TECHNOLOGIES
DRUG DISCOVERY
TODAY
Metabolomics in the natural products field – a gateway to novel antibiotics
Changsheng Wu
1,2, Hye Kyong Kim
1, Gilles P. van Wezel
2, Young Hae Choi
1,*
1NaturalProductsLaboratory,InstituteofBiology,LeidenUniversity,Sylviusweg72,2333BELeiden,TheNetherlands
2MolecularBiotechnology,InstituteofBiology,LeidenUniversity,Sylviusweg72,2333BELeiden,TheNetherlands
Metabolomicsisahighthroughputanalyticaltechnique usedtogloballymeasurelowmolecularweightmetab- olites,allowingsimultaneousmetaboliccomparisonof differentbiologicalsamplesandthushighlightingdiffer- entiallyproducedcompoundsaspotentialbiomarkers.
Althoughmicrobesarerenownedasprolificsourcesof antibiotics,thetraditionalapproachfornewanti-infec- tivesdiscoveryistime-consumingandlabor-intensive.In thisreview,theuseofNMR-orMS-basedmetabolomics isproposedasanefficientapproachtofindantimicro- bialsinmicrobialsingle-orco-cultures.
Sectioneditor:
PascaldeTullio– UniversityofLie`ge,Lie`ge,Belgium.
Introduction
Thenumberofmulti-drugresistantbacteriaisrisingalarm- ingly and the treatment of infections caused by these microbes is extremely challenging [1]. Actinomycetes and filamentous fungiarethemajorsourceofthenaturalanti- biotics. Thetraditional methodsused fordrugdiscovery is time-andlabor-intensiveandtherediscoveryratesbecame increasingly demotivating [2,3]. Yet, genome sequencing revealedmanypreviouslyunsuspectedbiosyntheticclusters fornatural productsin long-studiedmodelstrains [4–6]. A major challenge lies in finding the appropriate chemical triggersorecologicalcuestoelicittheproductionofcryptic antibiotics[7,8].
Metabolomics isahigh throughputanalyticaltechnique usedtogloballymeasurelowmolecularweightmetabolitesin
biologicalsamples.Combinationofchemicalprofilesbuiltby LC–HRMS or NMR with multivariate data analysis (MDA) allowsscientiststocompareanddetectdifferentialmetabo- lites in active/inactive biological samples, narrowing the searchtopotentialbiomarkersandavoidingchemicalredun- dancy in an early stage. In this paper, we have reviewed examplesoftheuseofmetabolomicsasaneffectivetoolto increasetheefficiencyofantibioticdiscoveryfrommicrobial sources.
Metabolomics-guideddiscoveryofnewantibiotics Metabolomicsmethodology,eitherNMR-orMS-based,isan idealtoolforthechemicalscreeningandsubsequentdetailed comparisonofthesecondarymetabolomesofasetofbacte- rialfermentations,rapidlyrevealingthedifferences/biomark- ersamongexperimentalgroups.Thenextquestionishowto handlethecompoundsresponsiblefortheseparationinthe scoreplotofsupervisedand/orunsupervisedMDA.Because NMRandMStechniquescangivestructuralinformationof compoundsinanearlystage,therearethreepossibilitiesfor theidentificationofvaluablebiomarkers.Firstly,itispossible tocomparethestructuralinformationprovidedbyNMR(1H NMRchemicalshifts)and/orhigh-resolutionmassspectrom- etry(molecularformulaandisotopicpattern)withliterature and/ordatabases.Ifknownstructures,discriminatorscanbe identified without the need for time-consuming isolation process and thus metabolomics serves as an approach for Editors-in-Chief
KelvinLam–SimplexPharmaAdvisors,Inc.,Boston,MA,USA HenkTimmerman–VrijeUniversiteit,TheNetherlands
Metabolomics in Medicinal Chemistry
*Correspondingauthor:Y.H.Choi(y.choi@chem.leidenuniv.nl)
dereplication; alternatively, new compounds may possess known structural core but unknown functional groups –
‘knownunknowns’.Inthiscase,theirstructuralcharacteri- zationcanbedonewithinthecontextofthecrudebiological matrix with the aid of various 2D NMR techniques as reviewedin[9,10]. Besidesthis,thefragmentationpatterns observedintheMS/MSspectrainconjunctionwithmolecu- lar networking evaluation can enable the identification of previouslyunknowncloserelativeswithinthesamechemical familiesthroughspectralcorrelations[11,12].Finally,ifthe compounds have unprecedented nuclei, chromatographic isolationisinevitableforfulldenovoNMRstructuralcharac- terization, butatthesametimearethemostpromisingin termsoftrulynewantimicrobialsdiscovery.Forthisstep,the discriminatingsignalsgeneratedinthecomparativeanalysis (MDA)couldbeusedasprobestotrackthetargetofinterest (‘starantibiotic’)throughoutthechromatographicisolation, whichisreferredtoasNMR-[13,14]and/orMS-guided[15,16]
separationmethod.Thiscanpreventlosingtrackofthetarget thatis unfortunately quite commonin conventional TLC- guidedisolation.Furthermore,somedegreeofstructureiden- tification, albeit incomplete, can be achieved beforehand withthe2DNMRmeasurementsbecausetheyallowapartial prediction ofthenovel compounds. Thiscan also provide information onchemicalcharacteristics andhelpoptimize isolationprocedure,whichmakessensesforminimizingthe lossofcompounds(e.g.rulingouttheuseofbaresilicagelfor polyphenolicpolyketidesduetotheirpracticallyirreversible adsorptionasinthecaseofclostrubin[17]).Takentogether, thestrategyofmetabolomics-guideddiscoveryofnewanti- bioticsisillustratedinScheme1.
Theuseofmetabolomicsasamethodologyforde-replica- tionofknownmicrobialsecondarymetabolitesandprioriti- zation of promising candidates is exemplified in the following cases. The LC–MSmetabolome profiling of nine Myxococcusspeciesallowedtherapididentificationofknown myxobacterial metabolitesand thesubsequentcompound- basedprincipal componentanalysis (PCA) wassuccessfully highlightedtheputativenovelcompounddeterminedbyits exactmassandisotopicpattern[18].TimBugniandcollea- gues[19] usedUHPLC–HRMS-based metabolomics todere- plicate knownstructures andpredict novel compounds in microbialcrudeextract.TheythenappliedMS-guidedsepa- rationapproachtotargetthemoleculeshighlightedbymeta- bolomics analysis, and successfully discovered a series of antibiotics with novel skeletons, such as microtermolides [20], bottromycin D [21] and more recently forazoline A [22].ForazolineA(Fig.1)isapotentpolyketidewithahighly unusual skeleton, exhibiting in vivo antifungal efficacy against Candida albicans (MIC 16mg/mL) – comparable to thatofamphotericinBinamousemodelofC.albicansandno obviouscytotoxicity [22].Furthermore,they utilizedaLC–
MS–SPE–NMRhyphenationsystemthatintegratesMS-guided
separation and de novo NMR structure elucidation [23] to facilitate rapid structure elucidation of rare phenyl-acetyl- desferrioxaminesindicatedbymetabolomicsanalysis[19].In caseoflowlyabundantcompounds,targetedLC–MSmicro- isolationstrategy reportedby Jean-Luc Wolfenderandcol- leagues [24,25] for thepurification ofnovel metabolites is necessaryforfullstructuredeterminationbymeansofmicro- flow NMR analysis that just requires micrograms of com- pounds[26,27].
Metabolomicsappliedtomicrobialcocultivation Conventionally, scientists isolate pure strains of microbes collectedfromvariousbiological samples,suchas soil,ma- rine sediments,symbionts ofplants orsponges. Fermenta- tionsarethenscaleduptoaccumulatesufficientmaterialfor the next-stage systematical or bioassay-guided isolation.
However, the isolation of novel antibiotics from microbes bymeansofthiswell-establishedroutineisdifficult.Oneof the reasons is many putative gene clusters remain silent understandardlaboratorygrowthconditionswhenmicrobes arecultivatedsingly.Thus,itmightbefruitfultoconsidernot only single microorganism but more complex biological systems consisting of different organisms. This novel ap- proach in which microbes are grown together (co-culture orconfrontationexperiments)hasreceivedincreasinginter- est foritspotential toproduce newleads, andto decipher specificbiosyntheticpathwaysmainlyrelatedtodefense[28].
Thefocusshouldbesetonantagonisticinteractionsamong thedifferentmicrobes(fungiand/orbacteria)coexistingin thesamedefined environment, because they use chemical warfareconsistinggenerallyofsmallorganiccompoundsfor defense.Insomecases,clearlong-distancegrowthinhibition canbeobservedduringpairwise co-cultureonsolidmedia.
Thisphenomenaisalsoexpectedtobelinkedtotheproduc- tionofantimicrobialcompoundsasadefensemechanismby oneofthetwoco-inhabitingmicroorganisms[25,29].Cocul- turing the fungal endophyte Fusarium tricinctum with the bacterium Bacillus subtilis168 trpC2 onsolid rice medium resultedinupto78timestheproductionofenniatinsA1and B1[30]. Microbialcocultivation isalso effectiveforthein- ductionofnewmoleculeswithantimicrobialeffect,asveri- fiedby thediscoveryofthenew antibioticspestalone[31], emericellamides A and B [32], O-methylmellein [33]. It is quite probably that one microbe produce molecules that could be precursors for biotransformation into other final productsbyitspartner,whichcanincreasethechemodiver- sityofinducedmetabolitesandthusthechanceoffinding novelantibiotic[34].
Consideringthegreatpotentialofcocultivationasafertile sourcefornewantibiotics,itisnecessarytodevelopeffective methodsfor theirdetection andidentificationof induced metabolites. Metabolomics is particularly appropriate for this purpose as indicated in [29]. Untargeted generic
Microbial extracts (liquid or solid culture)
Chemical profiling (NMR, LC-MS)
Antimicrobial assessment (agar diffusion assay)
Bacillus subtilis and/or Escherichia coli as indicator
Chemometrics analysis (PLS, OPLS, etc)
Targets isolation (silica gel, Sephadex, C18-HPLC, etc)
Structure elucidation (2D NMR, HRMS, X-ray diffraction)
Antimicrobial test (96 well plate, MIC)
New antibiotics
Multi-drug resistant pathogens Validation
Design rational isolation procedure for wanted targets 1. Dereplicate known 2. Indicate structural novelty
Prioritized NMR or MS signals as probe;
Drug Discovery Today: Technologies Scheme1. Metabolomics-guideddiscoveryofnewantibiotics.
O
O O
O
S N H
O HN
O O
bottromycin D forazoline A
N N
NH
H N H
HN
O O
O S
N S
CI O
O O
O O
HN N
O OH
N
Drug Discovery Today: Technologies Figure1. Structurallynovelantibioticsdiscoveredthroughtheframeworkofmetabolomicsmethodology.
fingerprintingusing1HNMR,butmorebyhyphenatedMS methods(consideredbysometobemorepowerfulbecauseof theirsensitivity) isaimed to capture metabolites compre- hensively.Thecomparisonofthemetabolomeofthecocul- ture with that of the individual microorganisms can highlighttheinducedmetabolitespresentincoculturebut absent in monocultures, because these de novo-produced moleculeshavehighchancesofbeingnewcompounds(or evennewskeletons)withantimicrobialactivity.However,it isimportanttobearinmindthattheconventionalchemo- metricmethods,suchasPCAand(O)PLS-DA,arenotabso- lutelyidealforcoculturemetabolomicsstudies,becausethey mightnoteffectivelyfiltertheup-regulatedmetabolitesin monoculturesandthusnotalwayshighlightthecoculture- inducedmetabolites specifically. A noveldata mining ap- proach referred to as projected orthogonalized chemical encountermonitoring(POChEMon)wasrecentlydeveloped andhasprovedtobemoreappropriateforthispurpose[28].
Thedetectionofantibioticsusingthemicrobialcocultiva- tionstrategymaybefurtherrevolutionizedbytheapplication ofadvanced analyticaltechnologies introduced into meta- bolicprofiling,oneofwhichisimaging massspectrometry (IMS)[35–37].Thistechniquecanprovidedistinctchemical/
structural data on the spatial distributions of metabolites directly on theagar Petri dishes avoidingcomplex sample pre-treatment[38–41].Forthis,theantagonisticinteraction betweentestedmicroorganismsisfavored.Aftertheantago- nistic phenotypes are observed, theinhibition regions are subjectedtoIMSscreeningtovisualizethemetabolites.The microbialmetaboliteswithaspatialdistributionthatissuper- imposed with the zone of inhibition are considered to be potentialanti-infectivecandidates.Theexactmassandiso- topepatternprovidedbyhighresolutioninstrumentssuchas MALDI-TOF-IMS,incombinationwithMS/MSnetworkanal- ysiscanhelpestimatethestructuralnoveltyoftheinduced metabolites[11,42].Thistechniquewasappliedinindividual co-culturesofthepathogensStaphylococcusaureusandStaph- ylococcus epidermidis with Streptomyces roseosporus NRRL 15998. Using matrix-assisted laser desorption ionization (MALDI)imagingmassspectrometry,itwaspossibletoob- serve three molecules (m/z 863, 877 and 891) that were responsiblefortheinhibitionofbothStaphylococcuspatho- gens.Thesestress-inducedionsweresubsequentlyidentified as arylomycins, a class of broad-spectrum antibiotics that targettypeIsignalpeptidases[43].
Metabolomicsreveals naturallyoccurringmicrobial interactions
Throughout thousands of years of evolution and survival adaptation, microorganisms have established all types of closerelationships,includingsymbiosis,parasitism,compe- titionandantagonism.Revealingtheunderlyingmolecular mechanismsinvolvedinthese‘invisible’interactionswould
presumably contribute to the discovery of new chemical entities with antibiotic activity, because virulence factors intheirnatural contextsmight turnout tohavebeneficial applicationsinhumananti-infectivetherapy[44].Particular- ly,decipheringthemicrobialpathogenesis ofsomeecolog- ically relevant systems, such as associations with plants, mushrooms,insects andsponges [45], opens new perspec- tivesfortheexplorationfornewantimicrobials,asexempli- fiedbythediscoveryofthenovelantibiotics,rhizoxin[46]
anddesoxyhavannahine[47](Fig.2).Averypromisingbut yetquiteunexploredreservoirfornewantibioticsdiscoveryis theinsect-associatedmicrobiome.AsreviewedbyHelgeBode [48], insects could be true pioneers as providers of anti- infectivesfor human beings. Paenilamicins (Fig. 2),a class ofnovelhybridnonribosomalpeptide/polyketideantibiotics, werediscoveredinthebeepathogen,Paenibacilluslarvae.Bee larvalco-infectionassaysrevealedthatthepaenilamicinsare employedbyP.larvae tofightecologicalnichecompetitors andarenotdirectlyinvolvedinmortalityofthebeelarvae [49].
Forcultivablesymbionts,secondarymetabolitesproduced inanatural contextareprobably differentfromthosein a standard laboratorysetting, because thegrowth condition considerably influences microbial secondary metabolism.
Manycrypticbiosyntheticgeneclustersencodingmolecules, remain silent in the laboratory setting due to the lack of appropriateenvironmentalstimuli,anditisextremelychal- lengingtoactivatetheirexpressioninthelaboratory. More so, many symbiotic microbes are not even amenable to artificialcultivation.Inotherwords,antibioticsthatwould occurduetoinsitumicrobialinteractionsareimpossibletobe discovered in a traditional culture-dependent approach.
Therefore,weneedtomimicthenaturalsettingtoactivate
‘cryptic’antibiotics.Theadvancesinmetabolomicsprovide an opportunity to make what appeared to be impossible possible,using forexampletheabovedescribed IMS meta- bolomeprofilingtechniques.ThemotileGram-negativebac- terium Janthinobacterium agaricidamnosum causes soft rot diseaseintheculturedbuttonmushroom Agaricusbisporus.
Driven by the hypothesis that the pathobiology of this mushroompathogenwaspotentiallycorrelatedtoantifungal search, MALDI-IMS was used for in situ profiling of the secondarymetabolitesharboredinthemushroom-associated ecologicalsystemofA.bisporus vs.J. agaricidamnosum.The visualization of a unique ion of m/z 1181 [M+H]+ in the diseased region of themushroom prompted the effortsto determinethestructureofthiscompoundanditwasidenti- fiedasanunprecedentedcycliclipopeptideproduct,jagaricin (Fig.2).Jagaricinturnedouttobeafungicidethatishighly activeagainsthumanpathogenicfungiincludingC.albicans, AspergillusfumigatusandAspergillusterreusatsubmicromolar concentrations.Notably,jagaricinproductionby thebacte- rium J.agaricidamnosumtendedtooccurexclusively inthe
mushroom(A.bisporus)andwasotherwiseimpairedundera varietyoflaboratorycultivationconditions[50].Thisexam- pleservesasastrongsupportforourargumentsthatunder- standing naturally occuring microbial pathogenesis is beneficialforantibioticsearch;andthatIMS-basedmetabo- lomicscanbeappliedtorevealthecrypticantibioticsthatare onlyproducedinaparticularnaturalenvironment.
Revealingcrypticnew antibioticsinenvironmentalsam- plesmight benefit fromsinglecell metabolomics, a newly developedtoolformetabolicanalysisatcellularandsubcel- lularlevel(recently reviewedin[51–53]).Ourfocusisonits potential use in unveiling the metabolic communication
betweensymbiontsandhosts.Based onthemorphological phenotypes or other properties such as fluorescence, the selectivemetabolicanalysisofcertainmicrobialcellsinen- vironmentalsamplescouldbeusedtoviewtheuniquesec- ondarymetabolitesgeneratedbymicroorganismsduringthe naturally occuring interspecies interactions. For instance, halogeninsituhybridization-secondaryionmassspectrosco- py(HISH-SIMS)hasallowedthesimultaneousidentification and quantitation of metabolic activities of environmental microbialassemblageatsingle-celllevelwithouttheneedfor traditional microbial separation and laboratory cultivation [54]. Single-cell mass spectrometry (SCMS) is a rapidly N
O
HO O
O O
O O O O
O
O O
O O
O O
O
rhizoxin
Paenilamicin B1
jagaricin
desoxyhavannahine
NH OH OH
OH OH
NH NH
HN
HN
NH HN
NH
N
O
O O OH O
O O O
O O
O O
OH H
HO N
HN H
N NH
O O OH OH
O O
O O
O
NH N
H
HN H
N N H2N H
H2N NH2
NH2 OCH3
N
H N
H
HN H
N
HN
NH2
O OH
Drug Discovery Today: Technologies Figure2. Structuresofseveralnovelantibioticsdiscoveredthroughtheinvestigationofmicrobialinteractions.
growing fieldin analyticalchemistry,andthere areseveral examplesofitsapplicabilitytodifferentcells[51].Inthiscase, thepossibilityofaccuratemassmeasurementandacquiring structuralinformationasprovidedbytandemMS,facilitate theidentificationofnumerousmetabolitesfromasinglecell.
Conclusion
Metabolomicsmethodsprovideaneffectivestrategyforthe discoveryofnewantibioticsfrommicrobialsources.Allowing thecomprehensive chemometric comparison ofsecondary metabolomes, it makes finding the proverbial needle in a haystack more realistic. The ability to measure the entire microbial metabolomes during symbiotic interactions can becrucialfortheexploitationoftheseuntappedreservoirs ofanti-infectives[55].Correlatinggeneexpressionprofilesto fluctuationsinthemetabolomeallowsrapididentificationof the gene cluster for the (known or unknown) bioactive molecule[56].Thus,metabolomicscomplements(meta-)ge- nomics methodology in accessing previously inaccessible naturalproducts[57],andhastheaddedadvantageofdelving intothebiosyntheticpotentialofunculturablemicroorgan- isms when applied to in situ analysis of live, undamaged biologicalsamples.
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