen28), two known alterporriol-type metabolites anthranoid[33]. Among dimers them, (two29 and new30 anthraquinone), as well as the derivatives known analogs, (27 and altersolanols 28), two alterporriol A–C (31–-33type), macrosporin anthranoid (dimers34), tetrahydroaltersolanol (29 and 30), as well B as (35 the), and known alterporriols analogs, B altersolanols (36), C (37), A D–C (38 (),31 and–33), E macrosporin (39), were found (34), totetrahydroaltersolanol possess moderate or B weak (35), and antibacterial alterporriols properties B (36), C when (37), D tested (38), againstand E (39 a) panel, were offound terrestrial to possess and pathogenicmoderate or bacteria, weak antibacterial such as Micrococcus properties tetragenus when ,testedE. coli against, Staphylococcus a panel albusof terrestrial, Bacillus and cereus pathogenic, S. aureus, Kocuriabacteria, rhizophila such as Micrococcus, and Bacillus tetragenus subtilis, (FigureE. coli, 8Staphylococcus). Metabolites albus28,, Bacillus31, 32, cereus34, and, S. 38aureuswere, Kocuria active againstrhizophila at, and least Baci threellus bacterial subtilis (Figure strains, 8) with. Metabolites minimum 28 inhibitory, 31, 32, 34,concentration and 38 were active (MIC) against values at in least the rangethree bacterial between strains 2.07 and, with 10 µ M.minimum Compounds inhibitory27, 33 concentration, and 37 exhibited (MIC) selective values activityin the range against betweenE. coli (MIC2.07 and = 9.8 10µ μM),M. B.Compounds subtilis (MIC 27 =, 33 8.8, andµM), 37 and exhibitedS. albus selective(MIC = 8.9 activityµM), respectively,against E. coli while (MIC compounds = 9.8 μM), 29B. ,subtilis30, and (MIC36 showed = 8.8 μM), selectivity and S. albus against (MICB. = cereus8.9 μM),strain respectively, with MIC while values compounds of 8.3, 8.1, 29 and, 30, 7.9 andµ M,36 correspondingly.showed selectivity In against addition, B. cereus compound strain39 withshowed MIC values antibacterial of 8.3, activity8.1, and against7.9 μM, two correspo testedndingly. strains, B.In cereusaddition,(MIC compound = 2.5 µM) and39 showedE. coli (MIC antibacterial = 5.0 µM). activity All aforementioned against two tested metabolites, strains, with B. cereus the exception (MIC = of2.533 μM)and and35, E. were coli investigated (MIC = 5.0 μ forM). cytotoxicity All aforementioned against mouse metabolites, melanoma with (B16F10) the exception and A549 of 33 cell and lines, 35, were investigated for cytotoxicity against mouse melanoma (B16F10) and A549 cell lines, however, however, showed no activity (IC50 > 10 µM). Moreover, these compounds were found to be inactive whenshowed tested no activity for brine (IC shrimp50 > 10 μM). lethality Moreover, using theseArtemia compounds salina, thus were suggesting found to be that inactive their antibacterial when tested activityfor brine is shrimp not due lethality to general using cytotoxicity Artemia salina [33]. , thus suggesting that their antibacterial activity is not due to general cytotoxicity [33].

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Figure 8. Chemical structures of 27–39. Figure 8. Chemical structures of 27–39. 2.1.3. Compounds with Inhibitory Activity towards NO Production 2.1.3. Compounds with Inhibitory Activity towards NO Production The endophyte R. rufulum AS21B (see Section 2.1.1) afforded six new spirobisnaphthalene derivatives,The endophyte namely, rhytidenonesR. rufulum AS21B A–F [ (see34]. SectionAmong 2.1.1) the isolated afforded compounds, six new spirobisnaphthalene rhytidenone C (40) showedderivatives, the most namely, potent rhytidenones inhibitory effect A–F [3 on4]. NO Among production the isolated in lipopolysaccharide compounds, rhytidenone (LPS)-stimulated C (40) J774.A1showed macrophagesthe most potent with inhibitory an IC50 value effect of on 0.31 NOµM. production On the other in lipopolysaccharide hand, the anti-inflammatory (LPS)-stimulated activity ofJ774.A1 rhytidenone macrophages D (41) with was an 10-fold IC50 value lower of (IC0.3150 μ=M. 3.60 On µtheM), other indicating hand, the that anti the-inflammatoryα-orientation activity of the hydroxyof rhytidenone group D at ( position41) was 10 7- isfold favorable lower (IC for50 the= 3.60 anti-inflammatory μM), indicating activitythat the of α- theseorientation metabolites. of the Remarkably,hydroxy group40 and at position41 exhibited 7 is favorableno cytotoxicity for the in antithe respective-inflammatory cells, activity thus indicating of these that metabolites. they are potentialRemarkably, leads 40 for and the 41 development exhibited no of cytotoxicity anti-inflammatory in the respective agents (Figure cells, 9thus). indicating that they are potential leads for the development of anti-inflammatory agents (Figure 9).

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Figure 9. Chemical structures of 40 andand 4141.. Figure 9. Chemical structures of 40 and 41. Cultivation of the fungus L.L. theobromae ZJ-HQ1ZJ-HQ1 (see Section 2.1.1 ) on on solid solid rice rice media media with with differentCultivation salinitysalinity (0.3%)(0.3%) of the ledled fungus toto the L isolation. theobromae of the ZJ novel-HQ1 lactone (see Section lasiodiplactone 2.1.1) on AA solid (42) ) rice with media an unusual with tetracyclicdifferenttetracyclic salinity system, system, (0.3%) containing containing led to the a a 12isolation 12-membered-membered of the β novel-resorcylicβ-resorcylic lactone acid lasiodiplactone acid lactone lactone,, a pyran, aA pyran,(42 )and with anda anfuran unusual a furan ring ringtetracyclic(Figure (Figure 10) system, [35]. 10)[ Compound35 containing]. Compound 42 a showed1242-memberedshowed potent potentβ -inhibitoryresorcylic inhibitory activityacid activitylactone toward, towarda pyran,NO production NO and production a furan in LPSring in- (Figure 10) [35]. Compound 42 showed potent inhibitory activity toward NO production in LPS- LPS-activatedactivated RAW264.7 RAW264.7 cells cells with with an anIC50 IC value50 value of of23.5 23.5 μM,µM, comparable comparable to to that that of of the the positive positive control activated RAW264.7 cells with an IC50 value of 23.5 μM, comparable to that of the positive control indomethacinindomethacin (IC50 == 26.3 26.3 μµM).M). Interestingly, compound 42 did not show anyany cytotoxicitycytotoxicity againstagainst LPS-activatedindomethacinLPS-activated RAW264.7(IC50 = 26.3 cellscells μM). upup Interestingly, toto 100100 µμM,M, thusthus compound indicatingindicating 42 that thatdid thethenot NONO show inhibitoryinhibitory any cytotoxicity effect ofof 4242 against isis notnot dueLPS- toactivated cytotoxicity. RAW264.7 Moreover, cells upcompoundcompound to 100 μM, 4242 thusdisplayeddisplayed indicating strongstrong that αα-glucosidase- glucosidasethe NO inhibitory inhibitoryinhibitory effect activityactivity of 42 is with not due to cytotoxicity. Moreover, compound 42 displayed strong α-glucosidase inhibitory activity with an IC 50 valuevalue of of 29.4 29.4 μM,µM, which which was was stronger stronger than than the theclinically clinically used used anti- anti-diabeticdiabetic drug drug acarbose acarbose (IC50 an IC50 value of 29.4 μM, which was stronger than the clinically used anti-diabetic drug acarbose (IC50 (IC= 36750 =μM). 367 µM). = 367 μM).

Figure 10. Chemical structure of 42. Figure 10. Chemical structure of 42. 2.1.4. Compounds with α-GlucosidaseFigure Inhibitory 10. Chemical Activity structure of 42. 2.1.4. Compounds with α-Glucosidase Inhibitory Activity 2.1.4.The Compounds endophytic with fungus α-GlucosidaseAspergillus Inhibitorysp. 16-5B, Activity obtained from leaves of Sonneratia apetala (HainanThe Island,endophytic China), fungus was Aspergillus investigated, sp. and16-5B this, obtained resulted from in theleaves characterization of Sonneratia apetala of polyketides (Hainan withIsland,Thein vitroChina) endophyticα-glucosidase, was investigated fungus inhibitory Aspergillus, and activity this sp. resulted 16 (43-5B–45, obtainedin) (Figurethe characterization from11)[36 leaves]. Interestingly, of of Sonneratia polyketides the apetala new with metabolite (Hainan in vitro Island, China), was investigated, and this resulted in the characterization of polyketides with in vitro aspergifuranoneα-glucosidase inhibitory (43) showed activity signiﬁcant (43–45 inhibition) (Figure of 11) enzyme [36]. activity, Interestingly, with an the IC 50 newvalue metabolite of 9 µM, α-glucosidase inhibitory activity (43–45) (Figure 11) [36]. Interestingly, the new metabolite whichaspergifuranone is approximately (43) showed 60-fold significant more potent inhibition than the of clinically enzyme used activityα-glucosidase, with an IC inhibitor50 value acarboseof 9 μM, aspergifuranone (43) showed significant inhibition of enzyme activity, with an IC50 value of 9 μM, (ICwhich50 = is 554 approximatelyµM). Moreover, 60-fold a new more isocoumarin potent than derivativethe clinically (44 used), which α-glucosidase was isolated inhibitor as a racemate, acarbose which is approximately 60-fold more potent than the clinically used α-glucosidase inhibitor acarbose along(IC50 = with 554 theμM). known Moreover, metabolite a new pestaphthalide isocoumarin derivative A (45), displayed (44), which considerable was isolated inhibitory as a racemate, activities (IC50 = 554 μM). Moreover, a new isocoumarin derivative (44), which was isolated as a racemate, againstalong withα-glucosidase the known with metabolite IC50 values pestaphthalide of 90 and 70 AµM, (45 respectively.), displayed Subsequentconsiderable kinetic inhibitory analysis activities of the along with the known metabolite pestaphthalide A (45), displayed considerable inhibitory activities mostagainst active α-glucosidase compound with (43) revealedIC50 values that of it90 exhibits and 70 noncompetitiveμM, respectively. inhibition Subsequent characteristics. kinetic analysis A later of studyagainstthe most from α -activeglucosidase the samecompound researchwith IC(4350) group valuesrevealed led of that90 to theand it exhibits isolation70 μM, noncompetitiverespectively. of ﬁfteen isocoumarins Subsequent inhibition (kinetic 46characteristics.–60), analysis including ofA the most active compound (43) revealed that it exhibits noncompetitive inhibition characteristics. A sixlater new study natural from products, the same which research were group derived led from to theTalaromyces isolation amestolkiae of fifteen YX1, isocoumarins an endophyte (46–60 of), later study from the same research group led to the isolation of fifteen isocoumarins (46–60), Kandeliaincluding obovata six new(Guangdong natural products, Province, which China) were (Figure derived 11)[ 37 from]. Remarkably, Talaromyces all amestolkiae these derivatives YX1, an including six new natural products, which were derived from Talaromyces amestolkiae YX1, an exhibitedendophyteα-glucosidase of Kandelia obovat inhibitinga (Guangdong activity, with Province, IC50 values China ranging) (Figure from 11) 17.2 [37] to. Remarkably, 585.7 µM, which all these were endophyte of Kandelia obovata (Guangdong Province, China) (Figure 11) [37]. Remarkably, all these morederivatives potent exhibited than acarbose α-glucosidase (IC50 = 958.3 inhibitingµM). activity, with IC50 values ranging from 17.2 to 585.7 derivatives exhibited α-glucosidase inhibiting activity, with IC50 values ranging from 17.2 to 585.7 μM, which were more potent than acarbose (IC50 = 958.3 μM). μM, which were more potent than acarbose (IC50 = 958.3 μM).

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Figure 11. Chemical structures of 43–60. FigureFigure 11.11. Chemical structures of 4343––6060..

2.1.5.2.1.5. Compounds Compounds with with Mycobacterium Mycobacterium tuberculosistuberculosis ProteinProtein Tyrosine Tyrosine Phosphatase Phosphatase B BB (MptpB) (MptpB)(MptpB) InhibitoryInhibitory ActivityActivity Activity TheThe endophyticendophytic endophytic fungus fungus Diaporthe DiaportheDiaporthe sp.sp.sp. SYSU-HQ3,SYSU-HQ3, isolated from from the the mangrove mangrove plant plantplant Excoecaria ExcoecariaExcoecaria agallochaagallocha, , afforded afforded three three isoprenylisoindoleisoprenylisoindoleisoprenylisoindole alkaloids with with a a rare rare 1,4 1,4-benzodioxan1,4--benzodioxanbenzodioxan moiety, moiety, namely namely diaporisoindolesdiaporisoindoles A–C,A A––CC, ,in in addition addition toto thethe knownknown derivative tenellone tenellone C C ( (6262))[ )[38]. [38].38]. Diaporisoindole DiaporisoindoleDiaporisoindole A A ((61(61)) and)and and 6262 62 (Figure(Figure (Figure 1212) 12)) showedshowed showed strong strongstrong inhibitory inhibitoryinhibitory activityactivity towardtoward MptpBMptpB,MptpB, ,with withwith IC ICIC505050 values values of of 4.2 4.2 and and 5.25.2 µμ M,μM,M, respectivelyrespectively respectively (positive (positive (positive control control control oleanolic oleanolic oleanolic acid; acid;; ICICIC5050 22.122.1 μμµM).M). Interestingly, Interestingly, the the the co co-isolated co-isolated-isolated diastereomerdiastereomer diaporisoindole diaporisoindole B B,B,, possessingpossessing an 8R conﬁguration,configuration,, displayed displayed no no activity, activity, indicating indicatingindicating thatthat the the the SS S configuration conﬁgurationconfiguration at at at C C- C-8-88 is is essentialisessential essential for forthe theMptpB MptpB inhibiinhibi inhibitorytorytory effect effect effectof of 61 61. .In of In order61 order. In to toorder get get further further to get furtherinsightsinsights insights into into the the into mode mode the of modeof actionaction of actionofof ththeseese of compound these compounds,s,, enzymeenzyme enzyme kinetickinetic kinetic analysisanalysis analysis was was performed. performed. was performed. The The Theresultsresults results showed showed showed that that that 61 61 acted61actedacted asas an asan an uncompetitiveuncompetitive uncompetitive inhibitor, inhibitor, whereaswhereas whereas 626262 as asas a a acompetitivecompetitive competitive inhibitor. inhibitor. inhibitor. Moreover,Moreover, compoundscompound compoundss 6161 61 andand and 6262 62 showed showedshowed nono inhibitoryinhibitory activity activity against against protein protein tyrosine tyrosine phosphatase phosphatase 1B1B1B (PTP1B)(PTP1B) (PTP1B) atat at a a concentrationa concentration concentration of ofof 200 200200µM, μμM,M, suggesting suggesting that that they theythey are areare selective selectiveselective MptpB MptpB MptpB inhibitors, inhibitors, inhibitors, and and thusand potentialthusthus potential potential leads leads forleads anti-TB for for anti anti investigation.--TBTB investigation.investigation.

FigureFigure 12.12. ChemicalChemical structures of 6161 andand 6262.. Figure 12. Chemical structures of 61 and 62.

Mar. Drugs 2018, 16, 319 12 of 31

Further bioactive metabolites derived from mangrove endophytes that could serve as potential lead structures are presented in Table1, and Figures 13 and 14.

Table 1. Further bioactive compounds isolated from endophytic fungi (63–105) of mangrove origin.

Compound Name Source Type of Activity [Ref.] IC50 or MIC Values Peniphenone B (63) Penicillium dipodomyicola IC 0.16 µM MptpB inhibitory [39] 50 HN4-3A from Acanthus ilicifolius Peniphenone C (64)(Hainan Province, China) IC50 1.37 µM Mycobacterial Talaromyces sp. HZ-YX1 from IC 55 µM, serine/threonine protein 50 Talaramide A (65) Kandelia obovata (Guangdong positive control AX20017 kinase G (PknG) Province, China) (IC = 98 µM) 1 inhibitory [40] 50

IC50 66 µM; positive control ribavirin (IC50 94 µM); Neosartoryadin A (66) Neosartorya udagawae no cytotoxicity Anti-inﬂuenza A virus HDN13-313 from Avicennia against the human leukemia (H1N1) [41] marina (Hainan Province, China) (HL-60) cell line IC 58 µM; Neosartoryadin B (67) 50 -//- MIC 24.5 µM/8.0 µg/mL Citrifelin A (68) Co-culture of Penicillium (both E. coli and S. aureus) citrinum MA-197 Antibacterial against µ µ (from Lumnitzera racemosa) and E. coli and S. aureus [42] MICs 5.6 M/2.0 g/mL (E. coli) Citrifelin B (69) Beauveria felina EN-135 and 11.2 µM/4.0 µg/mL (S. aureus)

IC50 81.7 µM; Pinazaphilone A (70) positive control acarbose (IC50 = 446.7 µM)

IC50 28.0 µM; Pinazaphilone B (71) Penicillium sp. HN29-3B1 from α-Glucosidase -//- Cerbera manghas 0 0 inhibitory [43] µ 6 -Methyl-[1,1 -biphenyl]- (Hainan Island, China) IC50 2.2 M; 3,30,40,5-tetraol (72) -//- IC 16.6 µM; Sch 1385568 (73) 50 -//- IC 14.4 µM; (±)-Penifupyrone (74) 50 -//-

IC50 188.7 µM; Microsphaeropsisin C (75) positive control acarbose (IC50 = 703.8 µM)

(3R,7R)-7-Hydroxy-de-O- IC50 25.8 µM; methyllasiodiplodin (76) -//-

(3R)-5-Oxo-de-O- IC50 54.6 µM; methyllasiodiplodin (77) -//- Co-culture of Trichoderma sp. 307 (3R)-7-Oxo-de-O- (from Clerodendrum inerme; α-Glucosidase IC50 178.5 µM; methyllasiodiplodin (78) Guangdong Province, China) inhibitory [44] -//- with Acinetobacter johnsonii B2 (3R)-5-Oxolasiodiplodin IC50 176.8 µM; (79) -//-

(3R,4R)-4-Hydroxy-de-O- IC50 60.3 µM; methyllasiodiplodin (80) -//- IC 198.1 µM; (3S)-Ozoroalide (81) 50 -//-

(E)-9-Etheno-lasiodiplodin IC50 101.3 µM; (82) -//- Mar. Drugs 2018, 16, 319 13 of 31

Table 1. Cont.

Compound Name Source Type of Activity [Ref.] IC50 or MIC Values IC 0.16 (MDA-MB-435) and Secalonic acid A (83) 50 0.41 µM (SW-620) IC 0.18 (MDA-MB-435) and Penicillixanthone A (84) 50 Cytotoxic against 0.21 µM (SW-620) MDA-MB-435 IC 4.06 (MDA-MB-435) and Blennolide J (85) (melanoma) and SW-620 50 µ Plant endophyte (colon cancer) cell 6.14 M (SW-620) Setophoma terrestris from a leaf lines [45] IC 0.58 (MDA-MB-435) and Hypothemycin (86) 50 litter collected in 2.14 µM (SW-620) a mangrove habitat IC 5.20 (MDA-MB-435) and Penicillixanthone B (87) 50 5.55 µM (SW-620) Cytotoxic against MDA-MB-435 and IC50 3.27 (MDA-MB-435) and Secalonic acid G (88) SW-620 cell 3.67 µM (SW-620)/MIC 7.83 µM lines/antibacterial (/5 µg/mL) against M. luteus [45] Rhizovarin A (89) 9.6 µM (HL-60) Rhizovarin B (90) 6.3 (A549) and 5.0 µM (HL-60) Rhizovarin E (91) 9.2 µM (A549) Cytotoxic against A549 Mucor irregularis QEN-189 from Penitrem A (92) and/or HL-60 8.4 (A549) and 7.0 µM (HL-60) Rhizophora stylosa (promyelocytic leukemia) Penitrem C (93) (Hainan Island, China) 8.0 (A549) and 4.7 µM (HL-60) cancer cell lines [46] Penitrem F (94) 8.2 (A549) and 3.3 µM (HL-60) 10β-Hydroxy-13- 4.6 (A549) and 2.6 µM (HL-60) desoxypaxilline (95) 7-O-Methylnigrosporolide IC50 0.7 µM (L5178Y) (96) Cytotoxic against L5178Y Pestalotiopsis microspora from (murine lymphoma) cell Pestalotioprolide D (97) IC50 5.6 µM (L5178Y) Drepanocarpus lunatus line or human ovarian IC 3.4 (L5178Y) and 1.2 µM; Pestalotioprolide E (98) (Cameroon) (A2780) cancer cell 50 line [47] (A2780)

Pestalotioprolide F (99) IC50 3.9 µM (L5178Y) IC 0.5 (HeLa) and Penicisulfuranol A (100) 50 µ Penicillium janthinellum 0.1 (HL-60) M; HDN13-309 from Sonneratia Cytotoxic against HeLa IC 3.9 (HeLa) and 1.6 µM Penicisulfuranol B (101) 50 caseolaris (Hainan Province, and HL-60 cell lines [48] (HL-60) China) IC 0.3 (HeLa) and 1.2 µM Penicisulfuranol C (102) 50 (HL-60) Eupenicillium sp. HJ002 from Cytotoxic against A549 IC 5.5 (A549) and Penicilindole A (103) Xylocarpus granatum 50 and HepG2 cell lines [49] 1.5 (HepG2) µM (South China Sea)

epi-Isochromophilone II IC50 4.4 (ACHN), 3.0 (786-O) (104) and 3.9 µM (OS-RC-2)

Cytotoxic against renal IC50 14 (ACHN), 8.9 (786-O) and Diaporthe sp. SCSIO 41011 from carcinoma cell lines: 13 µM (OS-RC-2); induced Rhizophora stylosa (Hainan ACHN, OS-RC-2, apoptosis (in 786-O cells) in Province, China) Isochromophilone D (105) and 786-O [50] a dose- and time-dependent manner, whereas it did not induce cell cycle arrest at a concentration level up to 10 µM. 1 Positive control is indicated in case the IC50 value of the respective compound is higher than 10 µM. Mar. Drugs 2018, 16, 319 14 of 31 Mar. Drugs 2018, 16, x FOR PEER REVIEW 14 of 32

FigureFigure 13. 13. ChemicalChemical structures structures of compounds of compounds with MptpB with MptpB-- (63 and (6634), mycobacterialand 64), mycobacterial PknG (65), anti PknG- (65infective), anti-infective (66–69) and (66– α69-glucosidase) and α-glucosidase (70–82) inhibitory (70–82) activities inhibitory derived activities from derived mangrove from endophytic mangrove endophyticfungi. fungi.

Mar.Mar. Drugs Drugs2018 2018, 16,, 16 319, x FOR PEER REVIEW 15 of1531 of 32

(a)

Figure 14. Cont.

Mar.Mar. Drugs Drugs 20182018,, 1616,, x 319 FOR PEER REVIEW 16 of 3132

(b)

FigureFigure 14.14. (a(a)) Chemical Chemical structures structures of compounds of compounds with cytotoxic with cytotoxic activity activity (83–95) derived (83–95) from derived mangrove from mangroveendophytic endophytic fungi. (b) Chemicalfungi. (b) structures Chemical of structures compounds of compounds with cytotoxic with activity cytotoxic (96–105 activity) derived (96– from105) derivedmangrove from endophytic mangrove fungi. endophytic fungi.

2.2.2.2. Bioactive Bioactive Compounds Compounds Derived Derived from from Fungi Fungi Originating Originating from Mangrove ( (Rhizosphere)Rhizosphere) Soil/Sediment Soil/Sediment Samples Samples 2.2.1. Cytotoxic Compounds 2.2.1. Cytotoxic Compounds The fungal strain Aspergillus versicolor HDN1009 derived from mangrove soil that was collected in Guangzhou,The fungal strain China Aspergillus yielded six versicolor unusual HDN1009 heterogeneous derived dimers,from mangrove versixanthones soil that A–Fwas (collected106–111) inpossessing Guangzhou, a tetrahydroxanthone China yielded six unit unusual anda heterogeneous biogenetically-related dimers, chromanone versixanthones monomer A–F (106 coupled–111) possessingvia a biaryl a linkage,tetrahydroxanthone as well as a unit known and compound,a biogenetically secalonic-related acid chromanone D (112)[51 monomer]. All compounds coupled viawere a biaryl tested link forage, cytotoxicity as well as toward a known HL-60, compound, K562 (myelogenous secalonic acid leukemia), D (112) [51]. A549, All compounds H1975, MGC-803 were tested(human for gastric cytotoxicity cancer), toward HO8910 HL-60, (ovarian K562 (myelogenous cancer), and leukemia), HCT-116 (colorectal A549, H1975, carcinoma) MGC-803 cell (human lines. gastric cancer), HO8910 (ovarian cancer), and HCT-116 (colorectal carcinoma) cell lines. Interestingly, Interestingly, compounds 106–108 showed activity against at least two cell types, with IC50 values compoundsbetween 2.6 106 and–108 25.6 showedµM, and activity derivatives against109 at– least112 exhibited two cell types cytotoxicity, with IC against50 values at between least ﬁve 2.6 cancer and 25.6 μM, and derivatives 109–112 exhibited cytotoxicity against at least five cancer lines with IC50 lines with IC50 values ranging between 0.7 and 21 µM (Figure 15). Remarkably, among all new valuescompounds, ranging only between110 revealed 0.7 and topoisomerase21 μM (Figure 15) I inhibitory. Remarkably, activity, among as was all new previously compounds, shown only for 110 the revealedknown derivative topoisomerase secalonic I inhibitory acid D (112 activity,)[51]. as was previously shown for the known derivative secalonic acid D (112) [51].

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Figure 15. Chemical structures of 106–112. Figure 15. Chemical structures of 106–112. Addition of the DNA methyltransferase inhibitor 5-azacytidine to the fungus Penicillium variabile HXQ-H-1,Addition isolated of the from DNA the methyltransferase mangrove rhizosphere inhibitor soil 5 collected-azacytidine on the to coastthe fungus of Fujian Penicillium Province, variabile China, ledHXQ to alteration-H-1, isolated of the from fungal the metabolome, mangrove rhizosphere yielding a highly soil collected modiﬁed on fatty the acid coast amide, of Fujian varitatin Province A (113, ) China, led to alteration of the fungal metabolome, yielding a highly modified fatty acid amide, (Figure 16)[52]. Interestingly, 113 exhibited activity against the HCT-116 cell line with an IC50 value ofvaritatin 2.8 µM. A Moreover, (113) (Figure it inhibited 16) [52]. 50% Interestingly, and 40%of 113 the exhi proteinbited tyrosineactivity against kinases the PDGFR- HCT-β116and cell ErbB4, line respectively,with an IC50 at value a concentration of 2.8 μM. ofMoreover, 1 µM, suggesting it inhibited that 50% the cytotoxicityand 40% of ofthe113 proteinis probably tyrosine exerted kinases due PDGFR-β and ErbB4, respectively, at a concentration of 1 μM, suggesting that the cytotoxicity of 113 to its protein kinase inhibitory activity. Subsequent mixed fermentation of this mangrove fungal strain is probably exerted due to its protein kinase inhibitory activity. Subsequent mixed fermentation of with the deep-sea-derived fungus Talaromyces aculeatus (collected at a depth of 3386 m, Indian Ocean) this mangrove fungal strain with the deep-sea-derived fungus Talaromyces aculeatus (collected at a afforded four novel polyketides, penitalarins A–C, containing an unusual 3,6-dioxabicyclo[3.1.0]hexane, depth of 3386 m, Indian Ocean) afforded four novel polyketides, penitalarins A–C, containing an in addition to nafuredins A and B (114) (Figure 16), which were not detected in the axenic fungal unusual 3,6-dioxabicyclo[3.1.0]hexane, in addition to nafuredins A and B (114) (Figure 16), which cultures under the same conditions [53]. Compound 114 showed cytotoxicity against a panel of human were not detected in the axenic fungal cultures under the same conditions [53]. Compound 114 cancer cell lines (HeLa, K562, HCT-116, HL-60, A549, and MCF-7), with IC values in the range from showed cytotoxicity against a panel of human cancer cell lines (HeLa, K562,50 HCT-116, HL-60, A549, 1.2 to 9.8 µM, whereas penitalarins A–C and nafuredin A proved to be inactive. and MCF-7), with IC50 values in the range from 1.2 to 9.8 μM, whereas penitalarins A–C and nafuredin A proved to be inactive.

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Figure 16. Chemical structures of 113 and 114. Figure 16. Chemical structures of 113 and 114. In 2009, a study on the chemical constituents of the solid-phase culture of the fungus Aspergillus ustusIn094102 2009, a (from studyBruguiera on the chemical gymnorrhiza constituentscollected of inthe Hainan solid-phase Province, culture China) of the afforded fungus Aspergillus a series of ustuscytotoxic 094102 drimane (from sesqui- Bruguiera and gymnorrhiza meroterpenoids collected [54]. Toin furtherHainan investigateProvince, China) the biosynthetic afforded capacitya series of A.cytotoxic ustus, thisdrimane fungus sesqui was- cultured and meroterpenoids in both liquid [54]. and To on further solid media investigate to obtain the extracts biosynthetic enriched capacity with ofophiobolin A. ustus, derivatives.this fungus Eventually,was cultured the in chromatographic both liquid and work-upon solid ofmedi botha to extracts obtain yielded extracts seven enriched new withand elevenophiobolin known derivatives. ophiobolin Eventually congeners, the [55 chromatographic]. Interestingly, ophiobolins work-up of were both onlyextracts produced yielded during seven newstatic and cultivation, eleven known but not ophiobolin in the shaking congeners mode. In[55]. addition, Interestingly, the metabolic ophiobolins profile were of liquid only cultivationproduced duringunder staticstatic conditioncultivation, was but investigated not in the shaking at different mode. times/days; In addition, however, the metabolic no significant profile of effect liquid on ctheultivation production under of static ophiobolins condition was was found. investigated Compounds at different115–124 times/days;(Figure 17however,) showed no cytotoxicity significant effectagainst on the the human production gemcitabine-resistant of ophiobolins G3Kwas (pancreatic found. Compounds cancer) cell 115 line,–124 MCF-7, (Figure MD-MBA-231 17) showed (triple-negativecytotoxicity against breast the cancer)human cells,gemcitabine MCF-7/Adr-resistant (adriamycin-resistant G3K (pancreatic cancer) human cell breast line, MCF cancer)-7, MD cell- MBA-231 (triple-negative breast cancer) cells, MCF-7/Adr (adriamycin-resistant human breast line, MCF-10A (nontumorigenic breast epithelial cell line), A549, and HL-60 cells, with IC50 values cancer)in the rangecell line, from MCF 0.6-10A to 9.5 (nontumorigenicµM. Among the breast tested epithelial compounds, cell line), 21- A549,epi-ophiobolin and HL-60 O cells (121,) with was IC50 values in the range from 0.6 to 9.5 μM. Among the tested compounds, 21-epi-ophiobolin O (121) found to be the most active analog, with IC50 values of 0.6 and 0.8 µM toward the A549 and wasHL-60 found cell to lines, be the respectively, most active thus analog suggesting, with IC50 that values the of 2,5-dimethoxyl-2 0.6 and 0.8 μM towardH,3H,5H the-furan A549 moiety and HL is- 60a key cell structurallines, respectively, feature for thus cytotoxicity suggesting against that the the 2,5 tested-dimethoxyl cell lines-2H [,355H].,5H It-furan should moiety be noted is a thatkey structuralophiobolin feature O (122 for) has cytotoxicity previously against been the shown tested to cell induce lines cell[55]. apoptosis It should be in noted human that breast ophiobolin cancer OMCF-7 (122) cellshas previously via activation been ofshown mitogen-activated to induce cell apoptosis protein kinase in human (MAPK) breast signaling cancer MCF pathways-7 cells [ 56via]. activationMoreover, of inverse mitogen docking-activated analysis protein suggested kinase (MAPK) that 122 signalingcould bind pathways to glycogen [56]. Moreover, synthase inverse kinase docking3 beta (GSK3 analysisβ), whichsuggested is an that upstream 122 could regulator bind to ofglycogen G1 phase synthase [57]. Accordingly, kinase 3 beta it(GSK3β), was shown which that is anGSK3 upstreamβ knocked-down regulator of MCF-7 G1 phase cells [57]. were Accordingly, not sensitive it was to ophiobolin shown that OGSK3β (122) treatment,knocked-down indicating MCF- 7that cells the were latter not may sensitive target to GSK3 ophiobolinβ to induce O (122 G1) phasetreatment, arrest indicating in MCF-7 that cells. the In latter addition, may 122targettreatment GSK3β toresulted induce in G1 decreased phase phosphorylation arrest in MCF-7 levels cells. of In AKT addition, (protein 122 kinase treatment B) and resulted GSK3β , in as decreasedwell as in phosphorylationthe protein expression levels level of AKT of cyclin (protein D1, kinase whereas B) pre-treatmentand GSK3β, as with well phosphatase as in the protein inhibitor expression sodium levelorthovanadate of cyclin D1, blocked whereas122 -inducedpre-treatment G1 phase with arrest. phosphatase These results inhibitor indicated sodium that orthovanadate the anti-proliferative blocked 122effect-induced of 122 G1in MCF-7 phase arrest. cells may These be results mediated indicated through that interaction the anti-proliferative with the Akt/GSK3 effect of β122/cyclin in MCF D1- pathway.7 cells may Besides, be med122iatedsuppressed through tumorigenesisinteraction with in the a mouse Akt/GSK3β/cyclin xenograft model, D1 pathway. whereas itBesides, showed 122 no suppressedapparent cytotoxicity tumorigenesis [57]. in Furthermore, a mouse xenograft122 significantly model, whereas reversed it showed adriamycin no apparent resistance cytotoxicity in human [57].breast Furthermore cancer MCF-7/ADR, 122 significantly cells (11-fold) reversed at low adriamycin micromolar resistance concentrations in human (0.1 breastµM; lesscancer than MCF 20%- 7/ADRinhibition cells concentration) (11-fold) at [ 57 low]. The micromolar reversal effect concentrations of 122 was suggested (0.1 μM; to less be via than elevated 20% expression inhibition concentration)of pro-apoptotic [57]. proteins, The reversal as well effect as downregulation of 122 was suggested of resistance-related to be via elevated proteins, expression especially of pro of- apoptoticP-glycoprotein, proteins in, MCF-7/ADR as well as cells.downregulation Moreover, 122 of resistaenhancednce-related mitochondrial proteins, apoptosis especially pathway of P- glycoprotein,and G2/M cell in cycleMCF- arrest7/ADR caused cells. Moreover, by adriamycin, 122 enhanced due to increased mitochondrial level ofapoptosis ROS in MCF-7/ADRpathway and G2/Mcells [ 58cell]. cycle Remarkably, arrest caused combination by adriamycin treatment, due of to122 increasedand adriamycin level of ROS resulted in MCF in significant-7/ADR cells tumor [58]. Remarkably,growth suppression combination (70%) intreatment nude mice, of 122 suggesting and adriamycin122 as a promising resulted in lead significant structure tumor for multidrug growth suppressionresistance cancer (70%) chemotherapy. in nude mice, suggesting 122 as a promising lead structure for multidrug resistance cancer chemotherapy.

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FigureFigure 17. 17.ChemicalChemical structures structures of 115 of 115–124–124. .

2.2.2.2.2.2. Compounds Compounds with with Lipid Lipid-Lowering-Lowering Activity Activity ChromatographicChromatographic work work-up-up of ofsediment sediment-derived-derived PenicilliumPenicillium pinophilum pinophilum H608H608 (collected (collected from from the the XiamenXiamen coastline, coastline, China) China) extract extract resulted resulted in inisolation isolation of ofa series a series of ofphenolic phenolic compounds, compounds, which which were were evalevaluateduated for for their their inhibitory inhibitory effects effects against against oleic oleic acid acid-elicited-elicited lipid lipid accumulation accumulation in HepG2 in HepG2 cells cells [59]. [59 ]. AsAs a result a result of this of bioactivity this bioactivity screening, screening, eight compounds eight compounds (125–132 (125) (Figure–132) (Figure18) were 18 found) were to foundinhibit to lipidinhibit accumulation lipid accumulation at a dose at of a 10 dose μM, ofwith 10 µ noM, sig withnificant no significant cytotoxicity cytotoxicity (IC50 > 50 (ICμM).50 > Further 50 µM). investigationFurther investigation revealed revealed five compounds five compounds (125, 128 (125, and, 128 ,1 and30–132130)– 132 that) that significantly significantly suppressed suppressed intracellularintracellular total total cholesterol cholesterol and and triglycerides. triglycerides. Remarkably, Remarkably, the the analogs analogs 125125, 130, 130–132–132 werewere more more activeactive than than the thepositive positive control control simvastatin. simvastatin. A real A-time real-time quantitative quantitative PCR experiment PCR experiment indicated indicated that compoundsthat compounds 125, 128125, and, 128 ,130 and–132130 affect–132 affect the genes the genes responsible responsible for for enzymes enzymes involved involved in in lipid lipid metabolism,metabolism, including including downregulation of of the the expression expression of fatty of acid fatty synthase, acid synthas acetyl-CoAe, acetyl carboxylase,-CoA carboxylaseand 3-hydroxy-3-methylglutaryl-CoA, and 3-hydroxy-3-methylglutaryl reductase-CoA (inhibitionreductase (inhibition of lipogenesis), of lipogenesis), as well as upregulationas well as upregulationof carnitinepalmitoyl of carnitinepalmitoyl transferase-1 transferase (stimulation-1 of(stimulation lipid catabolism). of lipid Moreover, catabolism). metabolites Moreover,125 , metabolites127, 128, and125, 130127–,133 128reduced, and 130 oxidized–133 reduced low-density oxidized lipoprotein low-density stimulated lipoprotein lipid stimulated accumulation lipid in accumulationRAW264.7 cells. in RAW264.7 Among the cells. active Among derivatives the active in the derivatives latter assay, in compounds the latter assay,125 and compounds132 revealed 125 the andmost 132 pronouncedrevealed the effect most comparablepronounced to effect the positive comparable control to rosiglitazonethe positive control at a dose rosiglitazone of 10 µM. Further at a dosebioassays of 10 μ showedM. Further that compounds bioassays showed125, 128 , that and 131 compounds–133 significantly 125, 128 decreased, and 131 the–133 intracellular significantly total decreasedcholesterol the levels,intracellular although total congeners cholesterol127 levels,and 130 althoughwere inactive congeners in RAW264.7 127 and 130 macrophages. were inactive Further in RAW264.7mechanistic macrophages. studies revealed Further that mechanistic compounds studies128 and revealed131–133 thatsignificantly compounds inhibited 128 and cholesterol131–133 significantlyuptake in RAW264.7, inhibited cholesterol whereas 125 uptakeand 131 in– 133 RAW264.7,stimulated whereas cholesterol 125 effluxand 131 to– HDL.133 stimulated Compounds cholesterol125 and 132 effluxshowed to HDL. a cholesterol Compounds efflux 125effect and 132 comparable showed a to cholesterol rosiglitazone efflux and effect caused comparable upregulation to rosiglitazoneof mRNA levels and caused of key regulators, upregulation such of as mRNA peroxisome levels proliferator-activated of key regulators, such receptor- as peroxisomeγ (PPAR- γ), proliferatorliver X receptor-activatedα (LXR receptorα), and-γ ATP-binding(PPAR-γ), liver cassette X receptor G1 (ABCG1). α (LXRα Similarly,), and ATP compounds-binding cassette131 and G1133 (ABCG1).showed significantSimilarly, inhibitioncompounds of cholesterol131 and 133 influx, showed which significant was slightly inhibition weaker than of cholesterol that of rosiglitazone, influx, whichas well was as slightly stimulation weaker of cholesterolthan that of efflux. rosiglitazone, However, as both well compounds as stimulation did of not cholesterol affect transcription efflux. However,of the aforementioned both compounds cholesterol did not affect efflux transcription stimulators, suggesting of the aforementioned an unknown cholesterol mechanism efflux of the stimulators,action for suggesting regulation an of cholesterolunknown mechanism efflux. Furthermore, of the action congeners for regulation131–133 of decreasedcholestero CD36l efflux. and Furthermore,SR-1 (critical congeners scavenger 131 receptors–133 decreased for regulation CD36 of andcholesterol SR-1 (critical dynamics) scavenger transcription receptors [59]. Thus,for regulationthe aforementioned of cholesterol phenolic dynamics) compounds transcription represent new [59]. natural Thus, product the aforementioned leads that can be phenolic utilized for compoundsthe development represent of hypolipidemic new natural productand anti-atherosclerotic leads that can agents be utilized (Figure for 18 ). the development of hypolipidemic and anti-atherosclerotic agents (Figure 18).

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Figure 1 18.8. ChemicalChemical structures structures of of 125125–133–133. .

2.3.2.3. Cytotoxic Cytotoxic Compounds Compounds DerivedDerived from Bacteria Originating Originating from from Mangrove Mangrove (Rhizosphere (Rhizosphere)) Soil/Sediment Soil/SedimentSamples Samples TheThe culture culture broth broth ofof thethe actinomycete strain strain StreptomycesStreptomyces sp.sp. 219807 219807 derived derived from from mangrove mangrove soil soil collectedcollected in in Hainan Hainan Province,Province, China, revealed a a remarkably remarkably high high yield yield of ofglycosylated glycosylated 16- 16-memberedmembered macrolidemacrolide derivatives derivatives belongingbelonging to the elaiop elaiophylinhylin family family [60]. [60 ].Specifically, Speciﬁcally, StreptomycesStreptomyces sp.sp. 219807 219807 waswas cultured cultured on on 18 18 different different media, media, and and waswas shownshown toto produceproduce the highest yield of of elaiophylin elaiophylin (up (up to 4486to 4486 mg/L) mg/L) in in shake-ﬂasks shake-flask containings containing DO DO fermentationfermentation medium. medium. The The high high yield yield of ofelaiophylin elaiophylin was was attributedattributed to to both both the the strain strain of microorganism of microorganism and and the DOthe mediumDO medium containing containing complex complex carbon carbon sources. Subsequentsources. Subsequent chemical investigation chemical investigation of the respective of the respective fermentation fermentation extract afforded extract afforded a new elaiophylin a new elaiophylin metabolite, halichoblelide D (134), along with several known analogs (135–140). metabolite, halichoblelide D (134), along with several known analogs (135–140). Compounds 134–140 Compounds 134–140 exhibited potent cytotoxicity against HeLa and MCF-7 cell lines, with IC50 exhibited potent cytotoxicity against HeLa and MCF-7 cell lines, with IC values in the range from values in the range from 0.19 to 2.12 μM, which renders them promising50 lead structures for the 0.19 to 2.12 µM, which renders them promising lead structures for the development of anticancer development of anticancer agents (Figure 19). agents (Figure 19).

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Figure 1 19.9. Chemical structures of 134–140. Figure 19. Chemical structures of 134–140. Bioassay-guidedBioassay-guided investigation of the mangrove-derived mangrove-derived actinomycete actinomycete StreptomycesStreptomyces sp.sp. Q22, Q22, Bioassay-guided investigation of the mangrove-derived actinomycete Streptomyces sp. Q22, isolated from a sample of mangrove mangrove soil (Guangdong, (Guangdong, China), afforded afforded eight eight natural natural products, products, isolated from a sample of mangrove soil (Guangdong, China), afforded eight natural products, including the known bagremycin B (141) and a new derivative bagremycin C (142) that showed including the known bagremycin B (141) and a new derivative bagremycin C (142) that showed cytotoxic properties (Figure 20)[) [61].61]. Compound 142 was found to be the most active analog against cytotoxic properties (Figure 20) [61]. Compound 142 was found to be the most active analog against four human glioma cells,cells, withwith ICIC5050 values ranging between 2.2 and 6.4 µμM,M, followedfollowed byby 141141,, withwith ICIC5050 four human glioma cells, with IC50 values ranging between 2.2 and 6.4 μM, followed by 141, with IC50 values from 7.3 to 13.3 µμM,M, thusthus indicatingindicating thatthat thethe NN-acetyl-(-acetyl-(S)-cysteine)-cysteine moiety plays an important values from 7.3 to 13.3 μM, thus indicating that the N-acetyl-(S)-cysteine moiety plays an important role in the the cytotoxicity cytotoxicity of of these these metabolites. metabolites. Interestingly, Interestingly, bagremycin bagremycin C ( C142 (142) at) concentrations at concentrations of 2.2 of role in the cytotoxicity of these metabolites. Interestingly, bagremycin C (142) at concentrations of 2.2 2.2 and 4.4 µM was found to induce late apoptosis (after 48 and 72h) in U87MG cells in a dose- and andand 4.4 4.4 μ μMM was was found found to to induce induce latelate apoptosisapoptosis (after 48 and 72h) in U87MG87MG cellscells inin a a dose dose-- and and time time-- time-dependent manner. Furthermore, a U87MG cell cycle assay showed that the cell population at dependentdependent manner. manner. Furthermore, Furthermore, aa U87MGU87MG cellcell cycle assay showed thatthat thethe cellcell populationpopulation at at the the the G0/G1 phase was enhanced after 12 h of exposure to 4.4 µM bagremycin C (142), indicating that G0/G1G0/G1 phase phase was was enhanced enhanced afterafter 1212 hh ofof exposureexposure to 4.4 μM bagremycin CC ((142142),), indicatingindicating that that the the the latter might block the cell cycle at the G0/G1 phase. latterlatter might might block block the the cell cell cycle cycle atat thethe G0/G1G0/G1 phase.phase.

Figure 20. Chemical structures 141 and 142. FigureFigure 2020.. ChemicalChemical structures 141 and 142..

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In 2013 and 2015,2015, thethe StreptomycesStreptomyces sp. strain CHQ-64,CHQ-64, obtained from rhizosphere soil collected from the mangrove conservation area of Guangdong province, China, was reported to be a source of intriguing natural products [[62,63]62,63].. Chemical investiga investigationtion of the crude extract of this actinomycete led to to the the isolation isolation of of hybrid hybrid isoprenoids isoprenoids (indotertines (indotertines A and A andB, and B, drimentines and drimentines C and CH) and as well H) as wellskipped as skipped-polyol-polyol polyene polyene macrolides macrolides (reedsmycins (reedsmycins A–F) with A–F) unprecedented with unprecedented scaffolds scaffolds [62,63]. [62 In,63 a]. Insubsequent a subsequent study, study, a mutant a mutant strain strain ΔrdmF∆rdmF of Streptomycesof Streptomyces sp. sp.CHQ CHQ-64-64 was was obtained obtained by byknockout knockout of ofthe the regulatory regulatory gene gene rdmFrdmF involvedinvolved in reedsmycins in reedsmycins biosynthesis biosynthesis [64]. [64 Chemical]. Chemical investigation investigation of the of theΔrdmF∆rdmF mutantmutant strain strain afforded afforded an unusual an unusual 2,3,4- 2,3,4-trisubstitutedtrisubstituted pyrrole, pyrrole, namel namelyy geranylpyrrol geranylpyrrol A, along A, alongwith a withnew aalkaloid new alkaloid piericidin piericidin F (143). F Interestingly, (143). Interestingly, the latter the exhibited latter exhibited pronounced pronounced activity activitytoward towardHeLa, NB4 HeLa, (acute NB4 promyelocytic (acute promyelocytic leukemia) leukemia),, A549, and A549, H1975 and H1975cell lines cell, with lines, IC with50 values IC50 valuesin the range in the rangebetween between 0.003 to 0.003 0.56 to μM, 0.56 whereasµM, whereas geranylpyrrol geranylpyrrol A was A inactive was inactive (Figure (Figure 21). 21).

Figure 21. Chemical structure of 143. Figure 21. Chemical structure of 143. Hayakawa et al., in the course of screening for antitumor compounds using 3Y1 rat fibroblasts transformedHayakawa with et adenovirus al., in the course oncogenes, of screening reported for a novelantitumorN-acylated compounds undecapeptide, using 3Y1 thioviridamide, rat fibroblasts fromtransformed the culture with broth adenovirus of the actinomycete oncogenes,Streptomyces reported olivoviridis a novel[65 ].N Remarkably,-acylated undecapeptide, thioviridamide showedthioviridamide selective, from cytotoxicity the culture against broth of 3Y1 therat actinomycete fibroblast cellsStreptomyces transformed olivoviridis with [65]. adenovirus Remarkably, type thioviridamide showed selective cytotoxicity against 3Y1 rat fibroblast cells transformed with 12 (Ad12-3Y1) and adenovirus E1A gene (E1A-3Y1), with IC50 values of 2.3 nM (3.9 ng/mL) and 23.8adenovirus nM (32 type ng/mL), 12 (Ad12 respectively.-3Y1) and Significant adenovirus numbers E1A gene of Ad12-3Y1 (E1A-3Y1) cells, with treated IC50 values with thioviridamide of 2.3 nM (3.9 containedng/mL) and condensed 23.8 nM (32 chromatin ng/mL), andrespectively. fragmented Significant nuclei, numbers indicating of that Ad12 thioviridamide-3Y1 cells treated induced with apoptosis.thioviridamide In a subsequent contained study, condensed the gene cluster chromatin for the and biosynthesis fragmented of thioviridamide nuclei, indicating in S. olivoriridis that NA05001thioviridamide was identified induced andapoptosis. heterologously In a subsequent produced study, in Streptomyces the gene cluster lividans forTK23 the [ 66biosynthesis]. In addition, of duringthioviridamide genome in mining S. olivoriridis for thioviridamide-like NA05001 was identified biosynthetic and geneheterologously clusters, a novelproduced cryptic in Streptomyces biosynthetic genelividans cluster TK23 was [66]. identified In addition, from duringStreptomyces genomesp. mining MSB090213SC12 for thioviridamide strain, obtained-like biosynthetic from mangrove gene soilclusters, in Ishigaki a novel Island, cryptic Okinawa, biosynthetic Japan. In ordergene to cluster induce was the expression identified of from the crypticStreptomyces metabolite, sp. variousMSB090213SC12 fermentation strain, media obtained were from employed, mangrove including soil in theIshigaki production Island, mediumOkinawa, for Japan. thioviridamide. In order to However,induce the this expression proved to of be the unsuccessful cryptic metabolite, for the production various fermentation of the novel media analog. were Nevertheless, employed, heterologousincluding the expressionproduction of medium the respective for thioviridamide. biosynthetic gene However, cluster this in Streptomyces proved to be avermitilis unsuccessfulSUKA22 for strainthe production resulted in of the the production novel analog. of the Nevertheless, new derivative heterologous neothioviridamide expression (144 of) possessing the respective four thioamidebiosynthetic bonds gene and cluster the unusualin Streptomyces amino acidsavermitilisβ-hydroxy- SUKA22N1 ,strainN3-dimethylhistidinium resulted in the production (hdmHis) of andthe 3-methyl-new derivativeS-(2-aminovinyl)cysteine neothioviridamide (3-Me-avCys) (144) possessing [67]. four Interestingly, thioamide neothioviridamide bonds and the unusual (144) displayed amino 1 3 cytotoxicacids β-hydroxy activities-N against,N -dimethylhistidinium SKOV-3 (human ovarian (hdmHis) adenocarcinoma), and 3-methyl-S- Meso-1(2-aminovinyl)cysteine (malignant pleural (3- Me-avCys) [67]. Interestingly, neothioviridamide (144) displayed cytotoxic activities against SKOV- mesothelioma), and Jurkat cells with IC50 values of 2.1, 0.7, and 0.4 µM, respectively (Figure 22). 3 (human ovarian adenocarcinoma), Meso-1 (malignant pleural mesothelioma), and Jurkat cells with IC50 values of 2.1, 0.7, and 0.4 μM, respectively (Figure 22).

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Figure 22. Chemical structure of 144. Figure 22. Chemical structure of 144. Sun et al. reported the isolationFigure of 2four2. Chemical new macrolactone structure of 144 polyketide. natural products of the borrelidinSun et family, al. reported namely, the borrelidin isolation F of (146 four), borrelidin new macrolactone G (147), borrelidin polyketide H natural (148), and products borrelidin of the I, Sun et al. reported the isolation of four new macrolactone polyketide natural products of the borrelidinin addition family, to the namely, known borrelidin analogue F borrelidin (146), borrelidin A (145 G) (Figure (147), borrelidin 23) [68]. H These (148 ), metabolites and borrelidin were I, borrelidin family, namely, borrelidin F (146), borrelidin G (147), borrelidin H (148), and borrelidin I, inobtained addition from to the Streptomyces known analogue rochei borrelidin SCSIO ZJ89, A (145 which) (Figure was 23 originated)[68]. These from metabolites a mangrove were obtained-derived in addition to the known analogue borrelidin A (145) (Figure 23) [68]. These metabolites were fromsedimentStreptomyces sample rocheicollectedSCSIO in Yalongwan, ZJ89, which wasChina. originated All compounds from a mangrove-derived were investigated sediment for cytotoxicity sample obtained from Streptomyces rochei SCSIO ZJ89, which was originated from a mangrove-derived collectedagainst A549, in Yalongwan, CNE2 (nasopharyngeal China. All compounds carcinoma), were HeLa, investigated HepG2, for and cytotoxicity MCF-7 cell against lines, A549, as well CNE2 as sediment sample collected in Yalongwan, China. All compounds were investigated for cytotoxicity (nasopharyngealagainst normal hepatic carcinoma), L02 and HeLa, normal HepG2, umbilical and MCF-7vein endothelial cell lines, Huvec as well-12 as againstcells. Compounds normal hepatic 145– against A549, CNE2 (nasopharyngeal carcinoma), HeLa, HepG2, and MCF-7 cell lines, as well as L02148 were and normal active toward umbilical the vein respective endothelial cell lines Huvec-12, with IC cells.50 values Compounds in the range145–148 fromwere 0.12 active to 22.75 toward μM. against normal hepatic L02 and normal umbilical vein endothelial Huvec-12 cells. Compounds 145– theAmong respective them, cellcompounds lines, with 145 IC 50andvalues 148 were in the the range most from active 0.12 with to 22.75 IC50µ vaM.lues Among between them, 0.12 compounds and 2.19 148 were active toward the respective cell lines, with IC50 values in the range from 0.12 to 22.75 μM. 145μM,and stronger 148 were than the those most of active the positive with IC controls50 values doxorubicin between 0.12 (IC and50 = 2.19 1.02µ–M,3.52 stronger μM) and than cisplatin those of (IC the50 Among them, compounds 145 and 148 were the most active with IC50 values between 0.12 and 2.19 positive= 2.30–12.85 controls μM). doxorubicinHowever, compound (IC50 = 1.02–3.52 148 was µfoundM) and to be cisplatin less active (IC50 toward= 2.30–12.85 the nonµ-Mcancerous). However, cell μM, stronger than those of the positive controls doxorubicin (IC50 = 1.02–3.52 μM) and cisplatin (IC50 compoundlines than 145148, waswhich found might to bebe lessattributed active towardto both thethe non-cancerous α-OH configuration cell lines and than cis145 geometry, which of might the = 2.30–12.85 μM). However, compound 148 was found to be less active toward the non-cancerous cell beC14 attributed–C15 olefin to in both its thestructureα-OH compared configuration to the and latter. cis geometry Due to its of selectivity the C14–C15 toward olefin cancer in its structurecells, 148 lines than 145, which might be attributed to both the α-OH configuration and cis geometry of the comparedwas further to the investigated latter. Due on to its tumor selectivity cell migration toward cancer, employing cells, 148 anwas in further vitro wound investigated-healing on tumorassay. C14–C15 olefin in its structure compared to the latter. Due to its selectivity toward cancer cells, 148 cellRemarkably, migration, 148 employing effectively an inhibitedin vitro wound-healing tumor (HeLa and assay. A549) Remarkably, cell migration148 effectively, even at low inhibited micromolar tumor was further investigated on tumor cell migration, employing an in vitro wound-healing assay. (HeLaconcentrations and A549) (1/2 cell IC migration,50). Moreover, even it at exerted low micromolar little influence concentrations upon non (1/2-malignant IC50). Moreover, human umbilical it exerted Remarkably, 148 effectively inhibited tumor (HeLa and A549) cell migration, even at low micromolar littlevein endothel influenceial upon (Huvec non-malignant-12) cells, which human renders umbilical 148 a vein potential endothelial new antitumor (Huvec-12) lead cells, compound which renders with concentrations (1/2 IC50). Moreover, it exerted little influence upon non-malignant human umbilical 148botha cytotoxic potential and new antimetasta antitumortic lead properties. compound Interestingly, with both the cytotoxic known and analog antimetastatic borrelidin A properties. (145) has vein endothelial (Huvec-12) cells, which renders 148 a potential new antitumor lead compound with Interestingly,been reported the to knownbe an allosteric analog borrelidin inhibitor of A threonyl(145) has-tRNA been reported synthetas toe (ThrRS), be an allosteric thus preventing inhibitor both cytotoxic and antimetastatic properties. Interestingly, the known analog borrelidin A (145) has ofnormal threonyl-tRNA protein synthesis synthetase [69]. Moreover, (ThrRS), thus its cytotoxic preventing effect normal is associated protein with synthesis induction [69 ].of G0/G1 Moreover, cell been reported to be an allosteric inhibitor of threonyl-tRNA synthetase (ThrRS), thus preventing itscycle cytotoxic arrest and effect caspase is associated-mediated with cell induction death via of the G0/G1 MAPK cell signaling cycle arrest pathway and caspase-mediated [70]. Interestingly, cell in normal protein synthesis [69]. Moreover, its cytotoxic effect is associated with induction of G0/G1 cell deatha recent via study the MAPK, 145 was signaling shown pathway to increase [70]. the Interestingly, levels of unfolded in a recent protein study, response145 was shown(UPR) toassociated increase cycle arrest and caspase-mediated cell death via the MAPK signaling pathway [70]. Interestingly, in thewith levels ER stress of unfolded, leading protein to C/EBP response homologous (UPR) associatedprotein (CHOP) with ER-dependent stress, leading cell death to C/EBP in oral homologous squamous a recent study, 145 was shown to increase the levels of unfolded protein response (UPR) associated proteincell carcinoma (CHOP)-dependent cells [71]. Therefore, cell death 148 in oral might squamous exert similar cell carcinoma mechanisms cells [71for]. s Therefore,electively 148 targetmighting with ER stress, leading to C/EBP homologous protein (CHOP)-dependent cell death in oral squamous exertcancer similar cells. mechanisms for selectively targeting cancer cells. cell carcinoma cells [71]. Therefore, 148 might exert similar mechanisms for selectively targeting cancer cells.

Figure 23. Chemical structures of 145–148. Figure 23. Chemical structures of 145–148.

Figure 23. Chemical structures of 145–148.

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FurtherFurther bioactive bioactive metabolites metabolites derivedderived from mangrove soil/sediment soil/sediment microorganisms microorganisms that that could could serveserve as as potential potential lead lead structures structures areare summarizedsummarized in Table Table 22 andand Figure 2424..

Figure 24. Chemical structures of bioactive compounds from mangrove fungi and bacteria derived fromFigure soil/sediment 24. Chemical samples structures (149 of–163 bioactive). compounds from mangrove fungi and bacteria derived from soil/sediment samples (149–163).

Mar. Drugs 2018, 16, 319 25 of 31

Table 2. Bioactive compounds isolated from soil-derived fungi (149–162) and bacteria (163) of mangrove origin.

Penicibilaene A (149) Penicillium bilaiae MIC 4.23 µM/1.0 µg/mL MA-267 from the Antifungal against rhizospheric soil of Colletotrichum gloeosporioides Penicibilaene B (150)Lumnitzera racemosa [72] MIC 0.45 µM/0.125 µg/mL (Hainan Island, China) MIC 15.1 µM/4.0 µg/mL (E. coli, P. aeruginosa, Vibrio harveyi, Vibrio parahaemolyticus Penicisimpin A (151) C. gloeosporioides) and 30.3 µM (8 µg/mL) (M. luteus, Penicillium Antibacterial and antifungal Vibrio alginolyticus) simplicissimum MA-332 [73] MIC 15.1 µM/4.0 µg/mL from the rhizospheric (C. gloeosporioides) soil of B. sexangula var. rhynchopetala (Hainan MIC 30.5 µM/8.0 µg/mL Island, China) (E. coli, P. aeruginosa, V. harveyi, Penicisimpin C (152) V. parahaemolyticus) MIC 30.5 µM (8.0 µg/mL) (C. gloeosporioides) Inﬂuenza neuraminidase Simpterpenoid A (153) IC 8.1 nM inhibitory activity [74] 50 Antibacterial against Penicilone B (154) MIC 6.54 µM/3.13 µg/mL Penicillium janthinellum methicillin-resistant and HK1-6, isolated from -susceptible S. aureus, MIC 11.8–23.5 µM Penicilone C (155) mangrove rhizosphere vancomycin-resistant (6.25–12.5 µg/mL) soil (Dongzhaigang, Enterococcus faecalis, and Enterococcus MIC 6.1–24.4 µM Penicilone D (156) Hainan Island) -susceptible faecium strains [75] (3.13–12.5 µg/mL) Rubrumazine B (157) Eurotium rubrum MA-150 LD50 2.4 µM Dehydroechinulin (158)from mangrove-derived Cytotoxic in brine shrimp LD50 3.5 µM rhizospheric soil Neoechinulin E (159)assay [76] LD 3.9 µM (Andaman Sea coastline, 50 Variecolortide C (160)Thailand) LD50 9.8 µM

IC50 4.6–7.6 µM; HeLa, HEK Penicitol A (161) 293, HCT-116, and A549 Penicillium chrysogenum Cytotoxic against several cell lines HND 11–24 from the cancer cell lines and HEK rhizosphere soil of IC50 3.4–9.6 µM; HeLa, 293 [77] BEL-7402 (hepatocellular Penicitol B (162) Acanthus ilicifolius carcinoma), HEK 293, HCT-116, and A549 cell lines Actinomycetospora chlora Cytotoxic toward SNC-032 from mangrove Thiasporine A (163) non-small-cell lung cancer IC 5.4 µM swamp sediment sample 50 H2122 cell line [78] (Vava’u, Tonga)

3. Conclusions and Outlook Mangrove-associated microorganisms have gained considerable attention as a rich source of structurally diverse secondary metabolites with pronounced biological activities, which could be utilized in the discovery of new drug leads [79]. In this review, 163 compounds have been presented from mangrove-associated microorganisms, the majority of which show remarkable activities, such as the potent cytotoxic penicisulfuranols A–C (100–102)[48], phomoxanthone A (19)[32], and piericidin F (143)[64], as well as the anti-inﬂammatory rhytidenones C and D (40 and 41)[34]. Nevertheless, these metabolites represent only a small fraction of the biosynthetic capacity of the source microorganisms, as predicted through genomic studies [80]. This is partially due to the fact that most of the biosynthetic gene clusters expressing novel bioactive metabolites remain silent (or cryptic) under standard laboratory culture conditions, and thus the metabolic potential of these microorganisms remains untapped [81,82]. Taking this fact into account, new methods and technologies are warranted to activate cryptic pathways and explore the secondary metabolome of microbes [83]. Mar. Drugs 2018, 16, 319 26 of 31

The biosynthetic potential of mangrove-associated microorganisms has been associated with activation of silent genes by unique environmental stimuli imparted on this special ecological niche [84]. Thus, production of cryptic metabolites may be accomplished by altering culture conditions, such as temperature, media, pH, and light, or by adding elicitors/chemicals, e.g., DMSO, inorganic salts, and plant exudates—in the case of endophytes—to the culture [79,82]. Alteration of culture conditions, the so-called OSMAC approach, has been successfully exploited to generate novel compounds from mangrove-associated fungi, such as brocapyrrozin A (25) and lasiodiplactone A (42) from the endophytic fungi P. brocae MA-231 [26] and L. theobromae ZJ-HQ1 [35], respectively. Co-cultivation of microorganisms has likewise been exploited to enhance the accumulation of constitutively present natural products and/or to trigger the production of cryptic metabolites [85], which could not be achieved though fermentation of individual strains. It is assumed that microbial interactions imitate the natural habitat of microbes, either in a symbiotic relationship or in competition for nutrients and space, and thus play an important role in the activation of silent genes [84]. This methodology has led to the induction of several novel cryptic metabolites, including citrifelins A (68) and B (69) from co-culture of the mangrove endophyte P. citrinum MA-197 with the bryozoan-derived B. felina EN-135 [42], as well as microsphaeropsisin C (75) and lasiodiplodin derivatives (76–79) with α-glucosidase inhibitory activity from co-culture of the endophytic fungus Trichoderma sp. 307 with A. johnsonii B2 [44]. In the last few years, epigenetic manipulation has attracted great interest as a strategy for activation of silent biosynthetic pathways. Several regulatory proteins, such as chromatin-modulating agents and transcription factors, are known to control the secondary metabolome of microbes [84]. The epigenetic modifiers suberoylanilide hydroxamic acid (SAHA) and 5-azacytidine that inhibit the activities of histone deacetylases and DNA methyltransferases, respectively, have proven to be effective in activation of silenced gene clusters [83,84]. For instance, addition of 5-azacytidine to the mangrove-associated fungus P. variabile HXQ-H-1 afforded the cryptic metabolite varitatin A (113) with potent cytotoxic and protein kinase inhibitory activities [52]. Notably, it has been shown that secondary metabolite production in Aspergillus nidulans was triggered by co-cultivation with the bacterium Streptomyces rapamycinicus due to targeted histone modification, thus shedding light on the connection between epigenetic modification and microbial crosstalk [86]. The majority of compounds described in this review have been derived from common genera, such as Streptomyces and Penicillium. Even though these microorganisms still hold enormous biosynthetic potential, as demonstrated by recent genome sequence studies, a vast amount of novel fungal and/or bacterial taxonomic groups still lies unexplored [79]. To a large extent, this is due to limitations in traditional isolation procedures, as well as the non-culturable feature of many of these latter microorganisms [87]. To overcome this problem, molecular approaches, such as high-throughput sequencing and metagenomics, have become promising tools for unraveling the biosynthetic potential of hitherto uncultured microorganisms, evading the need for isolation of individual species [87,88], and thus providing an inexhaustible source of new microbial taxa. Genome mining and bioinformatics approaches also serve as powerful strategies towards the prediction of key biosynthetic clusters from genome sequence analysis data, providing a wealth of information that can be linked to cryptic secondary metabolites, as exemplified in the case of neothioviridamide (144) from Streptomyces sp. MSB090213SC12 [67,89,90]. These novel biosynthetic clusters can be genetically engineered and expressed in heterologous hosts, such as Saccharomyces cerevisiae, E. coli, or Streptomyces lividans for large-scale production of the desired lead compounds or derivatives thereof [66,91,92]. Overall, mangrove-associated microorganisms have gained considerable attention due to their unique ecological characteristics, diversity, and wealth of novel bioactive secondary metabolites. Nevertheless, pharmaceutical development of these metabolites is still in its infancy, with only the proteasome inhibitor, salinosporamide A, being hitherto in phase I clinical trials for the treatment of multiple myeloma [12,93]. In order to unravel the metabolic potential of mangrove endophytes, an integrative understanding of the principal molecular mechanisms involved in the regulation of Mar. Drugs 2018, 16, 319 27 of 31 natural product biogenesis is essential [88]. Strategies to activate silent genes for exploration of novel compounds, such as epigenetic modification, OSMAC, and microbial co-cultivation approaches, along with continuing advancements in modern “omics” methodologies, including transcriptomics, proteomics, and metabolomics, is expected to open up an exciting area of research for the discovery of lead compounds from mangrove-associated microorganisms in the coming years.

Funding: This research was funded by the DFG (GRK 2158) and by the Manchot Foundation. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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