Marine-Derived 2-Aminoimidazolone Alkaloids. Leucettamine B-Related Polyandrocarpamines

15 Pages·2017·1.82 MB·English

marine drugs
Article
Marine-Derived 2-Aminoimidazolone Alkaloids.
Leucettamine B-Related Polyandrocarpamines Inhibit
Mammalian and Protozoan DYRK & CLK Kinases
NadègeLoaëc1,2,ElettaAttanasio1,BenoîtVilliers1,EmilieDurieu1,TaniaTahtouh1,
MorganeCam1,RohanA.Davis3 ID,AlineAlencar4,MélanieRoué5,
Marie-LiseBourguet-Kondracki5,PeterProksch6,EmmanuelleLimanton7,SolèneGuiheneuf7,
FrançoisCarreaux7,*,Jean-PierreBazureau7,*,MichelleKlautau4,*andLaurentMeijer1,* ID
1 ManRosTherapeutics,PerharidyResearchCenter,29680Roscoff,Bretagne,France;
[email protected](N.L.);[email protected](E.A.);[email protected](B.V.);
[email protected](E.D.);[email protected](T.T.);[email protected](M.C.)
2 StationBiologiquedeRoscoff,CNRS,‘ProteinPhosphorylationandHumanDisease’Group,PlaceG.
Teissier,29680Roscoff,Bretagne,France
3 GriffithInstituteforDrugDiscovery,GriffithUniversity,Brisbane,QLD4111,Australia;
r.davis@griffith.edu.au
4 UniversidadeFederaldoRiodeJaneiro,InstitutodeBiologia—DepartamentodeZoologia,Av.Carlos
ChagasFilho373-CCS-BlocoA-SalaA0-100,IlhadoFundão,21941-902RiodeJaneiro,Brazil;
[email protected]
5 MoléculesdeCommunicationetAdaptationdesMicro-Organismes,UMR7245CNRS,MuséumNationald’
HistoireNaturelle,57rueCuvier(C.P.54),75005Paris,France;[email protected](M.R.);
[email protected](M.-L.B.-K.)
6 InstitutfürPharmazeutischeBiologieundBiotechnologie,Universitätsstr.1,40225Düsseldorf,Germany;
[email protected]
7 UniversitédeRennes1,InstitutdesSciencesChimiquesdeRennes,ISCRUMRCNRS6226,GroupeChimie
OrganiqueetInterfaces(CORINT),Bât.10A,CampusdeBeaulieu,AvenueduGénéralLeclerc,CS74205,
35042RennesCEDEX,Bretagne,France;[email protected](E.L.);
[email protected](S.G.)
* Correspondence:[email protected](F.C.);[email protected](J.-P.B.);
[email protected](M.K.);[email protected](L.M.);Tel.:[phone](F.C.);
[phone](J.-P.B.);[phone](M.K.);[phone](L.M.)
Received:30August2017;Accepted:12October2017;Published:17October2017
Abstract: A large diversity of 2-aminoimidazolone alkaloids is produced by various marine
invertebrates,especiallybythemarineCalcareousspongesLeucettaandClathrina. Thephylogenyof
thesespongesandthewidescopeof2-aminoimidazolonealkaloidstheyproducearereviewedinthis
article. Theorigin(invertebratecells,associatedmicroorganisms,orfilteredplankton),physiological
functions, and natural molecular targets of these alkaloids are largely unknown. Following the
identificationofleucettamineBasaninhibitorofselectedproteinkinases,wesynthesizedafamilyof
analogues,collectivelynamedleucettines,aspotentinhibitorsofDYRKs(dual-specificity,tyrosine
phosphorylation regulated kinases) and CLKs (cdc2-like kinases) and potential pharmacological
leads for the treatment of several diseases, including Alzheimer’s disease and Down syndrome.
Weassembledasmalllibraryofmarinesponge-andascidian-derived2-aminoimidazolonealkaloids,
alongwithseveralsyntheticanalogues,andtestedthemonapanelofmammalianandprotozoan
kinases. PolyandrocarpaminesAandBwerefoundtobepotentandselectiveinhibitorsofDYRKs
andCLKs. TheyinhibitedcyclinD1phosphorylationonaDYRK1Aphosphositeinculturedcells.
2-Aminoimidazolones thus represent a promising chemical scaffold for the design of potential
therapeuticdrugcandidatesactingasspecificinhibitorsofdisease-relevantkinases,andpossibly
otherdisease-relevanttargets.
Mar.Drugs2017,15,316;doi:10.3390/md15100316 www.mdpi.com/journal/marinedrugs
Mar.Drugs2017,15,316 2of15
Keywords: marine sponge; Porifera; Calcarea; ascidian; Polyandrocarpa; 2-aminoimidazolone
alkaloids; leucettamineB;leucettine; polyandrocarpamines; proteinkinases; DYRK;CLK;kinase
inhibitor;Alzheimer’sdisease;Downsyndrome
Mar. Drugs 2017, 15, 316 2 of 15
1. Introduction
Keywords: marine sponge; Porifera; Calcarea; ascidian; Polyandrocarpa; 2-aminoimidazolone
Proteinkinasescatalyzethephosphorylationofproteinsonserine,threonineandtyrosineresidues[1].
alkaloids; leucettamine B; leucettine; polyandrocarpamines; protein kinases; DYRK; CLK; kinase
They represinehnitbiatolra; Arglzehefaimmeirl’sy doisfeainset;r Dacoewlnlu slyanrdrroemgue lators which, after their initial discovery by Edwin
Krebsand EdmundFisherinthe1950s[2],hasseenmassiveresearchinvestmentontheirfunctionsand
regulations.Thehumankinomecomprises518kinases,manyofwhichdisplaysomeabnormalactivityor
deregulationinhumandisease.Thispromptedbothacademicandpharmaceuticallaboratoriestosearch
1. Introduction
for,optimize,characterizeanddeveloppharmacologicalinhibitorsofselecteddisease-relevantprotein
Protein kinases catalyze the phosphorylation of proteins on serine, threonine and tyrosine
kinases. Arsesaidmueast t[e1r]. oTfhfeayc tr,epkriensaenste sa hlaarvgee bfaemcoilmy eof thinetrlaecaeldluilnagr rtehgeurlaaptoersu twichitcahr,g aeftteirn tthheeir pinhitaiarml aceutical
industry, bdeifsocorveerGy -pbyro tEediwninc oKurpelbesd arnedc eEpdtmoursn,da nFidshoerv einr 3th0e in19h5i0bsi t[o2r],s hhaasv seeeanlr emaadsysivree arcesheeadrcht heclinical
market[1,3in,4v]e.sAtmltehnot uong hthmeira fnuyncitniohnisb aitnodr srewguelraetioinnist. iTahlley hduemriavne kdinforomme cnomatpurriaselsp 5r1o8d kuincatsse,sa, lmldanruy gofc andidates
which display some abnormal activity or deregulation in human disease. This prompted both
arechemicallysynthesizedatsomestageoftheirdevelopmenttowardsatherapeuticdrug.
academic and pharmaceutical laboratories to search for, optimize, characterize and develop
Durinpghaarmgeacnoeloragilcsacl rineheinbiftoorrs ionf hsiebleicttoerds doisfecaesell-rseilegvnaanlti nprgotaenind kcinealslecsy. Aclse ac monatttreor lokf ifnacats, eksin,awsees identified
the marinheavsep obnecgoemep rthoed ulecatdinlegu tcheetrtaapmeuitnice taBrgaets ina twhee apkhairnmhacibeuittiocral oinfdvuastrriyo, ubsefoprreo Gte-ipnrotkeiinn ases [5,6].
Followingcotuhpisledi nreitcieapltodrsi,s acnodv oevreyr, 30a inlahribgietorns uhamveb aelrreoadfya rneaaclhoegdu thees calinnidcald mearrikveat t[i1v,3e,4s]. wAlethreousgyh nthesized
many inhibitors were initially derived from natural products, all drug candidates are chemically
andoptimizedaskinaseinhibitors,especiallyasinhibitorsoftheDYRKs(dual-specificity,tyrosine
synthesized at some stage of their development towards a therapeutic drug.
regulatedkinaDsuersin)ga na dgeCnLerKals s(ccrdeecn2 -folirk ienhkiibnitaosrse so)f kcienlla ssiegnfaalmingi liaensd. Lceellu ccyectltea cmonintreol Bk-idnaesreisv, ewde inhibitors
arecollectiidveenltyifineda mthee dmalreiunec espttoinngees p[r6o,d7u]c.tT lehuecsetetaimnihnieb Bit aosr as wheaavke inahpiboittoern otfi vaalraiosuds rpurogtecina nkidnaidseast esforthe
treatment[o5f,6c].o Fgonlliotwivinegd thisiso irnditeiarls daisscsoovceirayt, ead lawrgeit nhuDmobewr nof saynanldogruoems aen(dD dSe)riavantdiveAs lwzehree ismynetrh’essidzeidse ase(AD),
and optimized as kinase inhibitors, especially as inhibitors of the DYRKs (dual-specificity, tyrosine
butalsoforsomecancers,inflammation,diabetes,andsoforth.
regulated kinases) and CLKs (cdc2-like kinases) kinase families. Leucettamine B-derived inhibitors
Leuceatrtea cmolliencetivBelwy naasmiendi tlieaulcleyttiindeesn [6ti,7fi]e. Tdhienset ihnehimbitaorrsi nhea,vec aal pcoatreenotiuals assp dorungg ceanLdeiudcaetetsta fomr tihcreo raphis[8].
The physitorelaotgmiecnatl offu cnogcntiiotivne odfisothrdiesrsm asasroicniaeteda lkwaitlho iDdowisn usynnkdnroomwe n(D, Sa)s anisd tAhlzehebiimoesry’sn dthiseeatsice pathway
leading its(ApDr)o, bduut catlsioo nfo.r sFoumret hcaenrcmeros, rien,flawmhmeatthioenr, dleiaubceteetst,a amndi nsoe foBrthis. produced by the sponge itself or
Leucettamine B was initially identified in the marine, calcareous sponge Leucetta microraphis [8].
bysomeassociatedmicroorganismsremainstobedetermined. Interestingly,leucettamineBshowsa
The physiological function of this marine alkaloid is unknown, as is the biosynthetic pathway
central2-almeaidninogi mitsi dpraozdoulcotinone. sFcuarfthfoerlmdosrhe,a wrehdethbeyr sleeuvceetrtaamlminea rBi nise pprordoudcuedc tbsy pthreo dspuocnegde ibtsyelfv oarr bioy usmarine
sponges,msoomset aosfsowcihatiecdh mbiecrlooonrggainnigsmtso rtehmeaicnlsa tsos bCe adlectaerrmeain,eadn. dIntbeyresotitnhgelyr, mleuacreitntaemiinnve eBr stehobwras tae s,suchas
central 2-aminoimidazolone scaffold shared by several marine products produced by various
ascidians and nudibranchs (Figure 1). Several extensive reviews on these marine alkaloids [9–11]
marine sponges, most of which belonging to the class Calcarea, and by other marine invertebrates,
and 2-aminoimidazolones in general [12] have been recently published. We thought it might be
such as ascidians and nudibranchs (Figure 1). Several extensive reviews on these marine alkaloids
interesting[9–t1o1]o abntda 2in-amsoinmoimeiodafztohloensees ilne guecneetrtaal m[12i]n heavBe- breeelan treedcenntlayt upurballispherdo.d Wuec tthsoutoghet vita mluigahtte them as
potentialkbien ainsteeriensthinibg ittoo rosb.taTinh sisomwea osf athlseosea lneuocepttpaomritnue nBi-tryelatotedre nvaiteuwral tphreodsupcotsn tgoe esvpaleucaitee sthpermo dasu cingsuch
moleculesp,owteintthialp kairntaisceu ilnahribeitmorps. hTahsisi swoans aLlseou caent toappaonrdtunCitlya tthor rienvaieswp ethcei essp,oangsew speelcliaess ptrhoeduwciindge scopeof
such molecules, with particular emphasis on Leucetta and Clathrina species, as well as the wide scope
2-aminoimidazolonesproducedbymarineorganisms.
of 2-aminoimidazolones produced by marine organisms.
OH
O OR OH
O H2N N
H2N N HN H2N N
MeN O HN
O PolyandrocarpamineA(R=Me) O
LeucettamineB PolyandrocarpamineB(R=H) PhorbatopsinA
Br
H2NHNN HN ONHBr 2-amRin1oHiRmN2id3Na2zoNlOi14n5-4-one MHNeNNMOe HN
O scaffold Aplysinopsine
DispacamideAH2NHNNR1 R2NHNHO MReNN NH N OO
O O MeN
HSSppymoonneggniiaaiaccliidddiiisnninABe(((RRR111===BBHrr;;;RRR222===BHBrr))) CCllaatthhrriiddiimnein(eR(=RO=)NH)
Figure 1. Structures of a selection of marine natural products sharing the 2-aminoimidazolin-4-one scaffold.
Figure1.Structuresofaselectionofmarinenaturalproductssharingthe2-aminoimidazolin-4-onescaffold.
Mar.Drugs2017,15,316 3of15
2. Results
2.1. PhylogenyofCalcareousSpongeswithSpecialEmphasisonLeucettaandClathrina
Sincemostofthecompoundsdescribedhereareproducedbymarinecalcareoussponges,wefirst
present a phylogeny of these 2-aminoimidazolones-producing sponges, with specific focus on the
LeucettaandClathrinaspecies. Sponges(phylumPorifera)aresessile,filter-feedinganimalsthatutilize
flagellate cells(choanocytes) topump waterthroughtheir bodies. They are morphologicallyvery
simple, as they do not have, for example, organs, sensorial cells or a nervous system. They are
consideredtheoldestpresentmetazoan(600Ma)[13]. Fourclassesofextantspongesarecurrently
accepted: Demospongiae,Hexactinellida,Homoscleromorpha,andCalcarea.
TheclassCalcareaBowerbank,1864,consistsofamonophyleticgroupofmarinespongeswhose
skeletonisformedbyMg-calcitespicules,whiletheothershavespiculesmadeofsilica[14]. Thisclass
iscomposedoftwosubclasses,bothmonophyletic—CalcineaBidder,1898,andCalcaroneaBidder,
1898—which differ by cytological, embryological, ontogenetic and skeleton characteristics. In the
Calcinea subclass, spicules are mainly regular (equiangular and equiradiate) and the first spicule
tobesynthesizedduringontogenyisthetriactine, whileinCalcaronea, spiculesareirregularand
diactinesarethefirstspiculestobeproduced. Moreover,thelarvaeinCalcineaisthecalciblastulaand
thenucleusofthechoanocytesisbasal,whileinCalcaroneathelarvaeistheamphiblastulaandthe
nucleusofthechoanocytesisapical. Calcareousspongesareconsideredviviparousandtheirlarvae
are lecytotrophic. Calcinea has two orders, Clathrinida Hartman, 1958, and Murrayonida Vacelet,
1981,theformerbeingthemostdiverse. ThegeneraLeucettaHaeckel,1872,andClathrinaGray,1867,
arepartoftheClathrinidaorder(seeSupplementaryDataandSupplementaryFiguresS1–S4,and
SupplementaryTableS1fordetaileddescriptionandphylogenyofLeucettaandClathrinaspecies).
2.2. PolyandrocarpaminesArePotentInhibitorsoftheDYRKandCLKKinases
Leucettamine B [8] shares a 2-aminoimidazolone scaffold with various marine natural
products such as polyandrocarpamines [15,16], dispacamide [17–19], aplysinopsine [20,21],
clathridine/clathridimine [8,22], hymenialdisine/spongiacidin B [23–30] and phorbatopsin [31,32]
(Figure1). Asummaryofallmarineaminoimidazolonesdescribedsofar,andtheirnaturalsources
andstructures,ispresentedinSupplementaryTableS2andSupplementaryFigureS5.
Wetestedaselectionofthese2-aminoimidazolones(Figure1)onabatteryof27purifiedkinases
(14mammalianand13unicellularparasites(SupplementaryTableS3)). Dose-responsecurvesrun
withtheactivecompoundsprovidedIC valuesthatarereportedinTable1. Asexpected[29,30],
50
hymenialdisineandthecloselyrelatedspongiacidinBwereverypotentinhibitorsofmostkinases(IC
50
valuesinthe1–10nMrange). Yettheywerenotveryselective,inhibitingessentiallyalltestedkinases.
WiththeexceptionofPfGSK-3,thePlasmodiumfalciparumorthologueofglycogensynthasekinase3
(GSK-3)[33],andLmCK1,theLeishmaniamajororthologueofcaseinkinase1(CK1)[34],unicellular
parasitekinasestendedtobelesssensitivethantheirmammaliancounterparts.
Aplysinopsine, dispacamide, clathridine and clathridimine were essentially inactive in the
kinase panel. As expected [6], leucettamine B was modestly active (sub-micromolar IC values),
50
andleucettineL41waspotentonmembersoftheDYRKsandCLKsfamilies. Again,littleinhibitory
activitywasdetectedwithkinasesfromunicellularparasites. Finally,bothpolyandrocarpaminesA
andBwerefoundtodisplaysignificantlyselectiveinhibitoryeffectsonDYRKsandsomewhatless
on CLKs (Table 1). To investigate the selectivity of polyandrocarpamines, polyandrocarpamine A
wasscreenedonthelarge-scaleDiscoveRxKinomeScanpanel(Figure2, SupplementaryTableS4).
Thisinteractionassayprovidesasemi-quantitativescoringviewoftheaffinityofacompoundfor
anyof442humankinases[35]. Thiscomprehensivescreeningapproachconfirmedtherathergood
selectivityofpolyandrocarpamineAforDYRKs,anddetectedadditionalaffinityforCSNK2A1/A2,
CLK1/4(Table2).
Mar.Drugs2017,15,316 4of15
Mar. Drugs 2017, 15, 316 4 of 15

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TSaubpleplSe4m.entary Table S4.
Mar.Drugs2017,15,316 5of15
Table1.Proteinkinaseselectivityofaselectionof10marinenaturalproductssharingthe2-aminoimidazolonescaffold:leucettamineBanditssyntheticleucettineL41
derivative;polyandrocarpaminesA&B,aplysinopsine,dispacamide,hymenialdisine,spongiacidinB,clathridine,andclathridimine.Allcompoundsweretestedat
variousconcentrationsagainst27purifiedkinases(14mammalianand13unicellularparasites).IC values(concentrationsinducing50%inhibitionofmaximal,
50
non-inhibitedkinaseactivity),calculatedfromthedose-responsecurves,arereportedinµM.IC valuesbelow1µMareshowninbold.
50
Kinases LeucettamineB LeucettineL41 PolyandrocarpamineA PolyandrocarpamineB Aplysinopsine Dispacamide Hymenialdisine SpongiacidinB Clathridine Clathridimine
CDK1/cyclinB >10 >10 >10 >10 >10 >10 0.005 0.031 >10 >10
CDK2/cyclinA >10 >10 >10 >10 >10 >10 0.011 0.068 >10 >10
CDK5/p25 >10 >10 >10 >10 >10 >10 0.0061 0.042 >10 >10
CDK9/cyclinT >10 >10 0.93 5.2 >10 >10 0.011 0.2 >10 >10
CK1δ/ε >10 4.2 3.3 >10 >10 >10 0.0037 0.031 >10 >10
CLK1 0.1 0.039 1.0 2.7 >10 >10 0.003 0.022 6.9 7.2
CLK2 0.91 0.21 1.2 4.9 >10 >10 0.0041 0.0073 6.0 6.1
CLK3 9.7 2.4 2.6 8.6 >10 >10 0.0053 0.049 >10 >10
CLK4 0.12 0.031 0.32 0.82 >10 >10 0.0029 0.0036 3.9 3.5
DYRK1A 0.42 0.032 0.27 0.47 >10 >10 0.0033 0.78 7.8 5.2
DYRK1B 1.8 0.05 0.43 1.2 >10 >10 0.0032 0.0058 >10 5.6
DYRK2 0.49 0.088 0.42 0.76 >10 >10 0.0031 0.021 7.1 6.2
DYRK3 0.6 0.42 0.17 0.88 >10 >10 0.0049 0.044 >10 6.1
GSK-3α/β 7.3 1.1 >10 >10 >10 >10 0.0032 0.044 >10 >10
PfGSK-3 >10 8.5 >10 >10 >10 >10 0.0041 0.038 >10 >10
PfCLK1 0.79 0.42 0.65 3.1 >10 >10 0.011 0.085 >10 >10
LmCK1 >10 >10 >10 >10 >10 >10 0.0025 0.025 >10 >10
LmCLK >10 >10 >10 >10 >10 >10 0.4 5.2 >10 >10
LmDYRK2 4.2 2.9 5.9 >10 >10 >10 0.38 9.0 >10 >10
LdDYRK1B 6.9 0.82 1.1 >10 >10 >10 0.042 0.5 >10 >10
LdDYRK3 >10 >10 >10 >10 >10 >10 0.021 0.3 >10 7.9
LdDYRK4 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
TbCLK1 >10 >10 >10 >10 >10 >10 0.53 0.71 >10 >10
TcCLK1 >10 >10 >10 >10 >10 >10 0.51 1.1 >10 >10
CpLAMMER 1.8 0.1 0.17 0.2 >10 >10 0.016 0.13 >10 >10
GlCLK 3.1 >10 0.95 2.2 >10 >10 0.012 0.12 >10 >10
TgCLK 4.1 >10 >10 >10 >10 >10 0.041 0.48 >10 >10
Mar. Drugs 2017, 15, 316 6 of 15
Table 2. Protein kinase selectivity of polyandrocarpamine A in a kinase interaction assay (DiscoveRx
Mar.Drugs2017,15,316 6of15
KinomeScan®). Polyandrocarpamine A was tested at 1 μM on a 442 kinases interaction panel. A
semi-quantitative scoring of this primary screen was obtained. This score relates to a probability of a
Thaitb rlaeth2.erP rtohtaenin stkriincta aseffsineilteyc.t iSvciotyreosf >p1o0l,y baentdwreoecna r1p–a1m0 iannedA <1in inadkiicnaates ethinatte trhaec tpioronbaasbsialyity(D oifs bcoevinegR ax
Kfailnseo mpoeSsictaivne® )i.s <P2o0l%ya, n<d1r0o%c,a r<p5a%m, irneespAectwivaeslyt.e sTthede 1a1t 1beµstM scoonresa a4r4e2 pkriensaesnetsedin. tFeurallc trieosnulptsa naerel.
Aavsaeimlabi-lqeu iann Stiutaptpivleemsceonrtianrgy oTfatbhlies Sp4r.i maryscreenwasobtained.Thisscorerelatestoaprobabilityofa
hitratherthanstrictaffinity. Scores>10,between1–10and<1indicatethattheprobabilityofbeing
Kinases Abbreviation Score
afalsepositiveis<20%,<10%,<5%,respectively. The11bestscoresarepresented. Fullresultsare
Dual specificity tyrosine-phosphorylation-regulated kinase 1A DYRK1A 3
availableinSupplementaryTableS4.
Casein kinase 2 α CSNK2A1 12
Homeodomain interacting protein kinase 3 HIPK3 19
Kinases Abbreviation Score
Proviral Integrations of Moloney virus 2 PIM2 21
Dualspecificitytyrosine-phosphorylation-regulatedkinase1A DYRK1A 3
Ribosomal protein S6 kinase alpha-4 (Kin.Dom.2-C-terminal) RPS6KA4 22
Caseinkinase2α CSNK2A1 12
Dual specificity tyrosine-phosphorylation-regulated kinase 2 DYRK2 24
Homeodomaininteractingproteinkinase3 HIPK3 19
Dual specificPirtoyv itryarloInstienger-aptihonosspofhMoroylolanteiyovni-rruesg2ulated kinase 1B PIM2 DYRK1B 21 25
RibosomalproteinS6kCinaasseeinal pkhina-a4s(eK 2in α.D’ om.2-C-terminal) RPS6KAC4SNK2A2 22 26
Dualspecificitytyrosine-phosphorylation-regulatedkinase2 DYRK2 24
Interleukin-1 receptor-associated kinase 1 IRAK1 28
Dualspecificitytyrosine-phosphorylation-regulatedkinase1B DYRK1B 25
CCadseci2n-kliiknaes kei2nαa’se 4 CSNK2A2CLK4 26 30
Interleukin-1Crdecce2p-tloikr-ea sksoinciaasteed 1k inase1 IRAK1 CLK1 28 32
Cdc2-likekinase4 CLK4 30
Cdc2-likekinase1 CLK1 32
To confirm the effects of the active products on native DYRK1A in a cellular context, we made
use of a SH-SY5Y neuroblastoma cell line expressing human DYRK1A [36] and antibodies directed
ToconfirmtheeffectsoftheactiveproductsonnativeDYRK1Ainacellularcontext,wemadeuse
against cyclin D1 phosphorylated at Threonine 286, a reported DYRK1A phosphorylation site [36,37]
ofaSH-SY5YneuroblastomacelllineexpressinghumanDYRK1A[36]andantibodiesdirectedagainst
(Figure 3). SH-SY5Y-DYRK1A cells were exposed for 24 h to each product (30 μM, except for
cyclinD1phosphorylatedatThreonine286,areportedDYRK1Aphosphorylationsite[36,37](Figure3).
hymenialdisine and leucettine L41, which were tested at 3 μM), cells were harvested and their
SH-SY5Y-DYRK1Acellswereexposedfor24htoeachproduct(30µM,exceptforhymenialdisine
proteins resolved by SDS-PAGE, followed by Western blotting with antibodies against
andleucettineL41,whichweretestedat3µM),cellswereharvestedandtheirproteinsresolvedby
P-Thr286-cyclin D1, total cyclin D1 and GAPDH (loading control) (Figure 3). Phosphorylation of
SDS-PAGE,followedbyWesternblottingwithantibodiesagainstP-Thr286-cyclinD1,totalcyclinD1
cyclin D1 at Thr286 was partially inhibited by polyandrocarpamine A (better than
andGAPDH(loadingcontrol)(Figure3).PhosphorylationofcyclinD1atThr286waspartiallyinhibited
polyandrocarpamine B, leucettine L41 and hymenialdisine, confirming the potent effects on
bypolyandrocarpamineA(betterthanpolyandrocarpamineB,leucettineL41andhymenialdisine,
DYRK1A in a cellular context.
confirmingthepotenteffectsonDYRK1Ainacellularcontext.

FFiigguurree 33.. InhInibhiitbioitnio onf coyfclciny cDli1n TDh1r28T6h pr2h8o6spphhoorsyplahtoiorny.l aStHio-nS.Y5YSH ce-SllYs 5eYxpcreesllssinegx hpuremssainn gDYhRuKm1aAn
DwYerReK tr1eAatewde rfoert r2e4a hte dwiftohr d2i4mhetwhyitlhsudlfiomxiedthe y(lDsuMlfSoOx)id (e−)( oDrM 30S Oμ)M( −of) eoarc3h0 cµomMpoofuenadc h(+c) o(3m μpMou fnodr
(le+u)c(e3ttiµnMe Lf4o1r alnedu cheyttmineeniLal4d1isainned). hPyromteeinnisa lwdeisrien ree).solPvreodt ebiyn sSDwSe-rPeArGeEso alvnedd anbaylySzDedS -bPyA WGEestaenrdn
abnloatltyinzged bwyitWh estaenrntibbolodtiteisn g wdiirtehctaendt iboadgiaeisnsdti recPte-Td28a6g-aciyncsltinP -T2D816,- cytcolitnal D1c,ytcoltianl cyDcl1i n Do1r
ogrlygcelyraceldraelhdyedhey-d3-ep-3h-opshpohsapteh adteehdyedhryodgreongaesnea s(eG(AGPADPHD)H ()u(suesde dasa sloloaaddiningg ccoonnttrrooll)).. NNuummbbeerrss iinn
ppaarreenntthheesseess iinnddiiccaattee tthhee lleevveell ooff ccyycclliinn DD11 pphhoosspphhoorryyllaattiioonn rreellaattiivvee ttoo tthhaatt ooff ccoonnttrrooll,, DDMMSSOO--ttrreeaatteedd
cceellllss ((110000%%))..
To extend these results we synthesized both polyandrocarpamines, as well as a series of
To extend these results we synthesized both polyandrocarpamines, as well as a series of
derivatives [38] (Figure 4), and tested them on a panel of 16 purified kinases. Synthesized (Table 3,
derivatives[38](Figure4),andtestedthemonapanelof16purifiedkinases. Synthesized(Table3,
PAC 1 and 2) and natural (Table 3, PAC 12 and 13) polyandrocarpamines showed very similar
PAC1and2)andnatural(Table3,PAC12and13)polyandrocarpaminesshowedverysimilareffects
o nthetargetkinases. Incontrast,noneofthesyntheticanaloguesdisplayedsignificantefficacyinthis
kinasepanel.
Mar. Drugs 2017, 15, 316 7 of 15
effects on the target kinases. In contrast, none of the synthetic analogues displayed significant
Mar.Drugs2017,15,316 7of15
efficacy in this kinase panel.

FFiigguurree 44.. SSttrruuccttuurree ooff ssyynntthheettiicc aannaalloogguueess aanndd ddeerriivvaattiivveess ooff ppoollyyaannddrrooccaarrppaammiinneess ((PPAACC11--PPAACC1111))
uusseedd iinn tthhiiss ssttuuddyy..
TTaabbllee 33.. PPrrootteeiinn kkiinnaassee sseelleeccttiivviittyy ooff ssyynntthheettiicc pprroodduuccttss ddeerriivveedd ffrroomm ppoollyyaannddrrooccaarrppaammiinneess AA aanndd BB..
AAllll ccoommppoouunnddss wweerree tteesstteedd aatt vvaarriioouuss ccoonncceennttrraattiioonnss oonn 1166 ppuurriififieedd kkiinnaasseess ((1144 mmaammmmaalliiaann aanndd ttwwoo
uunniicceelllluullaarr ppaarraassiitteess)).. IICC5500 vvaalluueess,, ccaallccuullaatteedd ffrroomm tthhee ddoossee--rreessppoonnssee ccuurrvveess,, aarree rreeppoorrtteedd iinn µμMM.. IICC5500
vvaalluueess bbeellooww 11 μµMM aarree sshhoowwnn iinn bboolldd.. PPAACC11 aanndd PPAACC22,, PPAACC1122 aanndd PPAACC1133 aarree ssyynntthheettiicc aanndd nnaattuurraall
ppoollyyaannddrrooccaarrppaammiinneess AA aanndd BB,, rreessppeeccttiivveellyy..
Polyandrocarpamines (PAC)
Kinases Polyandrocarpamines(PAC)
Kinases 1 2 3 4 5 6 7 8 9 10 11 12 13
1 2 3 4 5 6 7 8 9 10 11 12 13
CDK1/cyclin B >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
CDKC2D/cKy1c/licny cAli nB >10> 10 >10> 10 ≥10> 10 ≥1>01 0 >1>01 0 >1>01 0 >>1100 >>1100 >>1100 >>1100 >1>010 >10>10 >10>10
CDK2/cyclinA >10 >10 ≥10 ≥10 >10 >10 >10 >10 >10 >10 >10 >10 >10
CDK5/p25 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
CDK5/p25 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10 >10
CDK9/cyclin T 1.1 2 >10 >10 ≥10 >10 >10 0.51 >10 >10 >10 0.9 1.8
CDK9/cyclinT 1.1 2 >10 >10 ≥10 >10 >10 0.51 >10 >10 >10 0.9 1.8
CK1CδK/ε1 δ/ε 5.2 5.2 >10> 10 ≥1≥0 10 ≥1≥0 10 >1>01 0 >1>01 0 >>1100 >>1100 33..66 >>1100 >1>010 5 5 >10>10
CLKC1L K1 0.430 .43 1 1 2.22 .2 ≥1≥0 10 2.22. 2 >1>01 0 >>1100 >>1100 ≥≥1100 >>1100 4.24.2 0.470.47 0.590.59
CLKC2L K2 1.2 1.2 2.5 2.5 6.36 .3 >1>01 0 7.57. 5 >1>01 0 >>1100 >>1100 >>1100 >>1100 ≥1≥010 0.640.64 2.422.42
CLKC3L K3 2.3 2.3 9.2 9.2 >10> 10 >1>01 0 >1>01 0 >1>01 0 >>1100 >>1100 >>1100 >>1100 >1>010 2.72.7 10 10
CLKC4L K4 0.4 0.4 0.950 .95 2.32 .3 ≥1≥0 10 2.42. 4 >1>01 0 >>1100 >>1100 >>1100 >>1100 3.23.2 0.470.47 0.910.91
DYRK1A 0.23 0.75 2.1 4 1.3 >10 >10 >10 >10 3 2.1 0.3 0.59
DYRK1A 0.23 0.75 2.1 4 1.3 >10 >10 >10 >10 3 2.1 0.3 0.59
DYRK1B 0.4 1.1 8.2 >10 6.1 >10 >10 >10 >10 >10 6.2 0.47 0.96
DYRK1B 0.4 1.1 8.2 >10 6.1 >10 >10 >10 >10 >10 6.2 0.47 0.96
DYRK2 0.14 0.48 2.2 4.5 0.64 >10 >10 >10 >10 3.4 1.3 0.24 0.38
DYRDKY2R K3 0.140 .280.480 .9 2.2> 10 4.55 .1 0.654.7 >1>01 0 >>1100 >>1100 >>1100 >31.04 2.41.3 0.340.24 0.680.38
DYGRSKK3-3 α/β 0.28> 10 0.9> 10 >10> 10 5.1> 10 5.>71 0 >1>01 0 >>1100 >>1100 >>1100 >>1100 22.4 >100.34 >100.68
GSK-P3fαG/SβK -3 >10> 10 >10> 10 >10> 10 >1>01 0 >1>01 0 >1>01 0 >>1100 >>1100 >>1100 >>1100 >102 >10>10 >10>10
PfGSLKm-3C K1 >10> 10 >10> 10 >10> 10 >1>01 0 >1>01 0 >1>01 0 >>1100 >>1100 0>.1603 >>1100 >1>010 >10>10 >10>10
LmCK1 >10 >10 >10 >10 >10 >10 >10 >10 0.63 >10 >10 >10 >10
3. Discussion
3. Discussion
CalcareousspongesoftheLeucettaandClathrinageneraandafewothermarineinvertebrates
produCcaelcaarweoeaulst hspoofnregleast eodf ythete dLievuecresteta2 -aanmd iCnloaitmhriidnaa zgoelonnereaa alknadlo aid fsew(S uopthpeler mmeanrtianrey iTnavbelreteSb2raanteds
Spuropdpulecme ean wtaerayltFhig oufr reeSla5t)e.dT hyeete dxiisvteernscee 2o-fatmheinseoiamlkiadlaozidosloinnes eavlkearaloliCdasl (cSaurepopulsemspeonntgareys rTaaisbelse sSe2v aenradl
qSuuepsptiloemnsernetgaarryd iFnigguthree irS5p)r.o Tdhuect ieoxnis.tWenhcaet oisf/ tahreesteh eablkioalsoyindtsh einti cspevatehrwala Cy/aslcianrveooluvse dsp(soenegreesf erraeinscees
1se9vfeorralfi qrsutehstyiopnost hreesgiasr)?dinAgr ethtehier speromdoulcetciuolne.s Wachtauta ilsly/arper othdeu cbeiodsybnyththeetics ppoanthgwesayo/rs binyvaoslsvoecdi a(tseede
mreifcerroeonrcgea 1n9is mfosr? fiIrnst ahdydpitoiothne,stihs)e?s eAmreo ltehceusele smmolaeycudleesri vaectfuraolmly aprpoladnukcteodn bsyo utrhcee stpakoenngeusp o(ra nbdy
pasossoscibialytedm emtaicbrooloizrgedan)ibsymtsh?e Isne afidltdeirt-ifoene,d tehress.eT mheoflaeccuttlehsa tmpaoyl ydaenrdivreo cfarorpma ma ipnleasnwkteorne sidouenrcteifi teadkeinn
auspc id(aiannds p(Pooslsyiabnlyd romcaertpaabospli.z)e[d1)5 ]bayn dthtehsaet mfilotestr-2f-eaemdeinrso.i mThidea zfaoclot ntehsatw peroelydainscdorvoecraerdpainmcianlecsa rweoeures
sidpeonntgifeiesdo ifnt haseciLdeiuacnest t(aPoalnydanCdrloactharrpinaa spg.e)n [e1r5a] a(nSudp tphaletm meonstta 2r-yamTainbloeimS2id)aszuoplopnoersts wtehree hdyispcootvheerseids
oinf cmailccraoreoorguasn sipsmon-gdeesr iovfe dthme eLteaubcoetltiate asn.dH Colawthervienra, gceenlleurlaa r(Sloucpaplilzeamtieonntaoryf cTlaabthler idS2im) sinueppreovrtesa ltehde
its presence in sponge cells rather than in associated bacteria [22]. Several 2-aminoimidazolones
Mar.Drugs2017,15,316 8of15
werefoundinNotodorisnudibranchsandinthecalcareousspongestheycrawlandfeedon[39–41].
Altogetherthesescatteredresultssuggestpossibletransfersof2-aminoimidazolones,theirprecursors
and/ormetabolitesfromoneorganismtoanother.
Manyquestionsremainopenregardingthephysiological/ecologicalfunctionsofthesenatural
products. Are they used as repellant for predators or to control the expansion of associated
microorganisms? Dotheyhavespecificfunctionsforthesponge/ascidian/nudibranchmetabolism
itself? Whataretheirnaturalmolecularandcellulartargets? Aretheysynthesizedaskinaseinhibitors
ordotheytargetotherenzymesorstructuralproteins?Someofthesemayhaveorthologuesinhumans.
Thebiologicalsource,functionsandnaturaltargetsofmarine2-aminoimidazolonealkaloidsshouldbe
furtherinvestigated.
Only a few of these 2-aminoimidazolones have been chemically synthesized (Supplementary
Table S2, right column) and are thus relatively easy to access for biological studies of their
pharmacological properties. Nevertheless, they have already proved to be promising scaffolds
for the design of potential therapeutic drug candidates. The identification of leucettamine B [6]
and polyandrocarpamines A and B [this article] as potent and relatively selective inhibitors
of disease-relevant kinases opens the way to the development of drug candidates for various
applications [7,42]. This could be guided by the crystal structures of leucettines in complex with
variouskinaseswhichhavebeensolved[7]. AtManRosTherapeuticswearecurrentlydeveloping
leucettinesasDYRK1Ainhibitorydrugcandidates[6,7,42,43]toaddresscognitivedeficitsassociated
withDS[Nguyenetal.,inpreparation]andAD[44,45],butapplicationsincancer,inflammationand
diabetestreatmentshouldbeinvestigated,too.
Besides kinase inhibition, polyandrocarpamine derivatives have been recently identified
as inhibitors of cystathionine β-synthase, another potential therapeutic target for DS [46].
2-Aminoimidazolones have been tested for evaluation of their antibacterial activity, anti-biofilm,
antifungal, anticancer [32,47], anti-tuberculosis, anti-protozoan, antiviral, anti-inflammatory,
adrenergic, anti-histaminic, anti-cholinergic, anti-serotoninergic, cardiac, immunosuppressive,
leukotrieneB receptorantagonistic[8,48]andADdrugactivities[12]. Itwouldbeofgreatinterestto
4
runmoremarinederived2-aminoimidazolonealkaloidsonwiderenzyme/cellscreeningpanelsto
identifynoveltargetsandpotentiallynoveltherapeuticapplications. Hopefullythesemoleculeswill
addtothegrowinglistofmarinedrugsanddrugcandidatesdevelopedinrecentyears[49,50].
4. ExperimentalSection
4.1. DNASequencing,AlignmentandPhylogeneticAnalyses
The methods used for molecular phylogenetic analyses of Leucetta and Clathrina species are
detailedintheSupplementaryData.
4.2. Chemistry: PurificationorSynthesisofPolyandrocarpaminesAandB,andAnalogues
Forthepurificationofclathridineandclathridimine,specimensofClathrinaclathruswerecollected
byscubadivinginthenorthwesternMediterraneanSea(Marseille,France)between10and15mdepth.
Lyophilizedsamples(4.2g)wereextractedwith1:1CH Cl /MeOH(3×20mL,sonicationfor15min)
2 2
atroomtemperature. The1:1CH Cl /MeOHextractwasconcentratedunderreducedpressureto
2 2
yield a dark brown viscous oil (208.1 mg), which was chromatographed on a C SPE column in
18
a vacuum chamber (H O, H O/MeOH 1:3, MeOH, CH Cl , 100 mL of each). The fraction eluted
2 2 2 2
withH O/MeOH,1:3(47.9mg)wassubjectedtosemi-preparativereversed-phaseHPLC(Gemini
2
C -phenyl10×250mm)withincreasingamountsofCH CN/0.1%formicacidinH O/0.1%formic
6 3 2
acidaseluent(flowrate: 5mL/min,wavelength: 254and280nm)andaffordedclathridimine(4.6mg,
0.11%ofspongedryweight)andclathridine(2.8mg,0.07%ofspongedryweight).
PAC1 to 11 were supplied by Rohan A. Davis The synthesis, purification and spectroscopic
characterizationofthisfocusedpolyandrocarpaminelibraryhasbeendescribedelsewhere[38].
Mar. Drugs 2017, 15, 316 9 of 15
PAC1 to 11 were supplied by Rohan A. Davis The synthesis, purification and spectroscopic
Mar.Drugs2017,15,316 9of15
characterization of this focused polyandrocarpamine library has been described elsewhere [38].
Aplysinopsine and leucettamine B were synthesized according to a previously described
protoAcopll y[5s1in,5o2p] sriensepeacntidvelleyu. cDeitstapmacianmeidBe wwearse sysynnththeseisziezded asa dcceoscrrdiibnegd itno [a53p].r eviously described
protoTchoel [5s1y,n5t2h]erseissp eocft ivpeollyy.aDnidsrpoaccaarmpaidmeinweass sAy n&th eBsi z(ePdAaCs1d easncrdib ePdAiCn2[)5 3w].as performed from
correTsphoendsiynngt hebseinszaoldfehpyodlyea ndderroicvaartpivaems,i nens-prAopy&lamBine( PAanCd1 2a-nadminPoAimC2id)azwoalisn-4p-eornfeo,r mthede
pfrroemparcaotirornes opfo wndhiinchg wbaesn pzraeldvieohuysdlye ddeescrirvibaetidv eins ,litne-rpatruorpey [l5a3m] i(nFeiguarned 5)2. -Gaumainniodiimneid (a2z.9o5l imn-m4-ooln, e1,
ethqe) apnredp HarCatlaioqn 6oNf w(9h micLh)w waesrpe rheevaioteuds layt d1e2s0c °rCib efodri n22l ihte. rWatautreer [w53a]s( rFeigmuorvee5d). uGnudaenr ivdaincueu(2m.9 5anmdm thoel,
c1reuqd)ea npdroHdCuclat qw6aNs (s9omluLb)ilwizeerde hine ahteodt aetth1a2n0o◦lC (f9o rm2L2)h. .DWieattheyrlwetahserre m(6o mveLd) uwnadse ravdadceudu,m thaennd tthhee
mcruixdtuerper owdausc tcowoalsedso tlou b0i l°izCe.d Ainftehro t6e hth, afnilotrla(t9iomnL w).aDs ieptehryfolertmheerd( 6anmdL t)hwe a2s-aadmdiendo,imthiednazthoelimn-i4x-tounree
wwaass icsooollaetdedt oas0 a◦ Cw.hAitfete pro6whd,efirl twraittiho na 4w9a%s pyeierlfdo.r mRMedNa n1Hd (thDe2O2-)a: mδ 4in.1o8im (si,d 2aHzo, lHin--14)-; oRnMeNw a1s3Cis (oDla2Ote)d:
δa s4a8.5w (hCit-e1)p; 1o7w4d.9e r(Cw=iOth); a[M49 −% HyCiel]ld+ =. R99M.0N4271H (9(9D.024O33): Cδ3H4.15N83(Os,);2 FH =, H20-01–);2R02M °NC. 1P3ACC(D1 2aOnd): PδA48C.25
w(Ce-r1e) ;t1h7e4n.9 p(rCo=dOuc);e[dM b−y mHiCxli]n+g= th99e. 0c4o2r7re(s9p9o.0n4d3i3ngC bHenNzalOde);hFyd=e2 0(v0a–n20il2lin◦C o.rP AcaCte1chanalddPeAhyCd2e w1e.4r8e
3 5 3
mthmenolp, r1o deuqc)e wdibthy mn-ipxrinopgytlhaemcionrer e(s2p.9o5n dminmgobl,e 2n zeaqld).e Thhyed eim(viannei lfloinrmoratciaotne cwhaalsd peheryfdoerm1.e4d8 munmdoerl,
m1eicqr)owwiathven -irprraodpiyatliaomni nate 6(20. 9°5Cm fomr o3l0, 2meiqn) .(Tmhaexiimmiizneedf oarumthaotiroiznewd apsopweerfro 6r0m Wed).u Andceetromniitcrriolew wavaes
airdrdadedia ttioo nthaet 6cr0u◦dCe fiomri3n0e mwiinth( m2-aaxmiminiozeimdiaduatzhoolirniz-4e-donpeo w(1e.r486 0mWm)o.lA, c1e teoqn),i tarinlde wthaes madidxteudreto wthaes
sctriurrdeedi mati nreeflwuixt hfo2r- a6m3 hin. oTihmei dsoazlvoelinnt- 4w-oasn ere(m1.4o8vemdm uonld,1ere rqe)d,auncdedt hperemssixutruer.e wasstirredatrefluxfor
63h.PTAhCe1s owlvaesn tiswolaasterdem aos vae dbeuingde eprorwedduecre dwpitrhe sas u7r1e%. yield. RMN 1H (DMSO-d6): δ 3.79 (s, 3H,
OCHP3)A; C6.123w (ass, 1isHol,a Cte=dCaHs)a; 6b.e7i6g e(dp,o 1wHd, eJr =w 8i.t0h Haz7,1 H%-y5i)e; l7d..10R M(slN, 21HH, (NDHM2S);O 7-.d365) :(sδl,3 1.7H9,( Hs,-36H); ,7O.5C6H (s3l),;
16H.23, H(s-,21)H; 9,.C24= (CsHl, )1;H6.,7 O6H(d),; 11H0.,3J8 =(s8l,. 01HH,z N,HH-)5;) R;7M.1N0 (1s3lC, 2(HD,MNSHO2-)d;67):. 3δ5 5(6s.l2, 1(OHC,HH-36));; 171.546.6( s(Cl,-12H);, 1H16-2.0);
(9C.2-45)(;s 1l,214H.0 ,(OC=HC)H;1)0; .13287(.s6l ,(C1H-6,);N 1H47).;1R (MC-N3);1 31C47(.D9 M(CS-4O);- d[6M):]+δ =5 62.323.(0O7C97H (32)3;31.1048.060 (CC1-12H);111N163O.03()C; F-5 >);
212640. 0°C(C. =CH);127.6(C-6);147.1(C-3);147.9(C-4);[M]+=233.0797(233.0800C H N O3);F>260◦C.
11 11 3
PPAACC22 wwaassi siosolaltaetdeda saas bae ibgeeigpeo wpdowerdwerit hwait8h0 %a 8y0ie%ld .yRieMldN. R1HMN(D M1HS O(D-dM6)S:Oδ-6d.16)4: (δs ,61.H14, C(s=, C1HH),;
C6.=7C0H(d),; 16H.70, J(d=, 81.H1H, Jz =, H8.1-5 H);z6,. 9H2-(5s),; 26H.9,2N (sH, 22H);,7 N.27H(2d);, 71.H27, (Jd=, 17H.5,H J z=, 7H.5- 6H);z7, .H53-6()s;, 71.H53, H(s,- 21)H;8, .H81-2()s;,
81.H81, N(sH, )1;H9.,1 N9H(s,);1 H9.1,9O H(s,) ;11H0.,4 O5H(s,);1 H10,.4O5H ()s;, R1MH,N O1H3C); (RDMMNSO 1-3dC6 )(:DδM11S2O.5-d(6C):= δC H11)2;.151 6(C.6=(CCH-5));; 111168..62
((CC--52));; 111283..21 ((CC--62));; 112283..21 ((CC--16));; 114268..02 ((CC--31));; 114466..60 ((CC--43));; F14>6.266 (0C◦-C4).; F > 260 °C.

FFiigguurree 55.. SScchheemmee ooff tthhee ssyynntthheessiiss ooff ppoollyyaannddrrooccaarrppaammiinneess..
44..33.. BBiioollooggyy
44..33..11.. BBuuffffeerrss
HHoommooggeenniizzaattiioonn bubfufefrf:er2:5 m25M MmOMP S;M15OmPSM; E1G5T Am;1M5 mMEGMTAgC; l21;56 0mmMM β-gMlygcCelr2o; ph6o0s phmaMte;
β1-5glmycMeropp-nhiotrsopphhaeten;y 1lp5 hmoMsp hpa-nteit;ro2pmheMnydlpithhoiospthhraetiet;o 2l (mDMTT d);it1himotMhreNitoa3l V(DOT4T;)1; 1m mMMN NaaF3;V1Om4;M 1
mdiM-so dNiauFm; 1p hmenMy lpdhi-ossopdhiuamte; p1Xhepnryoltpehasoespinhhaitbei;t o1rXc opcrkottaeials;e0 .2in%hiNbiotnoird ceotcPk-t4a0ils; u0b.2st%it uNteo.nidet P-40
substBituuftfee.r A:10mMMgCl ;1mMEGTA(MW380.4);1mMDTT(MW154.2);25mMTris/HCl(MW
2
121.1B),uafnfedr; A50: µ10g /mmML hMepgCarli2n; .1 mM EGTA (MW 380.4); 1 mM DTT (MW 154.2); 25 mM Tris/HCl
(MWB 1u2f1f.e1r),C a:n6d0; 5m0M μgβ/m-gLly hceerpoaprhino.s phate; 30mMp-nitrophenylphosphate; 25mMMOPSpH7.0;
5mMEGTA;15mMMgCl ;1mMDTT,and;0.1mMsodiumvanadate.
2
Mar.Drugs2017,15,316 10of15
AllchemicalswerepurchasedfromSigmaAldrich(St.QuentinFallavier,France),unlessotherwise
statedandtheproteaseinhibitorcocktailwasfromRoche(Boulogne-Billancourt,France).
4.3.2. ProductionandPurificationoftheParasiticKinases
The parasitic kinase genes (Supplementary Data, Table S3) were optimized for expression in
E.coli,synthesized,andclonedinpGEX-6P-1(GEHealthcare,Sigma-Aldrich,St. QuentinFallavier,
France) with the type II restriction enzymes BamHI and XhoI for glutathione S-transferase (GST)
fusionattheN-terminus(GenScript). PlasmidsweretransformedintochemicallycompetentE.coli
BL21(DE3),whichweregrownovernightat37◦Cin2×YTmediumcontaining100µg/mLampicillin.
Thesecultureswereusedtoinoculate1Lvolumesof2×YTmedium(containing100µg/mLampicillin)
in5Lflasks. Thecultureswereallowedtogrowat37◦Cbeforethetemperaturewasdecreasedto
18◦C.Atanopticaldensityat600nm(OD600)ofabout1.0,proteinexpressionwasinducedovernight
at18◦Cwith0.1mMisopropylβ-D-1-thiogalactopyranoside(IPTG).Thebacteriawereharvestedby
centrifugationandwerefrozenat−20◦C.
Cellswereresuspendedinalysisbuffer(PBSpH7.4,1%NP40,1mMDTT,1mMEDTA,1mM
PMSF)inthepresenceofproteaseinhibitorcocktail(Roche),andlysozymewasaddedto1mg/mL
andincubated1hat4◦Cwithgentleagitation. Cellswerethensonicatedonicebefore6mMMgCl
2
and 25 U/mL benzonase (Novagen, Merck Millipore, Fontenay sous Bois, France) were added to
degradenucleicacidsanddecreasesampleviscosity. Aftercentrifugationat4◦C,thesolublefraction
wascollectedandproteinswereboundtoGlutathionesepharose4Bbeads(GEHealthcare). Beads
werewashedthreetimeswithlysisbufferandoncewithmodifiedbufferC(25mMMOPSpH7.0,
60mMβ-glycerophosphate,15mMp-nitrophenylphosphate,15mMEGTA,15mMMgCl ,2mMDTT,
2
1mMsodiumvanadate). Proteinswereelutedwithelutionbuffer(bufferCcontaining30mMreduced
glutathione,15%glycerol,andpHadjustedto8.5).
4.3.3. ProteinKinaseAssays
KinaseactivitieswereassayedinbufferAorCat30◦CatafinalATPconcentrationof15µmol/L.
Blankvaluesweresubtractedandactivitieswereexpressedinpercentofthemaximalactivity,i.e.,inthe
absenceofinhibitors. ControlswereperformedwithappropriatedilutionsofDMSO.
The GS-1, CKS, CDK7/9 tide and RS peptide substrates were obtained from Proteogenix
(Oberhausbergen,France).
CDK1/cyclin B (M phase starfish oocytes, native), CDK2/cyclin A and CDK5/p25 (human,
recombinant)werepreparedaspreviouslydescribed[6,7]. Theirkinaseactivitywasassayedinbuffer
A,with1mghistoneH1/mL,inthepresenceof15µmol/L[γ-33P]ATP(3000Ci/mmol;10mCi/mL)
inafinalvolumeof30µL.After30minincubationat30◦C,thereactionwasstoppedbyharvesting
ontoP81phosphocellulosesupernatant(Whatman,DutscherSAS,Brumath,France)usingaFilterMate
harvester(PerkinElmer,Courtaboeuf,France)andwerewashedin1%phosphoricacid. Scintillation
fluidwasaddedandtheradioactivitymeasuredinaPackardcounter.
CDK9/cyclin T (human, recombinant, expressed in insect cells) was assayed as described
for CDK1/cyclin B, but using CDK7/9 tide (YSPTSPSYSPTSPSYSPTSPSKKKK) (8.1 µg/assay) as
asubstrate.
GSK-3α/β(porcinebrain,native)andPfGSK3(plasmodiumfalciparum,recombinant,expressedin
E.coliasGSTfusionproteins)wasassayed,asdescribedforCDK1with0.5mgBSA/mL+1mMDTT,
usingGS-1(YRRAAVPPSPSLSRHSSPHQSpEDEEE)(pSstandsforphosphorylatedserine),aGSK-3
specificsubstrate[54].
CK1δ/ε(porcinebrain,native)andLmCK1(leishmaniamajor,recombinant,expressedinE.colias
HISfusionproteins[34])wasassayedasdescribedforCDK1butinbufferCandusing25µMCKS
peptide(RRKHAAIGpSAYSITA),aCK1-specificsubstrate[55].
DYRK1A,1B,2and3(Human,recombinant,expressedinE.coliasGSTfusionproteins)were
purifiedbyaffinitychromatographyonglutathione-agaroseandassayedasdescribedforCDK1/cyclin