Medicinal Chemistry and Chemical Biology Chimia 2013, 67, Nr

medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 519

doi:10.2533/chimia.2013.519 Chimia 67 (2013) 519–543 © Schweizerische Chemische Gesellschaft

MedicinalChemistry Chemical Biology MC001 Medicinal ChemistryChemical Bioloy MC002

SOM230:Anew therapeuticmodality for Cushing’s disease Directed evolution of bicyclic peptides fortherapeutic application

Ian Lewis, Janos Pless, Rainer Kneuer, Antonio P. Silva, Daniel Hoyer, Gisbert Lisa Pollaro, Alessandro Angelini, KhanMaola andChristian Heinis Weckbecker, ChristianBruns and Herbert A. Schmid EPFL, BCH5305, 1015 Lausanne,Switzerland GlobalDiscovery Chemistry Department ExploratoryMedicinal Chemistry Unit andAutoimmune, Oncologyand Neuroscience Research Departments, Bicyclic peptideswith highbinding affinity andtargetselectivitycan be NovartisInstitutesofBiomedicalResearch, CH-4002 Basel,Switzerland isolated from large combinatoriallibrariesbyphage display as shown in the figurebelow. In brief,peptide librariesdisplayed on phage arecyclizedina The somatostatin (SRIF,somatotropinrelease inhibiting factor) field has chemical reactionand subjected to affinity selections. The bicyclic peptides been asuccess story in termsofmedicinal chemistryand drug discovery combine keyqualities of antibodytherapeutics (high affinity andspecificity) offering avariety of therapeutic opportunities, e.g. acromegaly, gastrointes- andsomeadvantagesofsmall molecule drugs. tinal neuroendocrine tumors, whole body imaging and radiotherapy. Indeed, arational medicinal chemistry approach capitalising on structure activity relationships led to the discovery of SOM230, astable cyclohexapeptide somatostatinmimic which exhibits unique bindingtohuman SRIF receptors (sst1-5). This approach involved transposing functionalgroups, in the form of unnatural amino acids, from SRIF-14 intothe stable,reduced size cyclohexapeptide template. Further, the hydroxyproline urethane extension of SOM230 has been functionalized with the chelators DTPA and DOTA, which is anecessary prerequisite forthe possibledevelopment of ligands which couldbeused for whole body imaging. Uniquely, SOM230exhibits binding with a30to40times higher affinity than Sandostatin® to the sst1 and sst5 receptors and exhibits higher efficacy in preclinical models in low- ering Growth Hormone, Insulin-Like Growth Factor-1, ACTH and corti- Potent andselective bicyclic peptide inhibitorsofthe cancer-associated costeronethanSandostatin®.Recently, phase III clinical studies have estab- proteasesurokinase-type plasminogenactivator (uPA) andmatrix lished thetherapeutic potential of SOM230 /Pasireotide (Signifor®), as the metalloproteinase 2(MMP-2) were recentlydeveloped. The properties of 1 first pituitary directedmedical therapyfor Cushing’s disease leadingto these inhibitorsand their structureswill be discussedaswellasfirst in vivo registration of SOM230 by both EMEA and FDA in 2012. resultspresented.

[1] Annamaria Colao, Stephan Petersenn, JohnNewell-Price,James W. [1]Heinis, C., et al., Nat. Chem.Biol.. 2009, 5,502. Findling,FengGu, MarioMaldonado,UlrikeSchoenherr, DavidMills, [2]Angelini, A., et al., ACS Chem.Biol. 2012, 18,817 Luiz RobertoSalgado and Beverly M.K. Biller for the PasireotideB2305 [3]Angelini, A., et al., J. Med. Chem., 2012, 26,10187 StudyGroup,A12-MonthPhase 3Study of Pasireotide in Cushing’s Dis- ease, NEngl JMed 2012;366:914-24.

MedicinalChemistryChemical Biology MC003 Medicinal Chemistry Chemical Biology MC004

Non-nucleosidicCOMTbisubstrate inhibitors Synthesisand testing of photoaffinity probes forthe site-selective chemical modificationsofthe 5-HT3 receptor Christian Lerner,[a] Caterina Bissantz,[a] BerndBüttelmann,[a] François Diederich,[b] AlainGast,[a] Roland Jakob-Roetne,[a] Thomas Jack*, Marc-DavidRuepp*,Oliver Mhlemann*, Andrew J. DorisRoth, [a] Markus Rudolph,[a] Daniel Schlatter[a] Thomsonǂ,Sarah C. R. Lummisǂ andMartinLochner*

[a]F.Hoffmann-La RocheLtd., 4070 Basel, Switzerland *University of Bern,DCB,Freiestrasse 3, CH-3012Bern [b]ETH Zurich,8093 Zurich,Switzerland ǂUniversity of Cambridge, TennisCourt Road,CB2 1QWCambridge, UK

Inhibitionofcatechol O-methyltransferase(COMT)inthe brainisexpected The5-HT3 receptor (5-HT3R) isanimportant ion channel responsiblefor to be an effectivetreatment of depression andinparticular of disturbedcog- thetransmission of nerve impulses inthecentral nervoussystem.1 It isdiffi- nitiveperformanceinschizophrenia.Tolcapone(1)with averylow culttocharacterizetransmembranedynamicreceptors with classical struc- brain/plasma ratio of ~0.01 hasbeenusedasatool compound to studythe tural biology approaches likecrystallization and x-ray.Theuse of central effectsofCOMTinhibitioninclinical trials andinanimalexperi- photoaffinity probes isanalternativeapproach to identify regions inthe ments[1].Research in collaborationwith theDiederich group at theETH proteinthat are importantfor thebinding of small molecules.Therefore we Zurich ledtothe discoveryofpotentnucleosidicbisubstrateinhibitors (2) synthesized asmall library of photoaffinity probes by conugating that bind to both, thecatechol substratepocketand S-adenosylmethionine photophores via various linkers to granisetron whichisaknownantagonist (SAM)cofactorpocketofCOMT[2].Inthispresentation, we shownon- of the5-HT3R. We were abletoobtainseveral compounds with diverse nucleosidicbisubstrateinhibitors (3)thatwere discovered using parallel linker lengthsand differentphotolabile moieties that show nanomolar bind- chemistrybased on virtualdocking andchemical intuition. ing affinities fortheorthostericbinding site.Furthermore weestablished a stable h5-HT3Rexpressing cell line andapurification protocol to yieldthe O receptor inahigh purity.Currently weare investigating thephotocrosslink- O N R1 2 NH O 2 ing of these ligands with the5-HT3R. NO 2 N N linker R2 N HO OH HO OH H N HO OH O N 1 2 3

HO OH Tolcapone nucleosidicbisubstrate inhibitor non-nucleosidicbisubstrate inhibitors

[1]J.A.Apud, D. R. Weinberger, CNS Drugs 2007, 21,535. [2]C.Lerner,A.Ruf,V.Gramlich, B. Masjost, G. Zürcher, R. Jakob- Roetne,E.Borroni,F.Diederich, Angew.Chem. 2001, 113,4164; Angew. [1]Thompson, A. J.;Lummis, S. C., Expert Opin.Ther. Targets 2007, 11, Chem.-Int. Edit. 2001, 40,4040. 527. 520 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

MedicinalChemistry Chemical Biology MC005 Medicinal ChemistryChemicalBiology MC006

DiscoveryofHighly Potent S1P1 Receptor Antagonists With In Vivo Structural Insights into E-selectin-Ligand Interactions Efficacy Roland C. Preston1,Florian P.C. Binder1,Mirco Zierke1,Roman Jakob2, Jean Quancard, Birgit Bollbuck, Daniela Angst, Philipp Janser, Frédéric Timm Maier2,Beat Ernst1 Berst, Peter Buehlmayer, Markus Streiff, Christian Beerli, Volker Brink- mann, Danilo Guerini, Paul A. Smith, TimSeabrook, Martin Traebert, 1Institute of Molecular Pharmacy, Pharmazentrum, University of Basel, KlausSeuwen, René Hersperger,ChristianBruns,Frédéric Bassilana,Marc Klingelbergstr. 50/70, CH-4056 Basel Bigaud 2Biozentrum, University of Basel, Klingelbergstr. 50/70, CH-4056 Basel

NovartisInstitutesfor BioMedicalResearch, Basel, Switzerland The lectin E-selectin is involved in avariety of inflammatory diseases by acting as leukocyte-adhesive protein on the cardiovascular endothelium. E- We presentthe identification of anovel S1P1 receptor antagonist scaffold by selectin has extensively been under investigation as drug target. HTS, followed by asuccessful optimization to afford single digit nanomolar We investigated the binding of sialyl Lewisx (1,sLex)and the affinity- tool compounds for subcutaneous administration. First in vivo studiesinrats improved glycomimetic 2 by X-ray crystallography. Co-crystallization of demonstratedimmunomodulatory properties, kicking-off abroad strategy to ligand and protein revealed significant conformational alterations of the addressthe scaffold inherent bioavailability issues. These efforts ultimately binding site upon binding, when compared to published apo- and sLex (1) led to the discovery of NIBR-0213. Oral administration of this highly potent soaked structures [1, 2]. In the induced conformation, additional hydrogen andselective S1P1 receptor antagonist induceslong lasting reductionofpe- bonds between the protein and the ligand are observed. We further investi- ripheral blood lymphocyte counts leading to efficacy in relevant animal gated the binding of antagonist 3 with afivefold improved affinity over 2. models of autoimmune diseases. The structural data reveals asigma-pi interaction of the benzoate moiety in the 2-position of the D-Gal moiety with the (S)-cyclohexyl lactic acid, leading to an improved preorganizationofthe carboxylic acid in its bioactive conformation. In addition, aminor shift of the (S)-cyclohexyl lactic acid moity was observed, allowing for closer contact of this moiety in antagonist 3 to the protein. These insights provide the basis for future rational in silico design of potent E-selectin antagonists. Flexibility 1,sLex 2 3 HO AcHN OH KD=878 µM KD=19µM KD =4µM Rigidity HO HO O OH H H OH OH O O O O O O O OSE O O O O O O O O O O O NHAc O HO O OH Me O Me OH HO O HO O OH OH OH OH OH Protein Surface OH OH OH HO HO HO [1] Graves, B.J. et al. Nature, 1994, 367,532-538. [2] Somers, W.S. et al. Cell, 2000,103,467-479.

Medicinal Chemistry Chemical Biology MC007 MedicinalChemistryChemical Biology MC008

Imidazolinesand Imidazoles as TAAR1Agonists Functionalized nanomaterials forcancer diagnosis

Guido Galley,AnnickGoergler,Marius C. Hoener, RogerD.Norcross and SolènePassemard1,DavideStaedler1,Giona Sonego1,Joachim Loup1, HenriStalder 2Lucienne Juillerat-Jeanneret, SandrineGerber-Lemaire1

F. Hoffmann-La RocheAG, pRED, CH-4070 Basel, Switzerland 1EPFL,SB, ISIC,LSPN,Batochime,CH-1015 Lausanne. 2UniversityInstitute of Pathology, CHUV-UNIL, CH-1011 Lausanne 2-Benzylimidazolineshavebeen identifiedassubmicromolar ligands for the Trace-Amine AssociatedReceptor TAAR1 by high-throughput screening. Medical imaging is amajor tool forthe preventionand detectionofcancer. Further optimization led to the discovery of various classesofhighlyactive Conjugationofsmall organic ligandstargetingspecificcancercells bi- imidazoles. An improvedselectivityagainst the adrenergic alpha 2receptor omarkers to inorganicnanoparticles(NPs) presenting versatile optical prop- wasobservedespeciallyfor 2-aminomethyl substituted imidazoles[1]. ertiescouldprovide increased levelofsensitivityand accuracyfor theearly diagnosisofmalignant diseases.Wehereinpresent thedesign, synthesisand

N N N biological evaluationofseveral targetingmolecules andtheir conjugationto NH polymer coated NPsfor thespecificlabelingofhuman cancercells. In par- N N H H ticular, fibroblastactivationprotein-α[1] wasselected as extracellularbi-

N 4 omarker forthe detectionofhuman-derived breast, lung andprostatecancer 1 2 R R R R3 cells.

The availabilityofselective ligands is regardedasanimportant step forward to studyand furtherunderstand the role TAAR1 in biologicalsystems [2].

[1]G.Galley, H. Stalder, A. Goergler,M.C.Hoener, R. D. Norcross, Bioorg. Med. Chem. Lett. 2012, 22,5244. [2]F.G.Revel,C.A.Meyer,A.Bradaia, K. Jeanneau,E.Calcagno, C. B. Andre, M. Haenggi, M. T. Miss, G. Galley,R.D.Norcross, R. W. In- vernizzi,J.G.Wettstein,J.L.Moreau,M.C.Hoener, Neuropsycho- pharmacology 2012, 37,2580.

[1]Lawandi,J.; Gerber-Lemaire, S.;Juillerat-Jeanneret,L.; Moitessier, N. J. Med.Chem. 2010, 53,3423. Juillerat-Jeanneret,L.; Gerber-Lemaire,S.Mi- ni Rev. Med.Chem. 2009, 9,215. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 521

Medicinal Chemistry Chemical Biology MC009 MedicinalChemistryChemical Biology MC010

Structure elucidation of twomajor metabolitesofCRTh2 antagonist Developmentand Evaluation of DNA-EncodedCompound Libraries as an Effective Drug-Discovery Method ACT-129968 (Setipiprant) RaphaelM.Franzini1,Willy Decurtins1,MorenoWichert1,Florent Samain2, Philippe Risch, Heinz Fretz, ThomasPfeifer, Julien Pothier Jörg Scheuermann1,and DarioNeri1

Actelion PharmaceuticalsLtd.,Gewerbestrasse 16,4123 Allschwil, 1InstituteofPharmaceuticalSciences, ETHZürich, Wolfgang-Pauli-Str.10, Switzerland 8093 Zürich; 2PhilochemAG, Libernstr. 3, 8112 Otelfingen

Two major metabolites M7 and M9 were detected afterincubating 14Cla- beled CRTh2 antagonist 1 with human hepatocytes. Analysis of the mass spectra (LC-MS/MS) indicated that the tetrahydropyridoindole core of 1 remainedintact whereasits naphthyl ringseemed to be convertedtotwo dihydroxy-dihydronaphthalene regioisomersasshown with 2.

DNA-encoded libraries(DELs)haveemerged as apromisingapproach for drug developmentbecause of auniqueprofile of speed andcost- efficiency.[1] DELs arecompoundcollections in whicheachstructure is unambiguouslydefined by aunique nucleicacidbarcode.Similar to phage- displaytechnology,panning theselibraries againstimmobilized target proteins provides enriched binders;high-throughput sequencingofthe corresponding DNA-barcodes enablesthe identification of thesestructures. In this presentation,wereportonour ongoingefforts in thedevelopmentof DELtechnology.Our design philosophy is to generate structurally compact DELs of high purityand chemical diversity. In particular,wedescribea librarycomprisingover100'000 encodedmolecules,based on pairsof650 Basedonliteratureprecedence, 1 wasassumed to be transformed in two structural fragments, which wasassembled by anovel solid/liquid-phase consecutive enzymatic stepstoM7 and M9,two distinct vicinal trans-X,Y- synthesisstrategy.Preliminary screeningexperiments againstvariedprotein dihydroxy-X,Y-dihydronaphthalene regioisomers. Consequently, thefour targetshaveestablished theeffectivenessofthislibrary forrapid andcost- most plausible regioisomers 2a-d were synthesized in racemic andenantio- effectiveidentificationofhit compounds. Selected examples of identified enrichedform. Structure andabsolute stereochemistryofthe two metabo- proteinbinders illustrate theutility of this method as adrugdiscovery tool. lites M7 and M9 could unequivocallybeassigned by comparinganalytical andspectraldata of the synthetic samples with thebiologicalextracts. [1]J.Scheuermann,and D. Neri ChemBioChem, 2010, 11,931.

MedicinalChemistry Chemical Biology MC011 Medicinal Chemistry Chemical Biology MC012

Identificationofnovel P3-P1’ macrocyclicinhibitors Applicationsofsilicon-rhodamine forthe imaging of living cells of HCV NS3/4A protease GražvydasLukinavičius,Keitaro Umezawa, LucReymond andKai Oliver Simić,Trixi Brandl, Pascal Rigollier, Ursula Bodendorf, Johnsson Nikolaus Schiering, Claus Ehrhardt,Ulrich Hassiepen, Kai Lin, Julie Flynn Ecole Polytechnique FédéraledeLausanne(EPFL),InstituteofChemical Expertise Platform Proteases/Novartis Institutes for Biomedical Research, Sciences and Engineering(ISIC), 1015 Lausanne, Switzerland 4002-Basel, Switzerland The idealfluorescentprobe for bioimagingisbright,absorbs at long wave- Hepatitis Cvirus(HCV) infection is amajorcause of chronic liver disease lengths andcan be flexiblyimplemented in living cells andinvivo. Howev- that subsequently can lead to cirrhosis, carcinomaand liver failure. Around er,the designofsynthetic fluorophores that combine allofthese properties 170 million people worldwide are chronically infectedwithHCV whichis hasproventobeextremelydifficult.Here, we introduceabiocompatible the leading cause of liver transplants.Approvaloftelaprevir and boceprevir near-infraredsilicon-rhodamine probethat can be specificallycoupled to in 2011 as direct acting HCV NS3/4Aprotease inhibitors complements the proteins using differentlabelingtechniques.Importantly, itshighpermeabil- previous standard combination therapy of injectable pegylatedinterferon-a ityand fluorogenic character permit imagingofproteins in living cells and (PEG IFN-a)and the antiviral drug ribavirin. tissues, while itsbrightness andphotostability make it ideallysuited for While both, telaprevir and boceprevir, actascovalent reversible inhibitors live-cell superresolution microscopy. The excellent spectroscopic properties bearing aketoamide warhead, our effortswere focused on the identification of the probe combinedwithits easeofuse in live-cellapplications make it a of non-covalent inhibitors. powerful newtool for bioimaging[1,2]. Based on x-ray crystallographic data analysis of acyclic ligands in complex with HCV NS3/4Athe discovery of novel macrocyclic acyl-sulfonamide based inhibitors will be highlighted and aconcise structure activity relation- ship along with optmization efforts to improve ADME properties will be described.

[1]G.Lukinavicius, K. Umezawa,N.Olivier,A.Honigmann, G. Yang,T. Plass, V. Mueller,L.Reymond, I.R. Correa,Jr., Z.G. Luo, C. Schultz, E.A. Lemke, P. Heppenstall,C.Eggeling, S. Manley, andK.Johnsson, NatChem. 2013,5,132-9. [2]K.Umezawa,G.Lukinavičius andK.Johnsson, International patent application. 2013, WO2011EP64750 522 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

Medicinal Chemistry Chemical Biology MC013 Medicinal Chemistry Chemical Biology MC014

Discovery of highly potent and selective covalent reversible cathepsinS Synthesis of inhibitorsofoncogenicfusion protein NPM-ALK for in inhibitors vitro and in vivo assays.

W. Haap,G.Hartmann, R. AlvarezSanchez,L.Anselm,D.W.Banner, R. Sébastien Tardya,William HBissona,LucaMolognib,Peter Goekjianc, Ecabert, U. Grether, H. Kuehne,B.Kuhn, T. Luebbers, J. U. Peters, J. M. Carlo Gambacorti-Passerinib,Leonardo Scapozzaa Plancher, A. Rufer,B.Spinnler aPharmaceutical BiochemistryGroup, School of PharmaceuticalSciences, Pharma Research andEarly Development, UniversityofGeneva, Quai E. Ansermet30, 1211 Geneva,Switzerland F. Hoffmann-La RocheAG, GrenzacherStr.124, bDepartment of Clinical Medicine,University of Milan-Bicocca, Italy 4070 Basel, Switzerland cLaboratoryofOrganic Chemistry, ICBMS, UniversityofLyon1,France

Cathepsin S(CatS)isacysteineprotease involvedinthe antigenpresenta- Anaplasticlarge cellLymphomas (ALCL) is acancer affectingmostly chil- tion processand in extra cellular matrix degradation. Dysregulated CatS dren andyoungadults. It is driven by the kinaseactivityofthe oncogenic activityisadriverfor chronic inflammation, destructive proteolysis,arterial fusion protein NPM-ALK resulting from t(2;5) (p23;q35) chromosomal calcificationand increasedneoangiogenesis.ThereforeCatSisaninterest- translocation. Usingstructure-based designwehaveshown that the skeleton ing target for severalindications such as autoimmune diseases, cancer or pyrido [2,3-b] indolecould be aleadscaffold for thepreparationofpotential cardio vasculardiseases. One approach to inhibit the proteolytic activityof tyrosine kinase inhibitorsofNPM-ALK.This core,commonlycalled - CatS is to block its active sitewith covalent reversibleinhibitorswhich react carboline,appearsinanumber of naturalproducts andmolecules of phar- with the cysteine presentinthe catalytic triad. This presentation will focus macological interest[1]. on the identificationand optimization of covalent reversible CatS inhibitors via X-raystructure elucidation andmolecular modeling to improve potency, selectivity, reversibility, andADMEproperties.

The objectivesofthisworkweretosynthesizealibraryoftyrosine kinase inhibitorsstarting from theazacarbazole scaffold by modifyingitwith many differentsubstitutions at position C-3 and C-6 or C-4 and C-6.Inorder to functionalize the aromaticring,the methodologyconsists in sequentially use chemo- andregioselectiveSuzuki-Miyaura,Sonogashira,and Buchwald palladium-catalyzed cross-coupling reactions [2]. The medicinal chemistry effort resulted in NPM-ALKinhibitors active in vitro and in vivo.

[1]Scapozza,L.et.al,Appl. Int. WO2010025872A2, 2010. [2]Goekjian,P., G. et.al, Eur. J. Org. Chem., 2010,6665-6677.

MedicinalChemistryChemical Biology MC015 MedicinalChemistry Chemical Biology MC016

TotalSynthesis of theAntimalarialAgent Cladosporin. Thermosome–acage proteinacting as aversatiledelivery platform

LaureC.Bouchez*,Marion Rusch, MaudePatoor,Madeleine Livendahl, Martin G. Nussbaumer,Alba Mascarin, Thomas Mindt &NicoBruns Christophe Bodenreider, Dominic Hoepfner. University of Basel, Klingelbergstr.80, 4056 Basel, Switzerland Novartis Institute forBiomedical Research,Fabrikstrasse 22, CH-4056, Basel, Switzerland Protein cages represent aversatile platformfor avariety of functions such as targeted delivery of drugs or contrast agents. These proteins protect the drug Cell-based screeninghas recentlybeenshowntobeanattractiveway to find from premature degradation and enhance uptakeindesired tissue 1.The ver- newleadsfor malariadrugdevelopment. Next generationantimalarials satilitystemsfrom the different modification sites of protein cages. Various needstobeactiveagainst drug-resistant parasitesand efficacious against cargoescan be packed in the interior of such protein cages. Theexterior can bothliver- andblood-stageinfections. We screened anatural productlibrary be modified with cell-targeting and/or –penetrating moieties. Furthermore, to identifyinhibitors of Plasmodium falciparum blood- andliver-stage pro- protein engineeringallows for the introductionofadditionallinking sites. liferation. [1] To this end, we use the thermosome Cladosporin,afungal secondary metabolite wasidentifiedasapotentan- (THS), achaperoninfrom T. aci- tiparasitic agentwith astrikingactivityagainst both blood andliver stages dophilum 2.THS features two cavi- (IC50 in thenanomolar range). ties that can accommodate guest Thus, we designed aroute fordenovo synthesisofCladosporin,with the molecules. Polyamidoamine flexibility to potentiallygeneratea(wide-ranging/diverse) collectionofana- (PAMAM) was covalently bound to logues.[2] and[3] thegenetically introduced cystein residuesinthe interior. This cationic polymer is able to bind and stabilise smallinterfering RNA (siRNA) by electrostaticinteractions 3.Oligonucleo- tides,such as siRNA, are normally rapidly degradedinserum. Ourexperi- ments showed the uptake of siRNA into our hybrid as well as the enhanced stability of siRNA to RNase. To achieve cell-targeting, THS was modified on itsouter surface with the cell penetrating peptide TAT. With TAT a75- fold increase of cell uptake was observed. Experiments are underway to elucidate thedelivery of siRNA to cells.Addi- tionally, we can show uptake of metal nanoparticle into THS, which can be used for MRI contrast purposes.

[1]Dominic Hoepfner et al., Cell Host &Microbe 2012, 11,654-663. [1]Y.Maetal., Adv. DrugDeliver.Rev. 2012, 64(9), 811-825. [2] Reported synthesis-Huaji Zheng et al., J. Org. Chem. 2012,5656-5663. [2]N.Bruns et al., Angew.Chem. 2009, 121,5776-5779. [3]Laure Bouchez et al.Article in preparation. [3] J.H. Zhou et al., Chem.Comm., 2006,2362. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 523

MedicinalChemistry Chemical Biology MC017 MedicinalChemistry Chemical Biology MC018

Inert Ruthenium Complexes as Anticancer Drug Candidates AntiprotozoalIsoflavan Quinonesfrom Abrusprecatorius Yoshie Hata1,Maria De Mieri1,Melanie Raith1,Samad Ebrahimi1,Stefanie Vanessa Pierroz,1,2Tanmaya Joshi,2,3Anna Leonidova,2 Cristina Mari,2 Leone Zimmermann1,2,TsholofeloMokoka3,Dashnie Naidoo3,Gerda Fouche3, 3 1 2 Spiccia, Stefano Ferrari and Gilles Gasser Vinesh Maharaj3,RetoBrun2,Marcel Kaiser2,MatthiasHamburger1

1Institute of Molecular Cancer Research, University of Zurich, 1University of Basel, Pharmaceutical Biology,Klingelbergstrasse 50,4056 Winterthurerstrasse190, CH-8057, Switzerland Basel, Switzerland. 2SwissTropical andPublic Health Institute,Socinstrasse 2Institute of Inorganic Chemistry, University of Zurich, Winterthurerstrasse 57,4002Basel,Switzerland. 3Councilfor Scientific andIndustrialResearch, 190, CH-8057, Switzerland P.O. Box395, Pretoria,0002, SouthAfrica 3School of Chemistry, Monash University, Victoria 3800, Melbourne, Australia ACH2Cl2/MeOH(1:1) extractofthe wholeplant of Abrusprecatorius (Fabaceae) showed strong in vitro activityagainst Trypanosomabrucei rho- Since its FDA approval in 1978, cisplatin has been one the most used anti- desiense. Theactivecompounds weretracked by HPLC-based activity pro- cancer drugs in the world. However, this particular platinum based complex filing. Tencompounds wereisolated, includingfivenew naturalproducts as well as its other derivatives display severe side-effects including ne- [1]. Theirstructuresand relative configurationwereestablished by NMR phrotoxicity.[1] To overcome these drawbacks, several metal-based com- (1H, 13C, COSY,HMBC, HSQC,NOESY). Theabsoluteconfiguration was plexes as drug candidates are being investigated; Ruthenium complexes determined by comparisonofelectroniccirculardichroism (ECD)spectra emerging as promising alternatives.[2,3] Recently, the in vitro behavior of with calculated ECDdata. AbruquinonesI(1)and B(8)showedstrong in two substitutionally inert Ru(II) complexes bearing aderivative of the lig- vitro activity(IC50sof0.30 ± 0.1 µMand 0.16 ± 0.1 µM, respectively).Se- and 2-(2'-pyridyl)pyrimidine (CppH) have been investigated in our groups. lectivityindices (SI) as calculated from cytotoxicity data in L-6 cellswere Complex 1,inparticular, shows cytotoxicity comparable to that of cisplatin 78.3 and61.3. Thesecompounds arepromising candidates for in vivo stud- on different cancer cell lines and, importantly, lower toxicity than cisplatin ies. on healthy cells. Using several biochemical assays, we could demonstrate that this Ru(II) complex exerts its cytotoxicity by triggering amitochondria- mediated apoptosis.[4]

[1] Dhar, S.; Lippard, S. J. In Bioinorganic Medicinal Chemistry;Alessio, E., Ed.; Wiley-VCH: Weinheim, 2011,351. [2] Keppler, B. K., Hartinger, C. et al., Bioinorganic Medicinal Chemistry, Alessio, E., Ed.; Wiley-VCH: Weinheim, 2011, 151. [3] Dyson, P. J. et al. Int. J. of Oncology 2006, 29, 261. [1]Y.Hata, M. Raith, S. Ebrahimi,S.Zimmermann, T. Mokoka,D. [4] Pierroz, V.; Joshi, T.; Leonidova, A.; Mari, C.; Schur, J.; Ott, I.; Spic- Naidoo,G.Fouche,V.Maharaj,M.Kaiser, R. Brun,M.Hamburger, Planta cia, L.; Ferrari, S.; Gasser, G. 2012, J. Am. Chem. Soc.134,20376. Med. 2013,DOI:10.1055/s-0032-1328298.

Medicinal Chemistry Chemical Biology MC019 MedicinalChemistry Chemical Biology MC020

Combinatorial chromophore assembly insideaDNAfour-wayjunction Specific Targeting of Cancer Cells using Cytotoxic Re(I) Complexes

Katarzyna Rajkowska, Markus Probst andRobertHäner Anna Leonidova,1 Vanessa Pierroz,2 Stefano Ferrari,2 Gilles Gasser1

DepartmentofChemistryand Biochemistry, UniversityofBern 1Institute of Inorganic Chemistry; 2Institute of Molecular Cancer Research, Freiestrasse 3, CH-3012 Bern University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich.

In 1964 first proposed by Robin Hollidayasamechanistic model to solve Minimizing the effect of anti-cancer drugs on healthy cells has become, the mysteryofhow geneticinformation is exchangedinyeast, the DNA nowadays, one of the major concerns in medicinal chemistry. Some strate- four-way junction or Hollidayjunction (HJ) wasproofedtobethe keyin- gies, such as in photodynamic therapy, involve the use of external triggers termediate in homologousrecombination andbecameanimportanttool in to select the area of drug activation, while other approaches rely on conju- the field of DNAorigami,computationand nanomachines. Herein we use gating the active compound to amoiety specifically targeting cancer cells. the assemblyoffour modifiednucleic acid strands intothe planarsquare In recent years, several Re compounds have been shown to be cytotoxic on conformation of thishigher orderDNA structure to demonstrate in aproof various cancer cell lines.[1] All of them, however, indiscriminately affect of principle mannerthe cumulativeeffectofpyrenemoietiesinteracting both cancer and healthy cells. In this study, we screened aseries of N, N- inside the junction.[1][2] bis(quinolinoyl) Re(I) tricarbonyl complexes for antiproliferative activity. The cytotoxicity of some of these compounds increases significantly upon light irradiation.

Due to the fluorescent properties of the Re(I) complexes employed, their localization in cells could be studied in cervical cancer (HeLa) cells. Impor- [1]R.Holliday, GeneticalResearch, 1964,5,282 -304. tantly, the metal complexes were attached to specific-targeting biomolecules [2]Deshmukh N. Gopaul et al., TheEMBOJournal, 1998,17, 4175 -4187 to further enhance the selectivity of bioconjugate towards cancer cells. [2]

[1] G. Gasser, I. Ott, N. Metzler-Nolte, J. Med. Chem., 2010, 54,3and references therein. [2] A.Leonidova et al., 2013, submitted. 524 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

MedicinalChemistry Chemical Biology MC021 Medicinal Chemistry Chemical Biology MC022

PhototoxicEvaluation of NovelDNA Intercalating Ru(II) Complexes Synthesis andIncorporation of Diazirine-Modified Uridine Triphosphate Cristina Mari,1 Vanessa Pierroz,1 MalayPatra,1 StefanoFerrari,1 Gilles Gasser1 Christine CSmith1,Christian JLeumann1

1Institutes of InorganicChemistry andofMolecularCancerResearch, 1DepartmentofChemistry andBiochemistry, UniversityofBern, University of Zurich,Winterthurerstr.190, 8057, Zurich,Switzerland. Freiestrasse 3, 3012 Bern Switzerland

Photodynamic Therapy(PDT) is amedical technique relyingonthe synergistic actionoflight andanon-toxicphotosensitizertoinducecelldeath Adiazirine-modifieduridine triphosphate analogue designed to detectRNA through theformation of reactiveoxygenspecies.[1] PDToffersthe oppor- –RNA binding protein (RBP)interactions wassynthesised andits use in T7 tunity to kill tumorcells with aspatial andtemporal control, without causing RNAtranscriptionassays is described. severe side-effects,asisobservedwith otherchemotherapeuticagentssuch as cisplatin.The currentsynthetic photosensitizers in themarket, though, are porphyrin basedand suffer from severaldrawbacks including tedious synthesis andpurification as well as prolonged lightsensitivity.Here, we report thephototoxic behavior of sixeasy-to-synthesizeRu(II)complexes (Fig.1), allofthemshowingstrong DNAbinding affinity andagood nuclear uptake. Moreover,the complexesexhibit luminescence enhancementwith DNA intercalationand ahighsinglet oxygen production in hydrophobic environ- ments. Thebiologicalbehaviorofthe complexes, evaluatedinthe dark and upon irradiationwith lightofdifferent wavelengths(essentialtomap their photoactivity),shows oneofthe compounds to rapidlyinduce tumorcell deathupon lightactivation.[2]

Figure 1.Synthesis of triphosphate Y. 3-(α-iodo-p-tolyl)-3-(trifluoromethyl)-3H-diazirinewas synthesized accord- ing to Bayley et al1. a) 4-pentyn-1-ol, NaH, THF, 65% ; b) DMTCl, Py, 77%; c) CuI, Pd(PPh3)2Cl2,Et3N, DMF,61%; d) Ac2O, DMAP, Py,71%; 2 Fig. 1. StructuresofRucomplexes andfluorescenceconfocal microscopy e)dichloroacetic acid, CH2Cl2,48%; f) Triphosphorylation . imageofHeLacells incubatedwithone of thecomplexes. [1]Shih, L. B.;Bayley, H. Anal.Biochem. 1985, 144,132-141. [1]Dolmans,D.et al., Nature Rev. Cancer 2003, 3,380-387. [2]Ludwig, J.;Eckstein, F. J. Org. Chem. 1989, 54,631-635 [2]Mari, C. et al., 2013, submitted.

MedicinalChemistry Chemical Biology MC023 Medicinal Chemistry and Chemical Biology MC024

Towards Novel Organometallic Antischistosomal Drug Candidates Synthesis of fluorescent probes for the study of the 5-HT3Areceptor : applicationsinimaging and fluorescence polarization 1 1 2 2 Jeannine Hess ,Malay Patra ,Katrin Ingram ,Jennifer Keiser , 1 1 1 2 1 Jonathan Simonin ,Thomas Jack ,Marc-David Ruepp ,Daniel Hothersall , Gilles Gasser Christopher Connolly2,Andrea Chicca3,Jürg Gertsch3,Andrew Thompson4,Sarah Lummis4 and Martin Lochner1 1University of Zurich, Institute of Inorganic Chemistry, Winterthurerstr.190, 8075 Zurich, Switzerland University of Bern, 1Department of Chemistry and Biochemistry, 3Institute of 2University of Basel, Swiss Tropical and Public Health Institute, 4002 Basel, Biochemistry and Molecular Medicine, CH-3012 Bern, Switzerland. Switzerland University of Dundee, 2Ninewells Hospital Dundee DD1 9SY, UK. University of Cambridge, 4Department of Biochemistry, Cambridge CB2 1QW, UK. Schistosomiasis is amajor human health problem caused by parasitic worms The 5-HT3 receptor is aligand-gated ion channel (LGIC) and amember of the Cys- of the genus Schistosoma.Annually, more than 280 000 deaths are reported, loop family of receptors. High affinity fluorescent probes can be used to study such a mostly in tropical regions of developing countries. In addition, more than membrane receptor which structure is difficult to solve by X-ray diffraction method. 207 million people are infected and nearly 800 millions are at risk of being In this work, asmall library of fluorescent probes was synthesized using granisetron[1] infected. The infective disease is currently controlled by an organic drug, as acore to attach diverse fluorophores. The fluorescent probes have been characterised by radioligand binding (K )and fluorescence spectroscopy (λ Abs, Praziquantel (PZQ), which, despite its success, suffers from several draw- i max backs (e.g. metabolic stability, inactivity against the juvenile stage of Schis- λmax Em, ε and Φf). They were tested in live cell imaging, resulting in two high affinity probes containing fluorescein, being specific for the 5-HT3Ahomomeric tosoma). With this in mind, our group has initiated aprogram to derivatize [2] PZQ with organometallic moieties using three different strategies (Scheme receptor and useful for fluorescent polarization. Research for more stable 1,2 fluorophores led to suitable probes for imaging studies. Our efforts to produce and 1). isolate the 5-HT Areceptor in micelles and in good purity were rewarded by O 3 O O O successful fluorescence polarization experiments. Based on these results, arobust OM N N Organometallic N N N N binding assay is currently being developed that could be used to determine binding N derivatization N N O affinities for compounds targeting the 5-HT R. O OM O 3 Praziquantel (PQZ) OM

Modification on thecyclohexylpart Modification on the aromatic part

OM =Organometallic Moiety Scheme 1. Strategies for the design of organometallic derivatives of PZQ.

Impressive in vitro activity, in the nanomolar range, against Schistosoma mansoni was reportedfor two of our organometallic derivatives of PZQ. Our recent in vivo results will be also discussed.1,2 [1] S.K.V.Vernekar; H.Y. Hallaq; G.Clarkson; A.J.Thompson; L.Silvestri; S.C.R.Lummis; M.Lochner, J. Med. Chem., 2010, 53,2324. (1) Patra, M. et al., Chem. Eur. J. 2013, 19,2232 and references therein. [2] J.Simonin; S.K.V.Vernekar; A.J.Thompson; J.D. Hothersall; C.N. Connolly; (2) Patra, M. et al. J. Med. Chem 2012, 55,8790 and references therein. S.C.R.Lummis; M.Lochner, Bioorg. Med. Chem. Lett., 2012,22, 1151. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 525

MedicinalChemistry Chemical Biology MC025 MedicinalChemistry Chemical Biology MC026

ReactionsofC-centered Amino AcidRadicals with Ascorbate Modified nucleoside triphosphates–agateway forthe generation of long chemically modified oligonucleotides AnastasiaS.Domazou1,JanuszM.Gebicki2 and WillemH.Koppenol1 Marcel Hollenstein 1SwissFederal Institute of Technology, CH-8093Zurich, Switzerland 2 Macquarie University, Sydney, NSW 2109, Australia University of Bern, Department of Chemistryand Biochemistry,Freiestras- se 3, CH-3012 Bern Aerobes are under constant exposure to partially reduced oxygen species (PROS).Proteinsare major potential biologicaltargets for PROS, because of their high reactivity. This causes formation of C- and O-centered radicals located on the amino acid residues of the proteins. Both radical types can propagatebiologicaldamage.Ascorbate, the principalendogenous low- molecular antioxidant, can repair tyrosine and tryptophan radicals in model compounds and in various proteins in vitro [1-3] and maythereby prevent reaction withO2.It also reduces the peroxyl radicals of glycine, alanine and prolinederivatives[4]. We studiedthe reactions of ascorbate with C-centered amino acid radicals in the model compounds -methylalanine (-metAla) and N-acetylamide de- rivativesofglycine (N-ac-Gly-amide), alanine (N-ac-Ala-amide) and proline (N-ac-Pro-amide). The radicals of -metAla and N-ac-Pro-amide were repaired by oxidizing ascorbate to the ascorbyl radical with rate constants close to 106 M1 s1;the Nucleoside triphosphates(dNTPs) have advanced as aversatile andconven- ient vector for supplying nucleic acidswith additionalchemical functionali- repair of the N-ac-Ala-amide radical was slower.The reactionwas followed 1,2 mainly at 360 nm,where ascorbylradicalhas an absorption coefficient of ties. Modified dNTPsbearingside-chains that can mediateorganocataly- sis3 or mimicthe residuesofthe activesiteofproteases4 areshowntobe 3300 M1 cm1.Incontrast, no repair of the radicals of N-ac-Gly-amide was excellent substrates in rolling circle amplificationand TdT-mediated observed, for which we estimated an upper limit of 104 M1 s1 for the cor- polymerization, for thegenerationoflong andheavilymodified single- responding rateconstant.Our results suggest that in living tissues, ascorbate stranded oligonucleotides. cannot inhibit the reaction of protein C-centered radicals with O2. [1]Hocek,M., Fojta, M., Org. Biomol. Chem. 2008, 6,2233-2241. [1] B.M. Hoey and J. Butler Biochim. Biophys.Acta 1984, 791,212-218. [2]Lauridsen,L.H., Rothnagel, J. A.,Veedu, R. N., ChemBioChem 2012, [2] R. Santus et al. Free Radic. Res. 2000, 33,383-391. 13,19-25. [3] A.S. Domazou et al Free Radic. Biol. Med. 2009,46, 1049-1057. [3]Hollenstein,M., Chem.Eur.J.2012, 18,13320-13330. [4] A.S. Domazou et al Free Radic. Biol. Med. 2012,53, 1565-1573. [4]Hollenstein,M., Org.Biomol. Chem. 2013,submitted.

Medicinal Chemistry Chemical Biology MC027 Medicinal Chemistry Chemical Biology MC028

Bloch SurfaceWave Based Platformfor Real-timeAmyloidAggrega- EffectsofA-minor interactionsinthe folding and catalytic activity of tion KineticsInvestigation DNAenzymes

S. Santi1,2,H.P.Herzig1 andR.Neier2 MichaelRäz,Marcel Hollenstein

1EPFL-IMT, RueA.-L. Breguet2,2000 Neuchâtel, Switzerland; 2UNINE, University of Berne, Department of Chemisty and Biochemistry, Frei- Av.DeBellevaux 51, Neuchâtel, Switzerland estrasse 3, 3012 Berne, Switzerland

The misfolding andaggregation of specificproteins hasbeenassociated with incurable diseasessuch as Alzheimer's or Parkinson'sdisease[1].In the specific case of Alzheimer's disease,recentstudies have shown that it is the soluble oligomeric formsofaggregatesappearing in the earlystagesof aggregationthat cause cell toxicity, rather than insoluble fibrils.Research on newstrategies of diagnosis is imperative to detect the diseaseprior to the onset of clinicalsymptoms[2]. Fig. 1A)DNAzymes 8-17 and 10-23 3.B)3-deazaadenine Here,wepropose the use of an optical method for protein aggregation kinetic studies using aBloch surface wave (BSW)sensor made of aperiodic DNAzymes aresingle stranded DNA molecules, which display catalytic stackofsilicon oxide andsilicon nitride layers [3]. The aimistodetect the functions similar to that of protein enzymes1.The DNAzyme 8-17 and 10- early dynamicevents of protein aggregation andfibrillogenesis of the amy- 23 (Fig.1 A) are two important members of artificially selected DNAzymes. loid-beta peptide Aβ42, whichplays acentralroleinthe onset of the Alz- To get abetter insight into the mechanism and their particular folding, the heimer’s disease.The detection principle relies on the refractive index adenine analogue shown in Fig. 1Bwas used and incorporated in the cata- changescausedbythe interactionofsoluble oligomeric aggregates with the lytic core of both DNAzymes.All adenine bases were exchanged as single sensor silicon nitride surface. modifications with N-3-deazaadenine and the obtained modified DNA- We demonstrate the efficacyofthe BSWsensor by monitoring in real-time zymes were used to assess the effect of A-minor interactions on the cata- the formation of the first toxicoligomeric forms of Aβ42.Furthermore, we lytic efficiency.2, 3 provide newinsights intothe complex mechanismofaggregationofthis protein system in the presence of small molecularprobesabletointerfere [1] Breaker, R. R.; Joyce, G. G., ADNA enzymethat cleaves RNA. Chem. with thekinetics of amyloid formation. Biol. 1994,1,223-229. As acontrol method, Transmission Electron Microscopy(TEM) hasbeen [2] Salandrian, K.; et al., Stability of DNA Containing aStructural Water used to morphologically characterize the sampleduring aggregation. Mimic in an A-TRich Sequence. J. Am. Chem. Soc. 2011,133, (6), 1766-1768. [1]P.T. Lansburyand H.A. Lashuel, Nature 2006, 443,774-779. [3] Bin Wang, et al., Probing the Function of Nucleotides in the Catalytic [2]H.Hampel et al., NatRev Drug Discov 2010, 9,560–574. Cores of the 8-17 and 10-23 DNAzymes by Abasic Nucleotide and C3 [3]V.Paeder et al., Sens ActB2011, 157,260-264. Spacer Substitutions. Biochemistry 2010, 49, 7553-7562 526 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

Medicinal Chemistry Chemical Biology MC029 MedicinalChemistryChemical Biology MC030

Anew inhibitor of extracellular prolyl-endopeptidases Toward NewInhibitors of E. coli PaPG-IIadhesins: fornano-theranostic applications Crystallography,ITC andModeling-AidedDrugDesign

aDavide Staedler, aSolène Passemard, aGuillaume Stéphane Schneiter, Giulio Navarra,Katja Stangier,RolandPreston, Pascal Zihlmann, Martin aGiona Sonego, aJoachim Loup, bLucienne Juillerat-Jeanneretand aSandrine Smieskoand Beat Ernst* Gerber-Lemaire Institute of MolecularPharmacy,University of Basel, Klingelbergstr.50, aEPFL, SB,ISIC, LSPN,Batochime, CH-1015 Lausanne. 4056, Basel, Switzerland bUniversityInstituteofPathology, CHUV-UNIL, CH-1011 Lausanne ThePapG-II adhesins of E. coli aresurface proteins involvedinadhesion to In thepastdecade, various nanotechnologies andtheir applications in biolo- hostcells in theurinary tract.Their expression is highlycorrelatedwith the gy have gained prominenceasnew options in cancer diagnosisand therapy risk of developmentofpyelonephritis in humans[1],apotentially life- (so calledtheranostic) [1]. Particularly, inorganic nanoparticles(NPs) com- threateningdisease. Sincebacterialresistancetoconventionalantibioticsis posed by optically versatile materials, showedinteresting characteristics for rapidlyincreasing, thereisaurgent need of newtherapeutic options.Inan cancertheranostic as probes for non-invasive imaging[2].Wepresenthere- effort to find newantagonistsasantiadhesive lead structures,wecritically in the functionalization of iron oxide (Fe3O4)NPs with acompetitive and examined allpreviously publishedstructureswith reportedaffinity for E. irreversible prolyl-endopeptidases(PEP)inhibitor for the specific labeling coli PapG-II adhesins[2].However,the best antagonist,4–methoxyphenyl α– of human-derived cancercelllines. D–Gal(1-4)β–D–Gal (1), exhibits onlymicromolaraffinity[1].Weco- crystallized 1 with purifiedPapG-II andsolvedthe structureofthe protein- ligandcomplex by X-ray. Basedonthisinformation, newantagonistswere rationally designed, synthesized andbiologically evaluatedinapolymer- basedaffinity assayaswellasinITC.Preliminaryresults arepresented. OR HO O HO OH OH O O The presented approach can be applied to othersmetal-basedNPs, such as HO O OH Bismuth ferrite(BiFeO3,BFO)SHG NPs, which areoptically versatile. 1 OMe [1]Zuo,L.; Wei, W.;Morris, M.;Wei, J.;Gorbounov, M.;Wei, C. Med Clin NorthAm [1]J.Ohlsson, A. Larsson, S. Haataja, J. Alajääski,P.Stenlund, J.S. Pink- 2007, 91,845. [2]Staedler,D.; Magouroux, T.; Hadji,R.; Joulaud, C.;Extermann, J.;Schwung,S.; ner, S.J. Hultgren, J. Finne,J.Kihlbergand U.J. Nilsson Org. Biol. Passemard, S.; Kasparian,C.; Clarke,G.; Gerrmann, M.;Dantec, R. L.;Mugnier,Y.; Rytz, Chem., 2005, 3,886-900. D.;Ciepielewski,D.; Galez, C.;Gerber-Lemaire, S.; Juillerat-Jeanneret, L.;Bonacina, L.; [2]A.Larsson, J. Ohlsson, K. W. Dodson, S.J. Hultgren, U.J. Nilsson, J. Wolf,J.P.ACSNano 2012, 6, 2542. Kihlberg Bioorg. Med. Chem. 2003, 11,2255-2261.

MedicinalChemistry Chemical Biology MC031 MedicinalChemistry Chemical Biology MC032

$ Stilbenoids from Pholidota chinensis as newscaffold forGABAA recep- Design andSynthesis of novel Matrixmetalloproteinase-Inhibitors tormodulators Peter Elmiger, Rainer Riedl* Diana C. Rueda,AngelaSchöffmann, Maria de Mieri, Melanie Raith, Stef- fenHering, MatthiasHamburger* $2013 Dr.Max Lüthi Award of the Swiss Chemical Society

Division of Pharmaceutical Biology, University of Basel, Klingelbergstrasse Zurich University of Applied Sciences, Institute of Chemistry and Biologi- 50, 4056,Basel,Switzerland cal Chemistry, Campus Reidbach, CH-8820 Wädenswil, Switzerland

2+ In asearch fornew naturalproduct-derivedGABAA receptor modulators, Matrixmetalloproteinase-13 (MMP-13) is aZn -dependent protease that we screened aplant extract libraryonXenopus laevis oocytes expressing catalyzes thecleavage of type II collagen, the main structural protein in ar- recombinant α1β2γ2S GABAA receptors, by means of atwo-microelectrode ticular cartilage. Excess MMP-13 activity causes cartilage degradationin voltageclamp assay[1].Adichloromethane extract of stemsand rootsof osteoarthritis; hence, MMP-13 is apromising therapeutic target protein. Pholidotachinensis (Orchidaceae) enhanced the GABA-induced chloride However, due to their lack of selectivity clinicalMMP inhibitors have been [1] current (IGABA)by132.75% ±36.69% at 100 µg/mL. associated with serious side effectslike the musculoskeletal syndrome.

By meansofanHPLC–based activity profiling approach [2], the threestruc- turallyrelated stilbenoids coelonin (1), batatasinIII (2), andpholidotol D(3) were identified.Dihydrostilbenoid 2 enhanced IGABA by 1512.19% ± 176.47% at 300 µM,withanEC50 of 52.51±16.96µM, while compounds 1 and 3 showed muchloweractivity, suggesting conformationalflexibilityas in 2 to be crucial forreceptor modulation.Thiswas confirmed by astudy on aseriesof11commerciallyavailable stilbenoids andtheir corresponding semisyntheticdihydro derivatives.Dihydrostilbenoids showedhigheractivi- ty in the oocyte assay than theircorresponding stilbenes. Thedihydro derivatives of tetramethoxy piceatannol andpterostilbene were themostactive, with modulationscomparable to thatofcompound 2.Dihydrostilbenoids representanewscaffoldfor GABAA receptormodulators. The aim of the presented study was the development of selective MMP-13 [1]I.Baburin,S.Beyl, S. Hering, PflugersArch. –Eur.J.Physiol. 2006, inhibitors. The potentialinhibitors were designed with cheminformatics 453,117. methods, subsequently synthesized and their biological activity determined. [2]H.J.Kim.I.Baburin,S,Khom, S. Hering, M. Hamburger, Planta Med. 2008, 74,521. [1] J. A. Sparano et al. J. Clin. Oncol. 2004, 22,4683-4690. [2] A. R. Johnson et al. J. Biol. Chem. 2007, 282,27781-27791. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 527

MedicinalChemistryChemical Biology MC033 Analytical Chemistry MC034

Synthesis of labelledpeptidestostudy themechanism of abacterial Monitoring intra- andinter-dayhuman breath metabolic signatures: oligosaccharyltransferase evidence forthe existenceofindividualphenotypes

1 1 1 2 Gaëlle Michauda,ChristianLizakb,SabinaGerberb,KasparLocherb,Tamis PabloM-L Sinues ,Lukas Meier ,ChristianBerchtold ,Noriane Sievi , 2 2 1 Darbrea andJean-LouisReymonda Giovanni Camen ,Malcolm Kohler ,RenatoZenobi

1 aDepartment of Chemistryand Biochemistry,UniversityofBerne, ETH Zurich,Department of Chemistryand AppliedBiosciences,Switzer- b land Freiestrasse 3, CH-3012 Berne; Institute of MolecularBiology and 2 Biophysics, ETH Zurich,Schafmattstrasse 20,CH-8093 Zurich University HospitalZurich, Switzerland AgroundbreakingNMR studyhas suggestedthe existenceofstableindi- N-linkedprotein glycosylationisanessentialpost translational proteinmod- vidualmetabolic phenotypes in urine[1].The analysis of breathisanattrac- ificationineukaryotes that is involvedinmanycellularprocesses. The en- tivealternativeapproach to confirmthe existenceofsuchindividualmeta- zyme oligosaccharyltransferase(OST) embeddedinthe ER membrane, ca- bolic signatures becausebreathanalysisiscompletelynon-invasive. talysesthe en-bloc transfer of alipid-linkedoligosaccharide(LLO) to an Asnside-chainlocated in therecognitionsequon Asn-X-Thr/Ser(X≠Pro). Four subjects breathed throughthe counterflow inletofacommercialmass With thefirst X-ray structureofafull-length bacterial OSTenzyme, the spectrometer. Thebreathsamples encountered an electrosprayofwater, PglB proteinfrom Campylobacterlari,essentialinsights into theprinciples wherebysomeexhaled compounds were ionizedand readilydetected.Ato- of glycosylationsequon recognitionand thecatalytic mechanism of OST talof341 mass spectracollected duringfourdayswereanalyzedstatistically were obtained. To studythe mechanism of PglB,linear peptides labelled (see figure). with 5-carboxyfluoresceinwere synthesizedbyFmocsolid-phase peptide synthesis(SPPS).Based on theacceptorsubstratesequence, linearpeptides with systematicmodificationofthe -2 (Asp),the 0(Asn) andthe +2 (Thr) positionwere designed.For furtherinsightofthe enzymatic mechanism, modified aminoacids were includedaswell. Most of them were obtainedby thedirect modificationofthe aminoacidside chains on solid support. In the othercases,pre-modified aminoacidbuildingblockswereintroduced duringthe SPPS. Usingfluorescence anisotropyand gelfluorescence, thesepeptides allowed to quantifyacceptorsubstratebinding affinitiesand glycosylationturnover rates by PglB.

[1]a)Lizak C.,Gerber S.,Numao S.,AebiM., Locher K.P., Nature 2011, 474,350. b) Gerber S.,Lizak C.,Michaud G.,BucherM., Darbre T., Ourresults supportthe existenceofindividualbreathmetabolic signatures. Aebi M., Reymond J.L., Locher K.P., JBC 2013, 288,8849. [1]M.Assfalg et al, PNAS 2008, 105(5),1420.

Medicinal Chemistry Chemical Biology MC035 Medicinal Chemistry Chemical Biology MC036

Protein-LigandAffinity usingLong-Lived States andLong-Lived Synthesesoftrans-X,Y-dihydroxy-X,Y-dihydronaphthalenederivatives Coherences forthe identification of twomajor metabolitesofCRTh2 antagonist ACT-129968 (Setipiprant) Roberto Buratto [a],AurélienBornet [a],Nicola Salvi [a],and Geoffrey Bodenhausen [a,b] Philippe Risch,Heinz Fretz, ThomasPfeifer, Julien Pothier

[a]Ecole Polytechnique Fédérale de Lausanne, Avenue Forel2,1015 Actelion PharmaceuticalsLtd.,Gewerbestrasse 16,4123 Allschwil, Lausanne,Switzerland. Switzerland [b]Ecole Normale Supérieure, 24 RueLhomond, 75231 Paris, France. Accordingtospectral resultsand mechanisticalhypotheses, metabolites M7 During the last decades, powerful NMR techniques have emergedthat can and M9 of Setipiprant 1 arepresumably among vicinal trans-X,Y- identifyleadcompounds for drug discovery andinvestigate the strengthof dihydroxy-X,Y-dihydronaphthalene (2a-d). protein-ligand interactions. In thiscontext, we areexploiting thepeculiar properties of the so-called Long-Lived States(LLS) [1]and Long-Lived Coherences (LLC)[2] in or- dertoincreasethe sensitivity of NMRfor drug screening[3]. The onlyrequirement is to have aweakligandwith an isolated AX spin system that can carryanLLS or an LLC. We have covalentlyattached ‘spy groups’likebromothiophene (thanks to S. Passemard, A. Laguerreand S. Gerber, LSPN,EPFL) to aweakpeptide ligand,inorder to renderLLS and LLCscreeningproceduresapplicable to arbitrary ligands.Wehavede- Theirsyntheses startfromtetralones 3 andinvolve an epoxyda- mostrated the possibilitytoperform drug screening by determining the best tion/hydratation sequencetobuildthe trans-diol moiety, abenzylicoxyda- inhibitorsfor trypsin among asmall libraryofcandidates. tion/Bamford-Stevens eliminationsequencetobuild the olefin moietyand a Currentlyweare exploring the possibilitytouse these techniques in combi- lithium mediated carboxylation. The use of Jacobsen’schemistryfor the nation with dynamicnuclearpolarization, in ordertoenhancethe signals intermediate epoxydationdeliversenantio-enriched compounds. anddecreasethe amountsofprotein andligands used.

[1]CarravettaM., LevittM.H. JAmChemSoc (2004) 126(20):6228-6229. [2]SarkarR., Ahuja P.,Vasos P.R.,BodenhausenG.PhysRev Lett. (2010) 104(5):053001. [3]Salvi N., Buratto R.,BornetA., UlzegaS., RenteroRebolloI., Angelini Finally, theconfrontation between the synthesized candidatesand the bio- A., HeinisC., Bodenhausen G. JAmChemSoc (2012) 134(27):11076- logical extractsallowedfor theidentification of metabolites M7 and M9 and 11079. the assignment of theirabsoluteconfigurations. 528 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

Medicinal Chemistry Chemical Biology MC037 MedicinalChemistryChemical Biology MC038

SpecificRNA LabelingStrategiesfor FluorescenceApplications Enhancementoforalbioavailability of FimH antagonistvia aprodrug approach Anita G. Schmitz,Gilles Gasser,Roland K. O. Sigel Wojciech Schönemann,SimonKleeb,Daniela Abgottspon, Anja Sigl,Beat Institute of InorganicChemistry, UniversityofZurich,Winterthurerstr. 190, Ernst* CH-8057 Zurich,Switzerland Institute of MolecularPharmacy,University of Basel, Klingelbergstrasse 50, The role of RNA in livingorganism is manifold.For example, riboswitches 4056 Basel, Switzerland and ribozymes canact as regulatory andcatalyticelements.They specifically bind to metabolites andthereby undergo structuralchanges.Inorder to Urinarytract infection(UTI) is themostcommonbacterialinfectionworld- studythe mechanism of functional RNAs,weuse fluorescence techniques wide.This, together with high risk of reoccurrenceafter initialacute infec- suchasFRET(Förster Resonance EnergyTransfer). We investigatethe tion, makesUTIssignificantly problematic in bothmedical andeconomical structural rearrangementsofthe btuB riboswitch of E. coli upon B12 binding terms. UTIispredominantly induced by uropathogenic Escherichiacoli (FigureA)and of thegroup II intron ai5γ of S. cerevisiae upon Mg2+ bind- (UPEC).FimH is amannose-specificadhesinlocated on thetip of type 1 ing(Figure B).[1,2] As thelabeling of such large RNAs is challenging,we pili, an organelle expressedbythe bacteria.Itmediatesbinding to theblad- developed novel strategies. We are currentlyusing non-natural nucleicacids dersurface andenables colonizationofthe host’surinary tract.FimH an- that haveanextremely strong and specificbinding affinity to RNA/DNA tagonistsare promisingalternativefor currently used antibiotic treatments. OH OH sequences. To studythe process of RNA folding upon binding of themetab- OH OH HO O HO O olite, we have also labeled theB12 itselfwith various fluorophores suitable HO Cl 1.Intestinalabsorption HO Cl O O forFRET.Thesestrategies present theopportunity to investigate thefolding 2.Hydroysis mechanism, and associatedligandbinding properties of functionalRNAs in 1(Prodrug) 2 unprecedented detail. COOR COOH Ourgroup haspublishedone of themostpotentFimH antagonist reported up-to-date(→2)[1].However, 2 suffers frompoorpharmacokinetic properties. Foranimprovement of thePKpropertiesaprodrug approach wasen- visaged[2].Here, we presentsynthesisand impact of thesemodifications on solubility,permeability andlipohilicity.

[1]Klein,T.; Abgottspon, D.;Wittwer,M.; Rabbani, S.;Herold, J.; Jiang, X.;Kleeb,S.; Lüthi, C.;Scharenberg,M.; Bezençon, M.;Gubler,E.; Pang, [1]M.Steiner, K. S. Karunatilaka, R. K. O. Sigel,D.Rueda, Proc. Natl. L.; Smiesko, M.;Cutting, B.;Schwardt, O.;Ernst,B.J. Med. Chem. 2010, Acad.Sci. USA 2008, 105,13853-13858. 53, 8627–8641. [2]S.Gallo, M. Oberhuber, R. K. O. Sigel,B.Kräutler, ChemBioChem [2]Beaumont,K.; Webster,R.; Gardner, I.;Dack,K.;, Curr drug Metab. 2008, 9,1408-1414. 2003,4,461-485.

Medicinal Chemistry Chemical Biology MC039 MedicinalChemistryChemical Biology MC040

Dendritic AntimicrobialPeptideagainst PathogenicBacteria BioisostericModifications to Extend theAntiadhesiveEffect of FimH Antagonists MichaelaStach,Tamis Darbre* andJean-Louis Reymond* Lijuan Pang,SimonKleeb,Daniela Abgottspon, Katrin Lemme,Beat Ernst* UniversityofBern, Freiestrasse 3, 3012 Bern, Switzerland

Institute of MolecularPharmacy,University of Basel Manyantimicrobialpeptides(AMPs)act by disruptingmicrobialmem- Klingelbergstrasse 50, CH-4056 Basel, Switzerland branes.[1] The discovery of antimicrobialpeptide dendrimers in whichposi- tive chargesare provided by the multiple aminotermini at the dendrimer Urinarytract infections (UTIs),primarily causedbyuropathogenic E. coli periphery wascarried outwith adiverse 6750-membered combinatorialli- (UPEC),affect millions of peopleand account forsignificantmorbidity and brary[2] of 3rd generation peptide dendrimers. These sequencetypes arepar- high medical costs. Thekey step in thepathogenesisofUTIsisthe bacterial ticularly resistant to proteolysis[3],and were prepared by split-and-mixsolid adhesion to urothelialcells,which is mediated by thevirulence factor FimH phase peptide synthesis (SPPS) on aphotolabileresin suitable for off-bead located on type 1pili. Blocking FimH andtherefore theadhesion with FimH assays.[4] antagonistsoffers anew therapeutic approach forthe preventionand treat- Third generation peptide dendrimers with hydrophobic aminoacids in their ment of UTIs.However,the antagonistsdevelopedsofar have hardly met branches andmultiple free aminotermini as the sourceofpositive charges therequirementsfor clinical applications due to poor pharmacokinetic (PK) possess significantantimicrobialactivities against Gram positive andGram properties. In vivo studies indicated that with biphenyl α-D-mannosides as negative bacteria. They show remarkablylow haemolytic activityand actas FimH antagonists, high doses were necessarytoachieve theminimalcon- membranetargeting agents on synthetic unilamellar vesiclesassembled centrations requiredfor antiadhesive effectsinthe bladder [1, 2].Toim- [5] from E. coli,phosphatidylglycerol andphosphatidylcholine lipids. prove oral bioavailability andrenal excretion, we chemically modified the Newapproachestodevelop even more potent antimicrobialpeptides den- aglycone by replacing thecarboxylatewith variousbioisosteres. Theaffinity drimersagainst the opportunisticpathogen P. aeruginosa areunderpro- andthe thermodynamic profile of theseFimH antagonistswereevaluated by gress. acell-freecompetitivebinding assayaswellasisothermaltitrationcalo- rimetry(ITC).Furthermore,the PK propertiesweredetermined by different [1]T.Darbre, J.-L.Reymond Org. Biomol. Chem. 2012, 10,1483-1492. in vitro and in vivo assays.Asaresult, this strategy ledtoFimH antagonists [2]N.Maillard, A. Clouet,T.Darbre, J.-L.Reymond Nature Protoc. 2009, with an excellent pharmacological profile regardingbothdurationand effec- 4,132-142. tivenessofthe treatment. [3]P.Sommer,V.S. Fluxa, T. Darbre,J.-L. Reymond, J.-L. ChemBioChem 2009, 10,1527. [1]Klein,T.et al.J.Med.Chem. 2010, 53,8627. [4]N.Maillard, T. Darbre, J.-L.Reymond J. Comb.Chem. 2009, 11,667. [2]Cusumano, C. K. et al.Sci.Transl. Med. 2011, 3,109ra115. [5]M.Stach, N. Maillard, R. U. Kadam, D. Kalbermatter,M.Meury, M. G. P. Page,D.Fotiadis, T. Darbre,J.-L. Reymond Med.Chem. Commun. 2012, 3,86. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 529

MedicinalChemistryChemical Biology MC041 MedicinalChemistryChemical Biology MC042

Arevised foldingpathway of groupIIintrons: First Cis-DichloroDithiolato-BridgedRh(III) Half-Sandwichcomplexes Assigningspecificstructurestothe individual FRET states GajendraGupta,a Benjamin S. Murrayb,Paul. J. Dyson,b Bruno Therrien*,a EricaFiorini1,Lucia Cardo1,2,Danny Kowerko1 andRolandK.O.Sigel1 aInstitute of Chemistry, University of Neuchatel, AvedeBellevaus 51, CH- 1UniversityofZurich, Institute of InorganicChemistry, Winterthurerstrasse 2000 Neuchatel, Switzerland; bInstitutdes Sciences et Ingénierie Chi- 190, Zurich,Switzerland; 2University of Birmingham, Instituteof miques, EPFL,CH-1015 Lausanne,Switzerland Biomedical Research,Birmingham,B15 2TT,UK Treatment of areneRh(III)and Ir(III)half-sandwichdichloridedimers with Group II introns belong to theclass of self-splicingribozymes,are mobile different thiols (HSR,R=CHPh,CHCH Ph,CHCH-p-tBu)leadstothe genetic elements, andare found within thegenes of bacteriaand lowereu- 2 2 2 2 6 4 formationofaseries of neutraland cationicthiolatobridged complexes. The karyotes.[1] We designed aderivativeofS. Cerevisiae ai5γ group II intron molecularstructuresofthe neutralcomplexes reveal that thetwo rhodium (~900 nts),labeled with theCy3-Cy5 fluorophorepairand biotin forsurface atomsare bridgedbytwo thiolato ligands without anymetal-metal bond and immobilization.Thisconstructenables adetailedcharacterisationofthe thepentamethylcyclopentadienyland chloride ligands are cis to each other, foldingpathway by smFRETusing TIRF microscopy, sinceitpreserves the an unexpected conformationfor such dinuclear complexesand seemstobe dynamicsand thecatalytic activityofthe parentribozyme.[2]Previous stud- thefirst exampleofacis-dichlorodithiolato Rh complex. Thecytotoxicity iesrevealed alinearthree-state foldingpathway fromthe unfoldedtothe of thecomplexes were determined in theA2780 andA2780cisRhuman native state devoidofkinetic traps.[2] Ourstudies revealed an additional ovarian cancer celllines andare found to be highly cytotoxic. In fact,the Ir foldingstep. Furthermore we made fourmutants each carrying adistinct complexesare found to be themostactive(IC <0.025 µM), even more mutation thatinterruptsaspecifictertiary interaction, knowntobeinvolved 50 than theruthenium andrhodium analogues. Interestingly, they appear to be in crucial inter-domaintertiary interactions. Thisenabled us to assign each themostactivepentamethylcyclopentadienyl iridiumcomplexes evaluated FRET statetoaspecificfolding intermediate. to date.Furthermore,computationalstudies to rationalizethe formationof the cis-isomerasthe most stable configurationare underway.

[1]A.M. Pyle, Crit.Rev. Biochem. Mol. Biol., 2010,45,215. [2]M.Steiner, K.S. Karunatilaka,R.K.O.Sigel,D.Rueda, Proc.Nat.Acad. Figure 1. Molecularstructure of cis-dichlororhodium dithiolato-bridged Sci., 2008, 105,13853. complexes

Medicinal Chemistry MC043 MedicinalChemistry Chemical Biology MC044

Phosphorylation-dependent Molecular Switches, the PleD Protein Case Bioactive compoundsfrom the Spondias tuberosa fruits

1 1 Juvenal Yosa ,Markus Meuwly M. L. Zeraik1,E.F.Queiroz2,I.Castro-Gamboa1,M.Cuendet2,J.-L. 1University of Basel, Klingelbergstrasse 80, CH-4056 Basel Wolfender2,M.Q.Paulo3,V.S.Bolzani1

PleD protein contains an intrinsic nucleotide cyclase activity,[1] which con- 1NuBBE, UNESP, 14800-900, Araraquara, São Paulo, Brazil. 2School of verts twomolecules of GTP into bis-(3’→5’)-cyclic diGMP (c-di-GMP). c- Pharmaceutical Sciences,UNIGE,1211, Geneva 4, Switzerland. di-GMP is amolecule of great interest which regulates surface-adhesion prop- 3LaboratoryofNatural Products,Dept.ofChemistry, UFPB, Paraiba, Bra- erties and motility in bacteria. In addition, c-di-GMP is involved in bacterial zil. bioﬁlm formation and persistence. Because c-di-GMP is found exclusively in bacteria makes it apotential target for medicinal applications. PleD is a The Northeast of Brazil is asource of greatvarietyofedible fruits used as homodimer protein, where each monomer contains tworeceivers domains; food by the local population. In most cases, these fruits have only been Rec1 and Rec2, Rec1 serves as aphosphoryl acceptor,whereas, Rec2 was poorly studiedfor their chemical constituents andtheir biological activities proposed to act as an adaptor for dimerization. Also PleD contain GGDEF [1]. The purpose of this study was to evaluate the chemical compositionof the methanolic pulp extract of Umbú (Spondias tuberosa Arr. Camara, Ana- domain or catalytic domain which is aconserved amino acid sequence mo- -1 tif with dyguanilate cyclase activity (DGC). [2] The signaling c-di-GMP is cardiaceae), as well as measure its anti-agingactivities.At40μgmL ,the extract presented highantioxidant activity in the DPPH (89%), ABTS (97%) synthesized from twoGTP molecules in asymmetric condensation reaction. and ORAC (64%) assays, as wellas61% inhibition of acetylcholinesterase Each monomer accommodate one GTP in the catalytic domain. It has been activity. The analytical HPLC-UV condition was geometrically transferredto demonstrated by cross-linking experiments that exclusively dimeric PleD is the preparative medium pressure chromatography(MPLC-UV) by chromato- catalytically active.[3] graphic calculations. In asecond step, MPLC-UV was used to isolate the Because dimerization is fundamental for the enzymatic activity,and this pro- active compounds. All of the MPLC fractions were monitored by cess is coupled with the phosphoryl transfer to REC-1, here we investigate, UHPLC/TOF/MSgiven a2DLC/LC plotofthe MPLC separation. In asin- using molecular dynamics simulations MD and bias molecular dynamics sim- gle stepsix compounds wereisolated for the first timeinthis plant and a ulation BMD, the pathway whereby the twomonomers are repacking, and newcompound derived from gallic acid wasfound. The structures of the speciﬁcally the role of the phosphorylation to form afunctional protein. Molec- isolated compounds were elucidated by classicalspectroscopic methods ular modeling on PleD protein can serveasamodel to understand the dynam- including 2D NMR and HR-MS. ical process and the molecular mechanism in bacterial cell cycle. Acknowledgement: BSJRPBRAZILIAN/SWISS Joint Research Pro- [1] Jenal, U. and Malone, J., Annu. Rev. Genet. 2006, 40,385. gramme grant to J.L.W., E.F.Q,I.C.G., M.C. and V.S.B. [2] Wassmann W.,etal. Structure. 2007, 15,915. [3] Paul, R., et al. J. Biol. Chem. 2007, 282,29170. [1] da Silva AR,deMoraisSM, Marques MM, de Oliveira DF, Barros CC, de Almeida RR, Vieira ÍG, Guedes MI. Pharm. Biol. 2012,50,740. 530 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

MedicinalChemistry Chemical Biology MC045 Medicinal Chemistry Chemical Biology MC046

Phytochemical investigation of the bark of Tetrapteris mucronata a Isolation of new antifungal compounds from Swartzia simplex plantuse in the ayahuasca preparation Q. Favre-Godal1,E.F. Queiroz1,L.Marcourt1,M.P. Gupta2,J.-L.Wolfender1 M. M. F. Queiroz1,E.F.Queiroz2,G.Marti2,L.Marcourt2, I. Castro-Ganboa1,V.Bolzani1,J.L.Wolfender2 1School of Pharmaceutical Sciences, EPGL, University of Geneva,Univer- sity of Lausanne, 30 quai Ernest-Ansermet,CH-1211 Geneva 4, Switzer- 1NuBBE, Department of Organic Chemistry, Institute of Chemistry, São land. 2Centerfor Pharmacognostic Research on PanamanianFlora - PauloStateUniversity (UNESP), P. O. Box 355,14800-900,Araraquara, CIFLORPAN, University of Panama, Apartado 10767, Estafeta Universitar- São Paulo, Brazil. 2School of Pharmaceutical Sciences, University of Gene- ia, Panama city, Panama. va, 1211, Geneva 4, Switzerland.

Tetrapteris mucronata Cav. (Malpighiaceae) is aplantused in someregions The dichloromethane extractofthe root bark of Swartzia simplex (Sw.) of Brazil in ayahuasca preparation apsychotropic plant decoction [1].To Spreng(Fabaceae) presented an interesting antifungal activity against Can- dida albicans in abioautography assay [1,2]. In ordertoisolate the active assessthe phytochemical compositionofT. mucronata andunderstand its compounds,bioguided isolation was undertaken using HPLC- traditional use, the aqueous and methanolic extracts wereinvestigated. Their microfractionationin96well plates andbioautography to localizethe active compounds in the HPLC-PADmetabolite profilingofthe crude extract. The HPLC-PDA-ESI-MS profiles have been determined.The isolation of the analytical HPLC-PAD conditions were geometrically transferred to aprepara- main compounds of the methanolic extractwas performed by adirect trans- tive medium pressure chromatography (MPLC-UV) by chromatographic calculations for the efficientisolation of the active compounds at the milligram ferofanalyticalHPLC conditionstomedium pressureliquid chromatog- scaleinone step. Usingthis approach ten compounds were isolated, six of raphy (MPLC). This resulted in the isolation of six alkaloids and one new them are new natural products. The structures of the isolated compounds were elucidated by classicalspectroscopicmethodsincluding UV, 2D NMR phenanthrene derivative. Since tryptamine and -carboline alcaloidsare and HR-MS. known to have powerful hallucinogenic activities, an HPLC-ESI-MS/MS Acknowledgements: SNFgrant CR2313-143733 to J.L.W.and E.F.Q. method has been developed for theirquantificationinT. mucronata water andmethanolic extracts.These results provide arational support forthe tra- [1] Q. Favre-Godal, E.F. Queiroz, D. Sanglard,J.-L.Wolfender. Planta Med 2012; 78-PD159. ditional useofT. mucronata stem bark in ayahuasca preparations,since - [2] Q. Favre-Godal, E.F. Queiroz, J.-L.Wolfender. JOAC 2013; in press. carboline alkaloids are presentinarelevantamount. [1] Ott, J., Ayahuasca analogues :Pangæan entheogens.1st ed.; Natural ProductsCo.: Kennewick, WA, 1994;p127.

MedicinalChemistry Chemical Biology MC047 Medicinal Chemistry Chemical Biology MC048

Vitis vinifera canes anew source of antifungal compounds against Plas- Synthesis, structure determination andbiological applicationsof mopara viticola, Erysiphe necator and Botrytis cinerea bicyclic bridgedpeptides

S. Schnee1,E.F. Queiroz1,L.Marcourt1,F.Voinesco1,L.Marcourt2,P.-H. M. Bartoloni,E.Blattes,T.Darbreand J.L. Reymond* Dubuis1,J.-L. Wolfender2,K.Gindro1 UniversityofBern, Departement of Chemistryand Biochemistry, 1Swiss FederalResearch Station Agroscope Changins Wädenswil ACW, Freiestrasse 3, 3012 Bern,Switzerland Route de Duiller 50,P.O.Box 1012,1260Nyon, Switzerland.2School of Pharmaceutical Sciences,EPGL, University of Geneva, University of Lau- Linear peptidesare poor drug candidates due to their low bioavailability and sanne, 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland. rapid proteolysis.These limitations can be overcome by rigidifyingtheir structure through head-to-tail or side chain-involving cyclizations. The use of multiple branchingamino acids in apeptide sequence, like diamino acids Worldwide the vast majority of grapevine areas are planted with Vitis vinif- (asused in peptide dendrimers1)oramino diacids, allows to synthesizepep- era cultivarsthat are all susceptible to various fungal diseases such as tidesresembling polycyclicalkanesusing an orthogonalprotectionscheme. downy(Plasmopara viticola)and powdery (Erysiphe necator)mildews and grey mould (Botrytiscierea). Since V. vinifera canes represent an unexploit- ed agronomical waste material, their biological activity against the major grapevinepathogens andtheir chemical contentwere investigated in order to determine if extracts or compounds could be potentiallyused in asustain- able manner to protect grapevines. Methanolic and ethanolic crudeextracts of Vitis vinifera canesexhibitedsignificant antifungalactivityagainst the three major fungal pathogens of grapevine: P. viticola, E. necator and B. cinerea.The activeextract was analysed by LC-PDA-ESI-MSand some compounds were dereplicated. Efficient targeted isolationusing medium pressure chromatography(MPLC-UV) afforded in onestep sixpure constit- These peptidesare structurally well-defined andcoveranalmost pristine uents. The structures of the isolated compounds wereelucidated by 2D 2 NMR and HR-MS. Six identified compounds presented interesting antifun- area of peptide topological space .Their conformationalrigidity wasinves- gal activities against P. viticola tigated by meansof2D-NMR andmay offeraplatform to designdrugs tackling protein-protein interactions. Moreover, by acombination of appropriate aminoacids,wedevelop cellpenetrating bicyclicpeptidesfor drug carrier design andcellularprotein targeting.

[1]T.Darbre, J.-L.Reymond, Org. Biomol. Chem.2012, 10, 1483. [2]M.Bartoloni,R.U.Kadam,J.Schwartz,J.Furrer, T. Darbre, J.-L. Reymond, Chem.Commun. 2011, 47, 12634. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 531

Medicinal ChemistryChemicalBiology MC049 MedicinalChemistry Chemical Biology MC050

“Bridging the Gap” -Adjusting the Acid Pharmacophore in sialyl Research on antagonists of Siglec-8 Lewisx MimicsbyRing Closing Metathesis

Mirko Zierke,# Martin Smiesko,# Norbert Varga,# Florian P. C. Binder, # Fan Yang, Beat Ernst* Mario Schubert,¶ Roland Preston,# and Beat Ernst#* Molecular Pharmacy, Pharmacenter, University of Basel, # University of Basel, Klingelbergstr. 50, CH-4056 Basel, Switzerland Klingelbergstrasse 50, CH-4056 Basel, Switzerland ¶ ETH Zürich, Schafmattstr. 20, CH-8093 Zürich, Switzerland Siglecs (sialic acid immunoglobulin-like lectins) are members of the immu- The selectins play akey role in the body’s defense mechanism against in- noglobulin gene family that contain sialoside binding N-terminal domains.[1] flammation.[1] They form aclass of three cell adhesion molecules (E-,P-, and L-selectin),which, in case of an inflammatory stimulus, are responsible They are cell surface proteins found predominantly on cells of the immune for the initial steps of the inflammatory response, that is, the tethering and system. Among them, Siglec-8isuniquely expressed by human eosinophils rolling of leukocytes on the endothelial surface of blood vessels. These steps and mast cells, as well as basophils. Targeting Siglec-8with glcomimetics are aprerequisite for the subsequent firm adhesion and the final extravasa- may thus provide ameans to specifically inhibit or deplete these cell types tion of leukocytes to the site of the inflammatory stimulus. However, exces- and be ideally suited for treatment of eosinophil and mast cell-related dis- sive infiltration of leukocytes into the adjacent tissue can lead to acute and eases, such as asthma or chronic rhinosinusitis.[2] chronic reactions, as observed in reperfusion injuries, stroke or rheumatoid [2] To study the interactions between Siglec-8 and its ligands, three sulfated arthritis. Therefore, the antagonism of selectins is regarded as avaluable x pharmaceutical option. sLe , 2A, 2B and 2C are synthesized starting from the common intermediate 1. For the development of efficient antagonists, the low binding affinity, high polarity and structural complexity of the physiological selectin ligand sialyl HO OH x x HO OH CO2H OH O Lewis (sLe )are challenges to be accomplished. In the past, we identified O O O NH x x [3] AcHN O O O 2 HO NHAc high-affinity sLe mimetics by stabilizing the Le -core structure. Based on HO O OH [4] 2A NaO3SO the structure of E-selectin co-crystallized with E-selectin antagonists, dif- OH HO ferent approaches with the aim to stabilize the orientation of the carboxylate HO AcO OLev OH CO H OSO3Na AcO OAc CO2Bn OBz HO 2 OH O O of the Neu5Ac moiety are explored. O O O O O O NHCbz AcHN O O NH2 AcHN O O O O AcO NHAc HO NHAc BzO HO O O OH TBDPSO OBn 2B HO 1 OBn OH [1] Physiology and Pathophysiology of Leukocyte Adhesion;D.N. Granger, BnO HO HO OSO Na HO OH CO2H OH 3 G.W. Schmid-Schönbein, Eds.; Oxford Univ. Press: New York, 1995. O O O O NH AcHN O O O 2 [2] B. Ernst, J.L. Magnani, Nat. Rev. Drug Discov. 2009, 8,661-677. HO NHAc HO O OH [3] D. Schwizer,etal. Chem. Eur. J. 2012, 18,1342-1351. NaO3SO 2C OH [4] Preston Roland, Abstract-SCS Fall Meeting Lausanne. 2013. HO [1] Crocker P.R. et al. Siglecs and their roles in the immune system. Nat. Rev. Immunol. 2007, 7,255-266. [2] Kiwamoto T. et al. Pharmacol. Therap. 2012, 135,327–336.

MedicinalChemistry Chemical Biology MC051 MedicinalChemistry Chemical Biology MC052

Synthesisand Evaluation of NovelCannabinoid Type 2Receptor Identification and analysis of the putative AMP-activated protein ki- Tracers forPET Imaging nase (AMPK) in blood stream form of Trypanosoma brucei

RogerSlavik1,DanielBieri1,Uwe Grether2,Stjepko Čermak1,StefanieD. Chiara Ambuehl, Patricia Graven, Leonardo Scapozza, Remo Perozzo Krämer1,Adrienne Müller1,LucaGobbi2,MarkusWeber1,Roger Schibli1, Simon M. Ametamey1,Linjing Mu1. University of Geneva, Quai Ernest-Ansermet 30, 1211 Geneva 4, Switzerland 1 InstituteofPharmaceutical Sciences,ETH Zürich,Wolfgang-Pauli-Strasse 10, 8093 Zürich, 2 F. Hoffmann-La RocheLtd., 4070 Basel AMP-activated protein kinase (AMPK) is aheterotrimeric complex well Thecannabinoidreceptortype2(CB2)has very lowexpression levelin conserved in all eukaryotic which acts as regulatory sensor of the energy braintissueunderbasal conditions,but it is up regulatedindiverse patho- status of the cells. Its role is based mostly on sensing the AMP/ATP ratio of logicalconditions.Todevelop abrain PETtracertowards CB2,weevaluat- the cells and to restore the adenine nucleotide homeostasis [1]. In the blood ed thepotential of apromising 4-oxo-quinolineleadstructure from literature stream form of Trypanosoma brucei,the parasite responsible for the Human [1]-designated KD2 -asanimaging agentfor CB2 sites. KD2has been AfricanTrypanosomiasis or sleeping sickness, the AMPK complex has nei- synthesizedand labeledwithan11C-isotope. Preliminary in vitro / in vivo ther been completely identified nor characterized to date. studies showed that [11C]KD2isapromisingPET tracerfor CB2.Withinan optimization programwithfocus on loweringlipophilicity andplasmapro- Previous investigations on the procyclic form of T. brucei allowed the iden- tein binding, severalnew KD2derivatives weresynthesizedand theirbind- tification of the β and γ subunits of TbAMPK [2]. It is the aim of this pro- ingaffinities towards hCB1/2 were determined. Kivaluesfor theCB2 re- ject to identify the unknown α subunit in the blood stream form of T. brucei ceptor ranged from 0.7–1220 nM,with aselectivity hCB2 over hCB1 from via genetic modification by homologous recombination to replace the β or γ 10 to >10’000. Furthermore,various novel CB2ligandsfromanovel struc- subunit by atagged variant, followed by subsequentisolation of the com- turalclass were synthesizedand tested in functional andbinding assays. The plex by affinity purification. After the identification of the α subunit the role most promising ligands will be radiolabeled with 11Cor18Fisotopesfor of the TbAMPK complex, but also of the identified subunitsalone, will be further in vitro and in vivo evaluation. investigated with respect to cell viability and cell metabolism using genetic an biochemical tools.

[1] David Carling, TIBS. 2004,29(1), 18. [2] Clemmens, C.S. et al., Exp Parasitol. 2009,123, 250. [1]Pasquini S. et al., J. Med. Chem., 2011, 54 (15),5444-53. 532 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

MedicinalChemistry Chemical Biology MC053 Medicinal Chemistry Chemical Biology MC054

Synthesis of Cell Penetrating Peptide-Peptide Nucleic Acid (CPP-PNA) Highly potent andselectiveMMP-13 inhibitors: Astraightforward Conjugatesand Evaluation of theirBiological Performance design approach SooHyeon Lee, Bastien Castagner, Jean-Christophe Leroux* Thomas Fischer, Rainer Riedl* ETHZ,Wolfgang-Pauli-Str. 10, CH-8093 Zürich,Switzerland Zurich University of AppliedSciences,InstituteofChemistry and Biologi- cal Chemistry, Campus Reidbach, CH-8820 Wädenswil, Switzerland PNAs areoligonucleotide analoguesinwhichthe sugar-phosphate backbone hasbeen replaced by peptide backbone formedfrom N-(2-aminoethyl)-glycineunitslinked by amidebonds (Fig.1A).PNA can Matrix Metalloprotease-13 (MMP-13) is one of the enzymesbelonging to bind to acomplementary mRNA anddisturbprotein expression via steric the zinc-depending endopeptidase family and is involved in angiogenesis as hindrance. We conjugatedaPNAto15differentCPPs to the increasecellular wellasintissue remodeling. An overactivityofMMPs can lead to various uptake andantisense activity(Fig. 1B)[1, 2].Their transfectionefficiencies pathological processessuch as rheumatoid arthritis or tumor growth and were evaluatedoncolon cancercells (HT-29) stably expressing luciferase by metastasis [1]. measuringthe expressionofthe latter.Weidentified3CPP conjugates that In our present study, we have discovered novel MMP-13 Inhibitors with displayed adose-dependentluciferase inhibition at concentrations that were nanomolar potency, as well as an attractive selectivity profile against aset not cytotoxic. These bio-reactive CPP-PNA conjugates with well-defined of antitargets within the sameenzyme family. structure will be furthermodifiedfor colon-specific delivery.This work was Starting from the co-crystal structure PDB 2OW9[2], we designedanovel financiallysupportedbyGebertRüf Stiftung (GRS-041/11). scaffold by maintaining the key attractiveinteractions to the protein backbone. Further, we targeted the loop which is buried deep in the S1’ pocket andisreferred to as theselectivity loop which furnished potent and selective MMP-13 inhibitors.

[1] M. G. Natchus et al. J. Med.Chem. 2001, 44,1060-1071. [2] A. R. Johnson et al. J. Biol. Chem. 2007, 282,27781-27791.

Figure 1. (A)StructureofPNA and(B) scheme of targetgenesilencing with PNAconjugatedtoCPPs. [1]F.A. Rogers, S.S. Lin, D.C. Hegan, D.S. Krause, P.M. Glazer, Mol. Ther. 2012, 20,109. [2]S.H. Lee, B. Castagner, J.C. Leroux, Eur. J. Pharm. Biopharm. 2013, DOI: 10.1016/j.ejpb.2013.03.021.

MedicinalChemistryChemical Biology MC055 Medicinal Chemistry Chemical Biology MC056

Synthesisofnovelfluorescent agonists,withselectivity Towardsthe site-specificmodification of thehERGK+channel forthe A1 adenosinereceptor SuradechSinghanat1,Marc-David Ruepp1,ThomasJack1,Jean-Sébastien J. L. Hemmings,1 G. Ladds,2 A. Knight,2 B. G. Frenguelli,2 M. Lochner1 Rougier2,HuguesAbriel2,Oliver Mühlemann1,Martin Lochner1

1Department of Chemistryand Biochemistry,Universität Bern,Switzerland. 1DepartmentofChemistry andBiochemistry 2School of Life Sciences,UniversityofWarwick,Coventry, CV47AL,UK. 2DepartmentofClinical Research UniversityofBern, Freiestrasse 3, 3012 Bern, Switzerland Theadenosine receptors aremembers of theGPCRfamilyand thereare four sub-types: A1,A2A,A2B andA3.The A1 adenosinereceptorisinvolvedina The human Ether-a-go-go Related Gene(hERG)encodesfor apotassium rangeofprocessesinthe CNS, includingepilepsyand ischaemia. Despiteits channelthatisexpressed in theheart muscle andiscriticalfor repolarization importantrole, relatively little is knownabout thetrafficking of this receptor of cardiac tissue during the heartbeat cycle[1].Ithas been shown that a in neurons.1 An agonistconjugatedtoafluorophoremay provideinsight. number of markettherapeuticdrugs have off-targetaffinity for the hERG channeland cause QT prolongation(LQT) which canleadtopotentially In collaboration with ProfessorBruno Frenguelli at theUniversityofWar- lethalcardiac arrhythmia.Inconsequence, every drug candidate hastobe wick we have startedtodesignand synthesise selectivefluorescentcom- testedinorder to avoid LQTside effects. The existing assays,including pounds.These arederived from knownA1adenosinereceptoragonistsand electrophysical patch clampassayand radioligand-basedcompetition bind- thestrategyistoattach alinkerand fluorophore via acycliccomponent at ing assays,havesome disadvantagesand arenot suitable for high- the6position of theadenine.Inthe initialstagesofthisproject we have throughput screening[1].The ultimategoalofthisprojectistocovalently prepared anumberofnovel intermediates, with interestingactivityatthe A1 attach asmall fluorophoretothe hERG channelusing photoaffinityprobes 1 adenosinereceptor(Figure1). Theinitial biological resultsand preparation basedondofetilide,the ligand whichisknown to bind to the channelwith of fluorescenttargets will be presented. highaffinity[2].Modifiable linkers will allow further post-photoaffinity labeling modificationofthe hERG channelinordertointroduceasmall fluorophorenearthe channelwhichwould provide ameans for sensitive and rapid detectionofpotential channelblockers.

Figure 1 Novelagonistsfor theA1adenosinereceptor.

[1]Baines, A. E.; Corrêa, S. A. L.;Irving, A. J.;Frenguelli, B. G., Neu- ropharmacology, 2011,61, 1. [1]D.H.Singleton et al., J. Med.Chem., 2007, 50,2931. [2]G.J.Diazetal., J. Pharmacol. Toxicol. Methods., 2004, 50,187. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 533

MedicinalChemistry Chemical Biology MC057 Medicinal Chemistry Chemical Biology MC058

Functionalized Oligoprolines As Multivalent Expression and purification of calcium-dependent protein kinase 2, a Scaffolds in Tumor Targeting potential target of triclosan in Plasmodium falciparum

Patrick Wilhelm and Helma Wennemers Lauciello, Leonardo1,Kappes, Barbara 2,Scapozza, Leonardo1,Perozzo, Remo1 Laboratory of Organic Chemistry, ETH Zürich, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland 1 School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30, Quai Ernest-Ansermet, 1211, Geneva Azidoproline containing oligoprolines are conformationallywell-defined, helical molecular scaffolds that can be functionalized for example by cop- 2 Friedrich-Alexander Universität Erlangen-Nürnberg, Paul-Gordan-Str. 3, per-catalyzed azide-alkyne cycloaddition (CuAAC) or Staudinger reduction 91052 Erlangen and subsequentacylation.[1] The structural integrity of this scaffold allows for conjugating targeting vectors in defined distances towards each other. Triclosan (TCL) is abiocide able to kill in vitro cultures of Plasmodium falciparum with an EC50 of less than 1 µM[1]. Despite enoyl-ACP reductase (FabI) has long been thought to be its primary target, there is strong evidence for TCL to act through amulti-target mechanism [2, 3]. By means of computational methods, we recently identified calcium-dependent protein kinase 2(CDPK2) as atarget of TCL in P. falciparum. Scheme 1 Azidoproline functionalized Oligoproline CDPKs are agroup of kinases involved in many cellular processes of the parasite. PfCDPK2 is considered to be essential, but its function is not yet known. We thus expressed and purified to homogeneity this enzyme in its Recent studies on radiolabeledoligoproline-bombesin conjugates that target non-phosphorylated form suitable for further biochemical characterization. the gastrin-releasing peptide receptor (GRP-R) showed in vitro and in vivo TCL is anon-competitive inhibitor of PfCDPK2 with an IC of 48 µM. A superior internalization compared to established monovalent ligands.[2] We 50 small library of TCL derivatives was subsequently used to perform a are currently expanding this concept to the integrin-ligand c(RGDyK)as structure-activityrelationship (SAR) study, showing that the B-ring could well as to [Tyr3]Octreotide derivedligands.[3] be modified in order to improve activity. This study represents afirst step towards the understanding of the [1] (a) Y.A.Nagel, M. Kuemin,H.Wennemers, CHIMIA. 2011, 65, 264. antiplasmodial effect of TCL in P. falciparum and astarting point for the (b) M. Kuemin, L.-S.Sonntag, H. Wennemers J. Am. Chem. Soc. 2007, development of new and potent CDPK2 inhibitors. 129, 466. [2] C. Kroll, R. Mansi, F. Braun, H.R. Maecke, H. Wennemers, 2013, sub- 1. Perozzo, R., et al., JBC, 2002.277(15): p. 13106-‐13114. mitted 2. Gomez Escalada, M., et al., Lett Appl Microbiol, 2005.41(6): p. [3] J.C. Reubi, Endocrine Rev. 2003, 24, 389. 476-‐481. 3. Russell, A.D., JAntimicrob Chemoth, 2004.53(5): p. 693-‐695.

MedicinalChemistryChemical Biology MC059 MedicinalChemistry Chemical Biology MC060

Firstinsightinto thestructure andmetal-ion bindingsites of Synthesis andBiological Activity of Analogs of theMicrotubule- thehuman CPEB3ribozyme Stabilizing NaturalProductZampanolide

Magdalena Rowińska-Żyrek&Miriam Skilandat,RolandK.O.Sigel M. Jordi, D. Zurwerra,F.Glaus, L. Betschart, J. Schuster,J.Gertsch, W. Ganci, K.-H. Altmann Department of InorganicChemistry,UniversityofZurich, Winterthurerstrasse190, 8057 Zurich,Switzerland ETH Zürich, Wolfgang-Pauli-Str. 10, 8093 Zürich

TheCPEB3 ribozyme is ahighly conserved, self cleaving RNAlocated in Zampanolide (1)isapolyketide-based macrolide that wasfirst isolatedin thesecond intron of the cpeb3 gene [1] in mammals. Most of theavailable 1996 by Tanaka andco-workers from themarinesponge Fasciospongia knowledge aboutthisribozymeisbased on comparativestudies with the rimosa [1]. The compound exhibits potent in vitro antiproliferative activity structurally andbiochemically similarHDV (Hepatitis DeltaVirus)ribo- against differenthuman cancer cell linesand wasdiscoveredrecentlytobea zyme;bothcan be folded in thesameoverall pseudoknot structure [1,2]and newmicrotubule (MT)-stabilizing agent(MSA) [2]. We have completeda show strong parallels in theircatalysis andmetal-ion requirements[1,3]. total synthesis of 1 andwehavecharacterized itsinteractions with the tubu- In this work,the solution structureofawell-conserved motifofthe CPEB3 lin/MT system in significantdetail[3]. ribozyme,the P4 region,isdetermined by NMRspectroscopy, andthe bind- 2+ 3+ ingsites andstructuralimpact of Mg and[Co(NH3)6] ions arediscussed. Thedetailedknowledge aboutthe P4 structureand metalion-binding prefe- rences thus brings us closer to understandingthe CPEB3ribozyme'sfunc- tion in thecell. Firstinformation on metal-ionbinding sitesofthe full- length ribozyme is provided by theresults of terbiumcleavageexperiments of thenativeCPEB3.NMR studies of asegmented,partially labeledcon- struct arealsodiscussed. This presentation will discuss the synthesis of aseriesofzampanolide analogs andtheir activityagainst apanel of human cancercelllines. This in- Financialsupportbythe SwissNationalScience Foundationand from aSciex postdoctor- cludes desTHP-zampanolide (2), whichwas obtained via aselectiveintra- al grant(Grant No.11.156) to MRZisgratefullyacknowledged. molecularHWE reaction with phosphonate/aldehyde 3 as the ring-closing step. Intriguingly, 2 retains profound antiproliferativeactivity. Building on thechemistry developed for the synthesis of 2 we have prepared aseriesof [1]K.Salehi-Ashtiani,A.Luptak,A.Litovchick, J.W. Szostak., Science, side chain-modifiedvariants of 2 which will also be discussed here. 2006,313,1788. [2]C.Webb, N.J. Riccitelli,D.J.Ruminski, A. Luptak, Science, 2009,326, [1]Tanaka, J.-i.; Higa, T. TetrahedronLett. 1996, 37,5535-5538. 953. [2]Field, J. J.;Singh, A. J.;Kanakkanthara, A.; Halafihi, T.; Northcote,P. [3]D.M.Chadalavada,E.A.Gratton,P.C.Bevilacqua, Biochemistry, 2010, T.; Miller,J.H.J. Med.Chem. 2009, 52,7328-7332. 49, 5321. [3]Zurwerra, D.; Glaus, F.;Betschart, L.;Schuster,J.; Gertsch, J.;Ganci, W.;Altmann, K.-H. Chem.Eur.J.2012, 18,16868-16883. 534 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

MedicinalChemistry Chemical Biology MC061 MedicinalChemistryChemical Biology MC062 Stereochemical controlofphosphorothioate linkagesinRNA synthesis InfluenceofmetalionsonRNA structureasrevealedbyX- controlled by the activator raycrystallography Hartmut Jahns1,Martina Roos1,RyanGilmour2,Jonathan Hall1 Michelle F. Schaffer,1 Joachim Schnabl,1 Guanya Peng,2 VincentOlieric2 1 1 Swiss FederalInstituteofTechnology (ETH), Wolfgang-Pauli-Str. 10, andRolandK.O.Sigel 8093 Zürich,Switzerland 1 Institute of InorganicChemistry, University of Zürich, 2 Westfälische Wilhelms-Universität, Corrensstraße 40, 48149 Münster,Germany Winterthurerstrasse 190, 8057 Zürich,Switzerland 2 SwissLight Source at Paul Scherrer Institute,5232Villigen,Switzerland The routine solid-phase synthesis of oligoribonucleotides (ORN), havinga fullyphosphorothioated (PS)backbone,yields 2n-1 differentdiastereoiso- [email protected] mers(ds),where n=number of nucleotides. We have performed adetailed studyofthe coupling mechanism andshowedthe influenceofdifferentacti- Duetothe polyanionicnatureofRNA, theprinciplesofchargeneutraliza- vators on the ds-ratio.Specifically,weanalyzedthe ds-ratiosofall possible tionand electrostatic condensationrequire that cations help to overwhelm 16 PS-dinucleotide motifs of RNAtogive apicture of Rp-toSp- distribu- therepulsive forces,inorder forRNA to adopt athree-dimensional structure tion. This dinucleotide model wasextendedtothe prediction of Rp-/Sp- [1,2]. Adetailedstructural knowledgeofRNA-metal ioninteractionisthus ratios of an octanucleotide containingasingle PS-linkage.Since Rp-/Sp- crucial to understand theunderlying mechanismofmetal ions in thecatalyt- diastereoisomershavedifferent chemical andbiologicalpropertiese.g.af- ic activityofribozymes or regulatory functions in riboswitches[1, 3]. finity, activityand stability[1][2], thisdinucleotide model represents a In our studyweuse an octamericRNA as modelsystemtosee apattern in means to predictablymodulate the ratios of Rp-and Sp-linkagesand there- coordinationofvarious metalions to specificbinding sitesand to under- foretheir biological properties. Severalcommonly-usedactivators were standthe interactions between metalions andRNA. testedinthe dinucleotide model.Thermaldenaturation experiments con- Newtechnical approaches of thesynchrotron radiationfacilityatthe Swiss firmed the resultsofswitchingthe Rp-/Sp-ratiosupon the destabilizing ef- LightSource (SLS)helps to obtainhigh-resolutiondatathatisrequired fora fectsofsulfur introductionintoPS-ORN/ORN duplexes. detailedstructural analysis.

Financialsupportbythe ERC, theSwiss NationalScience Foundationand theUniversityofZurichisgratefullyacknowledged.

[1]Sigel,R.K.O.Eur.J.Inorg Chem. 2005,12,2281–2292. [1]Maria Koziolkiewicz et al., Antisense &Nucleic Acid Drug Develop- [2]Freisinger, E., Sigel, R. K. O. Coord. Chem.Rev. 2007,251 , ment. 1997,7,43-48 1834–1851. [2]Agnieszka Krakowiak et al., Nucleosides&Nucleotides, 1998,17, 1823- [3]Roth, A.,BreakerR.R.Annu. Rev. Biochem. 2009,78, 305–334. 1834

MedicinalChemistry Chemical Biology MC063 MC064 MedicinalChemistry Chemical Biology HumanRNA G-quadruplex Structures as Potential Anticancer Targets Measurement of the tissue oxygenation in vivo by time-resolved lumi- Helena Guiset Miserachs,Daniela Donghi,Roland K.O. Sigel nescence spectroscopy of Ru(Phen), apoorly photosensitizing probe

UniversityofZurich, Institute of Inorganic Chemistry, Winterthurerstrasse V. Huntosova, S. Gay, P. Nowak-Sliwinska, G. Wagnieres 190, CH-8057 Zürich, Switzerland Swiss Fed. Inst. of Technology (EPFL), ISIC, Station 6, 1015 Lausanne, Guanine-rich nucleic acids fold into non-canonical helical structures known Switzerland as G-quadruplexes (GQ), stabilized by central metal ions [1].Human RNA GQsare relevant as potentialantitumor targets. Therefore, we study the GQ Measuring the tissue oxygen concentration (pO2) in vivo is of interest for in the mRNA of the NRASgene (neuroblastomaRAS viraloncogene homo- numerous fundamental and applied studies. One minimally-invasive ap- log), showntoinhibit protein synthesis in vitro [2], andthe TERRAGQ proach to assess pO2 is based on the measurement of the oxygen-dependent (telomeric repeat-containing RNA), involved in telomeremaintenance [3]. luminescence lifetime of molecular probes. The relation between the partial pressure of oxygen and this lifetime is governed by the Stern-Volmer equa- tion. Unfortunately,virtually all oxygen-sensitive probes based on this principle induce some degree of phototoxicity [1]. Thus, we assessed the phototoxicity (wavelength: 470±20 nm; light dose: 2.5-20 J/cm2;drug dose: 1-20 mg/kg; drug-light interval: 1min) and oxygen sensitivity of dichlorotris (1,10-phenantroline) Ruthenium (II) hydrate(Ru(Phen)) in the chicken embryo chorioallantoic membrane model (CAM). Luminescence lifetimes have been measured with adedicated time-resolved spectrometer [2]. We demonstrated that, after intravenous injection, Ru(Phen) presents an easily detecta- Currently, we are systematically assessing the stabilization of RNA GQsin ble, pO2-dependent, luminescence lifetime with "small" drug and light doses the presence of monovalent and divalent metal cations by means of circular (1 mg/kg and 120 mJ/cm2 @470 nm). With afluence rate of 65 mW/cm2, dichroism,UVmeltingand NMR. NRAS GQ formation and dynamicsare the phototoxic threshold was found to be at 10 J/cm2 with the same wave- beingstudied by single molecule FRET (Förster Resonance Energy Trans- length and drug dose. fer) spectroscopy. [1] Stepinac TK, Chamot SR, Rungger-Brandle Eetal., Invest Ophthalmol Financialsupport fromthe SBFI (COST ActionCM1105),ERC (R.K.O.S.), SNF(D.D. and Vis Sci. 2005, 46,956-966. R.K.O.S.) andUniversity of Zurich is gratefully acknowledged. [2] Piffaretti F, Santhakumar K, Forte Eetal., JBiomed Opt. 2011, 16, 037005. [1] J.L. Huppert, S. Balasubramanian, Nucleic Acids Res. 2007, 35,406. [2] S. Kumari, A. Bugaut, J.L. Huppert, S. Balasubramanian, Nat. Chem. Biol. 2007, 3,218. [3] B. Luke, J. Lingner, EMBO J. 2009, 28,2503. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 535

MedicinalChemistry Chemical Biology MC065 Medicinal Chemistry Chemical Biology MC066

Sequential combination of axitinibwith photodynamic therapy delays Arene-linkeddinuclear RAPTAanalogues tumor growth in preclinical model of ovarian carcinoma Benjamin S. Murray Rosario Scopelliti, Paul J. Dyson* Débora Bonvin, Weiss Andrea, Hubert van den Bergh, Patrycja Nowak-Sliwinska Institut desSciences et Ingénierie Chimiques, EcolePolytechniqueFédérale de Lausanne(EPFL),CH-1015, Lausanne, Switzerland Swiss Federal Institute of Technology (EPFL), 1015 Lausanne, Switzerland Organometallic"piano-stool"Ruthenium arenecomplexes areafamily of Vaso-occlusive photodynamic therapy (PDT) is considered as apromising promising drug compounds whichexhibitantitumouraland antimetastatic treatment for certain types of skin and lung cancer. However, the PDT- properties [1,2]. One seriesofthese,developed in the Dyson laboratory, 6 induced microvascular damages result in tissue hypoxia, activating the hy- arethe RAPTA compounds [Ru(η -arene)Cl2(pta)] whichexhibithighanti- poxia-inducible factor-1(HIF-1) that promotes VEGF-mediated angiogenic metastatic activitycombinedwith excellent clearancerates andlow general responsesand attenuates the overall PDT efficacy. As recent strategies have toxicity. clearly demonstrated increased therapeutic effectiveness of combining anti- VEGF drugs with PDT, we combined Visudyne®-PDT with axitinib, apo- tent small tyrosine kinase inhibitor (TKI) that specifically targets the VEGF receptors 1, 2and 3, in order to improve the clinical outcomes of the photodynamic treatment in cancer patients. In this preclinical study in the chorioallantoicmembrane (CAM) of the chicken embryo, we optimized the drug and light parameters, as well as the One attractivefeature of the RAPTA structure is the manner in whicheach relative schedules of the therapies. On the physiological CAM vasculature, ligand (arene,PTA, chlorides) maybemodulated andelaborated upon in injecting low doses of axitinib 4hours before PDT prolonged the vaso- ordertoconfergreaterbiologicalactivityonthe resultantcomplex [1]. occlusive effect of the treatment in astatistically significant manner. In addition, treatment of A2780 ovarian tumors grafted on the CAM with axitinib In thisworkwepresent thefirst examples of RAPTA compounds linkedvia caused increased intra-tumoral oxygenation, as measured between 24 and 54 organic tethers, through the areneligand, to yield complexes of significantly hours after drug injection. This so called “normalization window” was used enhanced activityrelativetomononuclear analogues. Aseriesofstereoiso- to improve the vaso-occlusive PDT outcome. Combination of low dose PDT meric dinuclear RAPTA compounds will be introduced andthe impact of (2.5 J/cm2 at 420 nm) with axitinib applied in various schedules resulted in the stereochemistryofthe linkeronthe biologicalactivityofthe dinuclear synergistic tumor growth suppression. RAPTA complexes will be discussed.

[1]Ang,W.H., Casini,A., Sava,G., Dyson, P. J., J. Organomet. Chem., 2011, 696(5),989-998. [2]Bruijnincx, P. C. A., Sadler.P.J., Adv.Inorg. Chem.,2009, 61, 1-62.

MedicinalChemistryChemical Biology MC067 MedicinalChemistryChemical Biology MC068

Amino Acid SequenceVariation in aMultivalentGlycopeptide Synthesis, biochemicaland structuralcharacterisation of novelthymine Dendrimer targetingLecA inhibiting Pseudomonasaeruginosa Biofilms derivatives as reporter probes forHSV1-tk gene expression imaging with positronemission tomography Myriam Bergmann,GabrieleGabrielli, Tamis Darbre andJean-Louis Reymond I. Novaković,L.Pernot Schiltz,L.Scapozza

Department of Chemistryand Biochemistry,University of Berne, Pharmaceutical Biochemistry group, School of Pharmaceutical Sciences, Freiestrasse 3, CH-3012 Berne UniversityofGenevaand UniversityofLausanne, Quai Ernest-Ansermet 30, CH-1211 Geneva 4, Switzerland The spread of antibiotic resistantbacteriaisone of themost pressing prob- lems in humanhealth today. Thegram-negativebacterium Pseudomonas aeruginosa is an opportunistic human pathogencausing lethalairwaysin- HSV1-TK as areporterprobe is themost studied systemfor thevisualiza- fections. It canformsurface-attached biofilms protectingitself from classi- tionofgeneexpression in vivo.C-6-alkylatedpyrimidinederivatives are cal antibiotic treatment.Biofilm formationismediatedinpart by the good substrates of HSV1-TK andexhibit no cytotoxiceffects[1],thus sev- galactose-specificlectin LecA (PA-IL) andthe fucose-specificlectin LecB eral C-6-substitutedanalogs were synthesizedand biochemically character- (PA-IIL).[1,2] Capitalizingonthe well-knowncluster effect observed on ized.Their phosphorylationpattern was monitoredinpresence of HSV1-TK binding of multivalentcarbohydrates to lectins, we have reportedthe first or hTK using aprotocolbased on HPLC-UV/DAD [2]. Synthesizedcom- cases of P. aeruginosa biofilm inhibitionwithmultivalentlectin inhibitors, pounds were phosphorylated in presence of HSV1-TK but not in presence of including thegalactosylated glycopeptidedendrimer GalAG2 (βGal- hTK.For thefirst time diphosphorylationofone non-natural substrateby KPL)4(KFKI)2 KHI-NH2 targetingLecA.[3] Herein we report thelectin HSV1-TK wasobserved.The crystalstructures of HSV1-TK in complex binding, biofilm inhibition anddispersal by an Alascan series of GalAG2 with thecompounds were solvedbymolecularreplacementusing thedata in ordertodeterminethe influenceofthe peptidic sequence. Some collected at theSLS synchrotron. Therefinedstructures unequivocally show galactosyl peptidedendrimers with variations in theterminal tripeptidic se- the detailed binding mode of thecompounds inside HSV1-TK. Theprolifer- quencewere also evaluated. ationofHSV1-TK transduced HEK293-cells wasselectivelyinhibitedby one of thecompounds with an IC50 valuecomparable to theone of ganciclovir. We have been using PK15 NTD celllines expressing human equilibrativeand concentrativenucleoside transporters accordingtothe pub- [1]V.E.Wagnerand B. H. Iglewski, ClinicAllerg Immunol 2008, 35, lished protocol [3]toassesscompounds transportability. The results of these 124 experimentsthatare in progresswill be shown. Concluding thepresented [2]Tielker et.al., Microbiology 2005, 151,1313 results indicatethatone of thesynthesized compounds hasapotentialtobe [3]R.Kadam,M.Bergmann, et al., AngewChemInt Ed Engl. 2011, 50, anew PETreporterprobe andfuturestudieswill be focusedonit. 10631 [1]U.Müller, et al., J. Nucl.Med.Bio. 2011, 39,256. [2]P.Schellingetal., J. Bio. Chem. 2004, 279,32832. [3]J.L.Wardetal., J. Bio. Chem. 2000, 275,8380. 536 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

Medicinal Chemistry Chemical Biology MC069 MedicinalChemistry Chemical Biology MC070

Interaction of platinum anticancer drugsand RNA Anti-Cancer Effect of Photodynamic Therapy Enhanced through the Combination with Low-Dose, Angiostatic Kinase Inhibitors MarianthiZampakou, Daniela Donghi Andrea Weiss1, Judy R. van Beijnum2,Débora Bonvin1, InstituteofInorganic Chemistry, UniversityofZurich, Hubert van den Bergh1,Arjan W. Griffioen2, Winterthurerstrasse 190, CH-8057, Zurich, Switzerland Patrycja Nowak-Sliwinska1,3

The interaction of platinum drugs with targets other than DNAcould be 1MedicalPhotonics Group, Swiss Federal Institute of Technology (EPFL), responsible for the toxicside effectsorcould playsome still unrevealed role Lausanne, Switzerland, 2Angiogenesis Laboratory, Department of Medical in their anticanceractivity. It hasalreadybeenreportedthatsome RNA Oncology, VU University Medical Center, Amsterdam, The Netherlands, dependent activities areinhibited upon administration of platinum drugs, but 3Department of Urology, University Hospital (CHUV), Lausanne, little is known on the effectsofplatinum on RNAbiology [1]. Moreover, Switzerland information on RNAstructuralchanges upon platinum binding is still very scarce.RNA structureismuch more complicated than DNAand interaction Photodynamic therapy (PDT) is used as an effective clinical treatment for a with platinum drugsmay leadtoadifferent scenario than the one observed number of different cancers; however, secondary tissue reactions to PDT with DNA-platinum interaction. such as hypoxia and inflammation have pro-angiogenic side effects which In ordertounderstand the effect of platinum drugsonRNA structure and may counteract the desired angio-occlusive effects of PDT. The combina- activityweuse as amodel RNAa27 nucleotide long hairpin derivedfrom tion of PDT with clinicallyused anti-angiogenic drugs may provide im- the mitochondrial group II intron ribozyme Sc.ai5γ[2].The chosen RNA proved anti-tumor outcome. We testedthe effect of three clinicallyapproved contains structuralfeatureswidespread in RNAs andits NMR structure in angiostatic tyrosine kinase inhibitors (TKIs) (sunitinib, sorafenib and ax- solution is known [3], making easieradetailedinvestigation of the itinib) and the VEGF neutralizing anti-body based compound bevacizumab structuralchanges upon platinum drug interaction. We arenow optimizing in two tumor models for the improvement of PDT outcome. The strongest the reaction conditions with both cisplatinand oxaliplatin. The platinated anti-cancer effects were seen for the combination of PDT and low dose ax- adducts will be characterized by different techniques like mass itinib or sorafenib which indicated synergistic potential. Real-time quantita- spectrometry, circular dichroism andUVthermalmelting studies as well as tive PCR indicated the suppression of VEGFR-2 expression in the vascula- NMR spectroscopy, in ordertoevaluate the structuralpreferencesofthe ture of tumors treated with the combination therapy of PDT and axitinib. binding. Pre-administration of angiostatic compounds did not improve PDT outcome and increased oxygenation in axitinib treated tumors with was not identified. Financialsupportbythe SwissNationalScience Foundation(Ambizione fellow- These result indicate that improved therapeutic outcome in combination shipPZ00P2_136726 to DD), by theUniversityofZurichand withinthe COST treated tumors was not due to avascular normalization effect. The current ActionCM1105isgratefullyacknowledged. data imply that there is future for certain anti-angiogenic agents to further improve the efficacy of photodynamic anti-cancer therapy. [1]E.G. Chapman, V. De Rose, J. Am.Chem.Soc., 2010, 132,1946 [2]R.K.OSigel, Eur. J. Inorg. Chem., 2005, 12,2281 [3]D.Donghi, M. Pechlaner, R.K.O Sigel, unpublished

Medicinal ChemistryChemical Biology MC071 MedicinalChemistry Chemical Biology MC072

CompoundswithaN+-C-C-O Backbone Catalyzethe Bis(benzoxaborole): organoboron antibacterial and antifungal agent Disproportionation of Peroxynitrite to Nitriteand Peroxynitrate Krzysztof M. Borys, Agnieszka Adamczyk-Woźniak, Andrzej Sporzyński Christian Molina,Reinhard Kissner and Willem H. Koppenol Faculty of Chemistry, Warsaw University of Technology, ETH Zürich, Wolfgang-Pauli-Str. 10, 8093 Zürich Noakowskiego 3, 00-664 Warsaw, Poland

The oxidizing and nitratingagent peroxynitrite,ONOO– and ONOOH is formed in vivo,which has stimulated studiesofits reactions with bio- Benzoxaboroles -internal, cyclic hemiesters of phenylboronic acids -have – molecules. Peroxynitrite is unstableand decaysbyisomerization to NO3 been recently earning growing research interest mainly in terms of their bio- – – and by disproportionation to NO2 andtothe artificial oxidant O2NOO [1]. actvity.[1] Along with their antifungal and antibacterial properties,[2] they The buffer tris(hydroxymethyl)aminomethane (Tris)catalyzes the dispro- present exceptional sugar receptor activity,[3] constituting an interesting portionation.2-Aminoethanol, choline, serine and 2-aminoethylphosphate class of organoboroncompounds to be investigated. acceleratethe disproportionation too while the amine analogue of Tris, the alcohol analogue of Tris and the mixture of both do not. The effect of cho- HO line indicatesthat the positively charged nitrogen of the N+-C-C-O substruc- B O ture –e.g. the protonatedamine of a1,2-aminol – may attractONOO– while N N the vicinal oxygen formsahydrogen bond withONOOH. When ONOO– O B and ONOOH both are coordinated to the same molecule, they are close OH – – enough to reactwith each other quickly under release of NO2 and O2NOO : O O O O N Preparation and biological evaluation of piperazine-derived N O O O H O O N bis(benzoxaborole) is reported. The bis(benzoxaborole) system can be R'' R O O N H straightforwardly assembled starting from 2-formylphenylboronic acid. Syn- + O N R'' thesis of the bis(para-fluoro) derivative, as well as methods for efficient R R' O R O R + preparation of bis(phenylboronic acid) analogues are presented. R' O N + R N H R R' O O R' H+ R'' - O R O NO2 [1] S. J. Baker, J. W. Tomsho, S. J. Benkovic, Chem. Soc. Rev. 2011, 40, O + R: H, Me + N R': H, CH OH 4279-4285. O N R 2 R' 2- O O R R'':H,Me, PO3 [2] A. Adamczyk-Woźniak, O. Komarovska-Porokhnyavets, B. Mis- R' terkiewicz, V. P. Novikov, A. Sporzyński, Appl. Organometal. Chem. The substructure N+-C-C-O is widespread amongbiomolecules, e.g. amino 2012, 26,390-393. acids,phospholipids (cephalin,lecithin,phosphatidylserine), glucosamine. [3] M. Bérubé, M. Dowlut, D. G. Hall, J. Org. Chem. 2008, 73,6471-6479. [1]D.Gupta et al, Dalton Trans. 2009, 29,5730 medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 537

MedicinalChemistry Chemical Biology MC073 MedicinalChemistryChemical Biology MC074

Amodular system forpoint-specificlabeling of native oligonucleotides Liganddevelopment forthe vesicularmonoamine transporter VMAT2: SynthesisofAnalogs of Tetrabenazine Igor A. Oleinich, Eva Freisinger

UniversityofZurich, Institute of Inorganic Chemistry, Winterthurerstr. 190, L. Radtke,M.Johannes, K.-H. Altmann 8057 Zurich,Switzerland ETHZürich, Wolfgang-Pauli-Str. 10, 8093 Zürich Functionalization of native ribonucleic anddeoxyribonucleic acids has paramount importanceinall research fields related to life sciences.Avarie- Tetrabenazine(TBZ(1), Fig. 1) is atetrahydroisoquinolinederivativewhich ty of bioorthogonalgroups andmodifications in biomolecules areinuse, is an approveddrugagainst dyskinesiaand hyperkinetic movement disor- e.g. for targeted drugdeliveryorimaging of DNAand RNA[1,2]. Re- ders (chorea Huntington).[1] TBZreversibly bindstoand blocks thevesicu- strictionofthe lengthsofsuchmodified oligonucleotides accessiblebyauto- larmonoaminetransporter 2(VMAT2),which is responsiblefor the mated phosphoramidite-basedsynthesis prompted us to devise an alternative transport of monoamines from thecytoplasmintogranular vesicles of pre- method. synaptic neurons.[2] In vivo TBZisconverted into its pharmacologically activemetabolite α-dihydrotetrabenazine (α-DHTBZ); onlythe (+)- enantiomerof α-DHTBZbinds to VMAT2with high affinity.[3]

R1 MeO MeO MeO R2 N HN MeO MeO N H MeO H 1 2 1 2= e : a- O O R , R M , H 3 d O The basic principlereliesonthe annealingofadonor DNAstrand that Thispresentationwill discuss anew stereoselectivesynthesis of 1 that en- carries areactivegroup to acomplementary template strand [3]. This posi- tails formationofthe macrocyclic keyintermediate 2 by RCM.[4] Follow- tions the reactive group close in space to thetargetnucleotide in thetem- ingthe same overallstrategywehavealsoprepared monomethylatedtetra- plate strand allowingits site-specific modification[3].The intermediately benazine derivatives 3a-d forbiological testing. The ultimate goal of this formed covalent cross-link between donor andtemplate strandcan be research is thediscovery of more selectiveVMAT2 blockers than TBZand cleaveddue to aspecifically designedlinkerbetween the reactive group and to assess whethersuchcompounds mayexhibit an improvedsideeffectpro- the donor DNA. This approach will be generallyapplicable fordifferent file in vivo. kind of modifications duetothe manifold combination possibilities of reactive groups andthe linkers. [1]P.Diana, Neuropsychiatric Disease andTreatment 2007, 5,545-551. Financial support by the Swiss NationalScienceFoundation (SNSF- [2]D.R.P.Guay, Am.J.Geriatr.Pharmacother. 2010, 8,331-373. Professorship PP002-119106/1 to EF)isgratefullyacknowledged. [3]M.R.Kilbourn, L. C. Lee, T. VanderBorght, D. Jewett, K. Frey, Eur. [1]J.Brunner, R. Kraemer, J. Am.Chem. Soc. 2003, 125,12410-12411. J. Pharmacol. 1995, 278,249-252. [2]D.M.Kolpashchikov, Chem.Rev. 2010,110, 4709-4723. [4]M.Johannes, K.-H. Altmann, Org. Lett. 2012, 14,3752-3755. [3]I.A.Oleinich,E.Freisinger,tobesubmitted.

MedicinalChemistry MC075 Medicinal Chemistry Chemical Biology MC076

PyridomycinAsLeadFor NewAnti-Tuberculosis Agents DevelopingProtein Containers with Tailored Recognition Properties Eita Sasaki1,TobiasBeck1,NeilP.King2,David Baker2 andDonald Hilvert1 Oliver Horlacher,RubenHartkoorn, StewartCole, Karl-Heinz Altmann 1LaboratoryofOrganic Chemistry, ETH Zürich, 8093 Zürich,Switzerland. ETH Zürich,Institute of Pharmaceutical Sciences,CH-8093 Zürich 2DepartmentofBiochemistry,UniversityofWashington, Seattle, WA 98195, USA. Pyridomycin (1)isabacterialnatural productthatwas firstisolatedfromthe Streptomyces strain no. 6706 in 1953 andwas showntoexhibitsignificant Macromolecule encapsulationbased on self-assemblyofhost proteins and in vitro anti-tubercular activity.[1] Only recently, themoleculartargetof1 guestmolecules plays important rolesinNature.These include virus capsids, hasbeen identifiedasthe NADH-dependent enoyl-reductaseInhA whichis ferritin proteins,and bacterialmicrocompartments such as the carboxysome. also thetargetofthe clinically used anti-TB drug isoniazid.[2] We have previously developed anovel protein container, AaLS-13, derived from lumazine synthase,whichwas engineeredtoencapsulatepositively chargedprotein cargo.[1] Creation of such unnatural proteincontainers is not onlyimportant for gaining adeeperunderstanding of natural encapsulation systems butalso for providing newtoolsfor variousapplications such as drug delivery,bioimaging, andnanoreactors. Here,weapplied the same strategyto acomputationallydesigned protein cage,O3-33.[2] O3-33forms a24-mer cage structure with octahedral symmetry,and itssize(13 nm in diameter)is considerablysmaller thanthat of AaLS-13(35 nm in diameter). First,we Theobjectiveofthisworkistoexplorethe biologyand medicinalchemistry introduced sixglutamic acid mutations on the internalsurfaceofeach O3-33 of pyridomycin with afocus on the de novo synthesisofanalogs of thenatu- subunit. Despitethe smaller luminal volume, andthus, highernegativecharge ralproduct. In this contextwehaveachievedthe stereoselectivesynthesisof density, O3-33-neg6 assembled intoasimilar cage structure as O3-33. Next, twotargetstructures 2 and 3 whichare structurally conservedanalogs of 1. we incubated O3-33-neg6 with apositively supercharged GFPvariant, Despite thelackofthe enol esterfunctionality,(R)-analog 2 retained [3] GFP(+36). As expected,upto4molecules of GFP(+36) associatedwith the most of theanti-tubercular activityof1,while (S)-analog 3 showed asignif- O3-33-neg6 cage.The crystal structure of thecomplex revealed that icantlyreduced activity. GFP(+36) molecules were likely locatedonthe internalside of the In ordertoinitiate astructure activity relationshipstudy around the O3-33-neg6 pore.The resultspresented here reveal that designstrategies pyridomycin scaffold,moreanalogs have been prepared,relying on satura- relyingonhost-guest electrostatic interactions constituteasimple andversa- tionofthe enol esterand on variationofthe hydroxypicolinic acidmoiety. tile method to create protein containers capable of encapsulatingcharged Thesynthesesofthese compounds,their biological activities andacrystal macromolecules. structureofananalogbound to InhA will be presentedatthe meeting. [1]Wörsdörfer,B.; Woycechowsky, K. J.;Hilvert, D. Science, 2011, 331, [1]MaedaK.et al., J. Antibiot. 6A 1953,140. 5894517. [2]HartkoornR.C.et al., EMBO Mol. Med. 2012, 4,1032. [2]King,N.P.et al.Science, 2012, 336, 11711174. [3]Horlacher O. P.etal., ACSMed.Chem. Lett. 2013, 4,264. 538 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

MedicinalChemistry Chemical Biology MC077 Medicinal Chemistry Chemical Biology MC078

Polymernanoreactorsasanefficient sourceofradicals“on demand” Design andre-iterative data mining of ahighlydiverse chemical library for photodynamictherapy used forantibacterial activity screenings

Patric Baumann1,MarianaSpulber1,Andrzej Sienkiewicz2,Cornelia G. Gianpaolo Chiriano,*a Liudas Slepikas,a AgataKranjc Pietrucci,a Ophelie Palivan1 Pattey,a Pierre Cosson,a Hajer Ouertatani-Sakouhi,a ThierrySoldati,a Sébas- tien Kicka,a Hubert Hilbi,b Christopher Harrison,b John McKinney,c and 1Department of Chemistry, University of Basel, Klingelbergstrasse 80, 4056 Leonardo Scapozzaa Basel, Switzerland 2 InstituteofPhysicsofComplex Matter,Faculty of Basic Sciences,École a b Polytechnique Fédérale de Lausanne, CH-1015 Lausanne UniversityofGeneva, Geneva-Switzerland; Ludwig-Maximilians Univer- sity, Munich-Germany; cEPFL, Lausanne-Switzerland. PhotodynamicTherapy (PDT) is gaining an increasing attention for treatment of various pathologic situations,such as cancer, age related macular In thiswork, we aimedatthe discovery of novelcompounds towards the degeneration,burns andulcer. However,there arestill open points that are prevention or treatment of bacterial infections caused by Klebsiellapneu- necessary to be solved, such as an increase of the efficiency of photosensi- moniae, Legionella pneumophila or Mycobacterium marinum using simple but experimentallyhighlytractable hosts, amoebae, among which Dictyoste- tizers,and adecrease their toxicity in otherbio compartments than the de- 1 sired ones. To solve these drawbacks, we introduced theconcept of polymer lium discoideum. To fulfill thisgoal, we designedapathway-basedhighly nanoreactor as asourceofradicals “ondemand”[1]. Nanoreactors based on diverse compounds library basedon18host and/or bacteria pathways cru- the encapsulation of ahighly efficient PDT photosensitizer (rose bengal- cial for the host-pathogeninteraction process. Knownligands/metabolites BSA) in polymervesicles serve for local generationofradicalsupon irradia- linkedtothesepathways have been used to launchacampaignofligand- basedvirtualscreening from ZINC lead-like database by the means of tion with aspecificelectromagnetic radiation. Polymer nanoreactors play a 2 dual role:theyprotect therose bengal-BSA conjugate, and allowittoact in ROCS. The resulting libraryincludes 1711 selected compounds (~100 per situ, insidethe cavity of vesicles. Nanoreactors produce under irradiation each pathway)and 2000 compounds from the Prokinase librarycontaining with appropriatewavelength reactive oxygen species inside cells and there- ligands binding to kinases involvedinseveral signalingpathways andthe fore induce cell death, whereas non-irradiated cells remain healthy, evenif NINDS. Ongoingscreening designedtoidentify compounds interferingwith they are in contact with the nanoreactors. host-pathogen interactions alreadyled to the discovery of several hit candidates with significant activityat30μM.Asresult,wedesignednew hits- basedlibrariesfor further testingand establishing primarySARs.Tovirtual- ly manage these librariesand performthe relateddata-mining,the Shared ResultsDatabase (SRD) wascreated by usingInstantJChem3 andwill be shared via internetamongthe scientific community. Librariesdesign, SRD structure andSAR studies for the most interesting hits will be presented.

1. Froquetetal., NatProtoc 2009;4(1): 25-30 [1] Patric Baumann, VimalkumarBalasubramanian, Ozana Onaca-Fischer, 2. http://www.eyesopen.com/rocs Andrzej Sienkiewicz,Cornelia G. Palivan, Nanoscale 2013, 5,247. 3. http://www.chemaxon.com/products/instant-jchem/

MedicinalChemistry Chemical Biology MC079 Medicinal Chemistry Chemical Biology MC080

The ionotropic serotonin receptor as scaffold for Pathogeninactivation lesionstoplatelets: comparison at thepeptide functional and structural studies levelofthe twomain techniques available

Ruud Hovius, Joachim Piguet, Horst Vogel Giona Sonego, Michel Prudent, Mélanie Abonnenc,Jean-Daniel Tissot, Niels Lion Laboratory of Physical Chemistry of Ploymers and Membranes EPFL ServiceRégional Vaudois de Transfusion Sanguine, Unité de rechercheet Développement,Lausanne, Switzerland Transmembrane proteins offer next to their own functionality, like transporter or signal transducer, the possibility to be tailored for other purposes, Pathogen reduction technologies (PRT) for platelets concentrates have a like scaffolds for the membrane-proximal attachment of proteins. Here, the centralroleinpreventing transfusion-transmitted infections. The principle serotonin-gated ion channel 5HT3R is used as central molecule for structur- of PRT is basedonthe photochemical modification of DNA/RNA strands, al and functional studies. This receptor is homo- or heteropentamericas- inactivating pathogens.Interceptprocess (adopted in Switzerland) uses sembly of subunits(~450 aa) that have each ahighly structured N-terminal amotosalenasaphotoactive compound whereas Mirasol uses riboflavin.In extracellular ligand-binding domain (~230 aa), four transmembrane helices addition of modifyingnucleic acids, theyalsoproducereactiveoxygenspe- and arather un-structured large intracellular loop (~125 aa) between the 3rd cies (ROS). AlthoughPRT areinroutine use in blood centers, theyare sus- and 4th TM. pected of reducingthe haemostatic activityofplatelets[1-3]. The mechanism Here, proteins were appended to the N-terminus of or inserted in the large giving rise to thisphenomenon is not clear. At the proteome level, we have intracellular loop of the receptor for imaging, e.g. fluorescent proteins (~250 shown that afew proteins areaffected by the Intercepttreatment[4].Here, aa) or SNAP-tag (~180 aa), or to engineer amembrane-bound calcium sen- the impact of PRT is investigated at single unit (amino acid) level. Different sor (~470/ 650 aa). Despite the large relative size of additions, the fusion sequences of aminoacids were treated with thephotochemically active proteins featured, in general, unperturbed ligand-binding and channel prop- techniques InterceptorMirasol,whichallowedtocomparethe impact of erties, showing that 5HT3R very tolerant to major modifications. these two PRT. Underthese oxidative conditions it waspossible to observe The subunit of heteropentameric 5HT3_A+B receptor was investigated us- andquantify,byliquid chromatography coupled to mass spectrometry, dif- ing Asubunits with appropriate fluorescent proteins in the large intracellular ferent kindsofoxidations, mainlyonThr,Tyr,Cys andHis moieties. More- loop enabling fluorescence resonance energy transfer (FRET). over, differences between thesetwo PRT were illustrated by the conversion Ligand binding to 5HT3R was quantified using aFRET assay employing a rates of oxidizedpeptidesafterthe treatment,where Mirasol exhibitsthe fluorescent labelled ligand and a5HT3R carrying afluorescent protein on greatest impact.Investigations on proteinsparticipating to platelet activa- its N-terminus. tion/aggregation andclot formation willfollow.

[1]Kerkhoffs J-LH et al., Br JHaematol 2010, 150,209. [2]CorashLandShermanCD, Br JHaematol 2011, 153,529. [3]Kerkhoffs J-LH, Br JHaematol 2011, 153,531. [4]Prudent Metal. JProteomics 2012, 76,316. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 539

Medicinal Chemistry Chemical Biology MC081 MedicinalChemistry Chemical Biology MC082

Highly Chemoselective andPractical Thio-Alkynylation Development and engineering of probes forbivalent epigenetic marks

Reto Frei andJérôme Waser Ahmed-HocineBoumezbeur &Beat Fierz, LCBM,EPFL

LaboratoryofCatalysis andOrganic Synthesis, EPFL,1015 Lausanne, Ecole polytechnique fédérale de Lausanne /LaboratoryofBiophysical Switzerland ChemistryofMacromolecules, CH B1 375,1015 Lausanne,Switzerland

Due to the tremendous capacity of acetylenechemistry,[1] methods that se- Nucleosomes of keydevelopmental genesinstem andprogenitor cells are lectively transfer alkyne moietiesonto vastlyfunctionalizedintermediates thought to be markedwith repressive (H3K27me3) as well as activating arehighlysoughtafter. Currentthio-alkynylation processes arerareinnum- (H3K4me3) post-translational modifications (PTMs). Theyhence formbi- berand generallyutilize nucleophilicacetylides understrongly basic condi- valent chromatindomains, where genesare repressed but believed to be tions.[2] Consequently,these harsh conditions have resulted in amoderate poised forinduction. Thispropertycould play amajor role in developmental scope andfunctionalgroup tolerance. In thiscontext,wehavedeveloped a anddifferentiationprocesses, andcould also be implicated in somecancers. highlychemoselective andpracticalthio-alkynylation reaction by utilizing The presenceofbivalentnucleosomes hasbeen inferred from methods such electrophilic, hypervalentiodine-based,alkyne-transferreagents.[3] The de- as genome wide chromatinimmunoprecipitation (ChIP), ChIP re-ChIPor veloped thio-alkynylation reactionreadilyproceeds at room temperature immunopurificationfollowedbyLCMS/MS experiments. The common (i.e.<5min)inanopenflask,using commerciallyavailable reagents. The limitation of these approaches is their requirement for celllysis andtheir scope of the reactionisbroad,with avariety of highlyfunctionalizedbio- reliance on antibodies.Further, no toolsare available to directly show co- logicallyand medicinallyrelevantphenolic-,benzylic-,heterocyclic-,ali- existence of H3K4me3and H3K27me3 on the same nucleosome. phaticand peptidic thiolsundergoingalkynylationinexcellent yield. Fur- In ordertodirectly visualize the colocalization of H3K4me3 and thermore,the utility of thethiol-alkynylation in post-synthetic elaboration H3K27me3 in cells andtherebytodetectand follow bivalent domains, we hasbeendemonstrated,for instance, via installmentoffluorophoretags. areengineeringgenetically encodedprotein probes. These probesare based on protein domains that specifically bind to H3K4me3 andH3K27me3 and bimolecular fluorescence complementation (BiFC) of asplitfluorescent protein for detection. In ordertovalidateand optimizeour probes, we are synthesizing chemically modifiednucleosomes carryingthe modifications as in vitro validation model. This will allowustooptimize probessensing propertiesthrough engineeringofefficientmarks binding domains. [1]Diederich, F.;Stang,P.J.; Tykwinski,R.R.Acetylene chemistry :chem- Finally, optimizedprobeswill allowdirect visualization of bivalent chroma- istry,biology,and material science;Wiley-VCH: Weinheim ;GreatBritain, tinwithinthe nuclei of living cells.Subsequently, thisapproach could be 2005. applied to develop arange of relevantmultivalent probestobeusedasa [2]Zheng,W.; Zheng, F.;Hong,Y.; Hu, L. Heteroat. Chem. 2012, 23,105 generaltool to studypost translational modifications. andreferences therein. [3]Frei, R.;Waser,J.Manuscript in preparation

Medicinal Chemistry Chemical Biology MC083 Medicinal Chemistry Chemical Biology MC084

Chromatinconformationaldynamics andheterochromatinprotein1 Visualization andCharacterization of theSplicing/Reverse-Splicing Equilibrium of singlepre-mRNAs with 3-coloursm-FRET spectroscopy Sinan Kilic, Beat Fierz MélodieHadzic,Danny Kowerko, Sebastian L.B. König,Erica Fiorini,Ro- ISIC,EPFL, 1015, Lausanne,Switzerland land K.O.Sigel

Chromatin,the complex of DNAand histonesineukaryoticorganisms, is UniversityofZürich, Winterthurerstrasse 190,8057Zürich, Switzerland involvedinthe regulation of cell specific retrieval andduplicationofgenetic information. Post-translationalmodifications of histonesserve as recruit- Group II introns arelargeself-splicing ribozymes that canbefound in bacte- ment platforms forchromatin effector proteins andtherebyexhibitaregula- ria andorganellargenes of plants, fungiand yeast. The splicedintron is able toryroleinthe orchestration of cellular processes like transcription, replica- to specificallyreinsertintoits original RNAsubstratebyaso-calledretro- tion andDNA-damagerepair. An important effector is heterochromatin pro- homing process, resulting in asplicing/reverse-splicing equilibrium.To tein 1(HP1),whichisassociatedwith geneticallysilenced chromatininpart catalyseboth reactions, the ribozymefolds intoseveral stable intermediates, throughthe recognition of histone H3 trimethylated at lysine 9(H3K9me3). minimizing the activationenergyofthe processes. The kinetics of the conformational transitions as well as the intron/exonbinding affinityplayan Currentknowledgeabout the higher-order organizationofnucleosomes into essentialrole in the catalytic activityastheygovernthe abilitytoreachthe fibers is limited, especially in complex with effectors, andlittleinformation native structure.Tocharacterise structurallyand kineticallythe splic- is available on the dynamics of the system.Our aimistoquantify the dy- ing/reverse-splicing equilibrium of the very large Sc.ai5γ group II intron by namics occurringwithinreconstituted chromatinfibers to understand the single molecule FRET spectroscopy(smFRET), we designhereasystem time-scales andthe distances associatedwith motions withinchromatin. labelled with threedifferentfluorophores.Suchalabelling scheme is essen- These investigations areextendedtostudythe interplaybetween the chro- tial to differentiatethe differentproducts andidentify the intron-exonbind- matin fiberand HP1. ing events. Furthermore, we encapsulateour single reactivesystems in phospholipid vesiclesinorder to allowthe productsand reactants to freely We designchromatin fibers with functionalities requiredfor biophysical diffuse inside the small reactors. By combiningthe free motion of the mole- characterizationbyemploying acombination of synthetic chemistry, en- cules to the presence of crowding agents andco-factors, we intendtoob- zyme-based andrecombinant methodologies. Histone H3 bearing the serve an in vivo-near behaviour, which remains unprecedented. K9me3markisproducedbyexpressed protein ligation. Modifiednucleo- somes areincorporated into chromatin fibers containing PCR-incorporated Financial support by the ERC,the SNFand the UniversityofZürichare fluorophores at definedpositions.This pavesthe wayfor detailed investiga- gratefullyacknowledged tions of localspatialand temporal fluctuations withinchromatin fibers as a function of the modificationstate andHP1 binding,usingfluorescence- [1]S.L.B.König, D. Kowerko, M. Khier, M. Hadzic, R.K.O.Sigel, submit- basedapproaches involving FRET andFCS. ted. [2]M.Steiner, K.S. Karunatilaka,R.K.O. Sigel, D. Rueda, Proc.Nat. Acad. Sci. USA 2008,105:13853–13858 540 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

MedicinalChemistryChemical Biology MC085 MedicinalChemistry Chemical Biology MC086

RNA foldingand splicing underphysiological conditions Evaluation of anew derivative of 5-aminolevulinic acid for vascular photodynamic therapy : in vitro stability study, in vivo fluorescence Susann Paulus,RolandK.O.Sigel spectroscopy and in vivo phototoxicity

InorganicChemistry, UniversityofZurich, Winterthurerstrasse 190, 8057 Sandrine Gay, Carla Martoccia, Georges Wagnières Zürich,Switzerland Swiss Fed. Inst. of Technology, EPFL, Station 6, CH-1015 Lausanne Oneofthe biggest challenges of structural biology nowadaysistoapplythe currentunderstanding about biological frameworks andinteractions gained Photodynamic therapy (PDT) based on photoactivable porphyrins (PAPs) is from in vitro studiestothe complexcellularenvironment. Thisisparticular- used to treat various dermatological conditions, including diseases associat- ly difficult forRNA, because of its functionaldiversityand its characteristic ed with vascular abnormalities. The production of PAPs can be induced in architecture. Ourresearchfocusesonaparticular processduringRNA pro- the skin by topical applicationoftheir precursor 5-aminolevulinic acid cessing calledsplicingand place emphasis on thecomplex foldingpathway (ALA). The penetration of ALA in the skin after topical application is of theinvolvedRNA undernativeconditions. Thesplicingmechanism is a suboptimal due to ALA’s hydrophilic nature. Moreover ALA is not selec- crucial stepduringRNA maturationand so farinvestigatedonlybyin vitro tively internalized by endothelial cells of the skin blood vessels. New ALA- studies. [1]Weestablished aseries of techniquestofluorescentlylabel the derivatives are developed by our group to optimize this selectivity. This RNAofagroup II intron from S. cerevisiae on specificregions whichwill approach is beneficial for treatment of conditions such as vascular malfor- be usedfor further in vivo studies(Figure). The foldingpathway is investi- mations or modifications (telangiectasia, port-wine stain, rosacea, etc). gatedbysingle-moleculeFörsterResonance Energy Transfer (smFRET) underphysiologicalconditions andthe obtainedresults are compared with We studied the stability of ALA-tyrosine ester in aqueous solution at in vitro data.[1, 2] Furthermore, bulkFRETexperiments in vitro are carried different storage conditions (pH and temperature) and at different times after out as afirst step towards in vivo studiesofthe group II intron splicingpro- solubilisation. We concluded that fresh solutions must used within 24h. In cess. addition they must be stored at low (4 °C) temperature to prevent hydrolysis of the ester bond, especially at physiological pH. We also studied in vivo the

intron fluorescence emission spectroscopy, the photostability, the photoproducts formation and the vascular phototoxicity of PAPs produced after atopical administration of ALA-tyrosine ester on the chick’s chorioallantoic exon 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ membrane model, and compared them with corresponding measurements FRET obtained with ALA. Our resuts suggest that the photobleaching constant of [1]M.Steiner, K.S. Karunatilaka,R.K.O.Sigel,D.Rueda, PNatlAcadSci PAPs is different with these two precursors (p <0.1). If confirmed, this small USA. 2008, 105,13853. difference could be explained by PAPs localization in different cells and/ or [2]M.Steiner, D. Rueda, R.K.O. Sigel, Angew Chem IntEdit. 2009, 48, cellular compartments, or by different relative proportions of the various 9739. fluorophores produced in the heme cycle.

Medicinal Chemistry Chemical Biology MC087 MedicinalChemistry Chemical Biology MC088

Heterochromatin Protein1association dynamics studied usingtotal CatalyticStrategies for Nucleic Acid Tagging internal reflection microscopy Kiril Tishinov, DennisG.Gillingham Marco Maggioni,Sinan Kilic, BeatFierz University of Basel, St. Johanns-Ring 19, 4056 Basel, Switzerland École Polytechnique FédéraledeLausanne, Route Cantonale,1015 Lau- sanne,Switzerland The field of nucleic acid research heavilyrelies on strategies for DNA and Post-translationalmodifications of histonetails play an important role in RNA tailoring in order to labeland expand their functionalpotential. The recruiting differentproteinstochromatin, including the chromatinarchitec- development of such methodologiesisakey step not only to the complete turalprotein HP1 (Heterochromatin Protein 1).This protein is considered understandingoftheir biological role, but also to directly perturbtheirfunc- one of the main actors in heterochromatin formation andinthe consequent tion for diagnostic and therapeuticuse. transcriptionalsilencing.However the dynamic of itsinteractionwith chro- matinhas not been yetstudied deeply.HP1 contains achromodomain responsiblefor recognitionofH3methylation at lysine 9(H3K9me3) anda chromoshadow domain that playsarole in the protein oligomerization.Us- ingsynthetic modifiedchromatin we aimatstudy the dynamics of HP1 association with chromatin, considering in particular how the abundanceof H3K9me3 markonhistonetails can affect the accumulationofthisprotein, itsaffinity andits residencetimeonthe chromatin. In our project we recon- stituteand immobilize chromatin fibers containingmultiple modifiednucleosomes. Using Total Internal ReflectionFluorescence Microscopy, apower- ful method to investigatesingle molecules dynamic, we want to determine the variation in the association anddissociationrates of HP1 andits resi- dencetimeonchromatin arrays as afunction of chromatin modification. In We haveshown thatavariety of nucleicacids canbecatalytically alkylated conclusion, our finalgoalistoset up abiophysical platformtomeasure the with rhodium(II) and copper(I)-carbenoids generated from -diazo carbonyl dynamicinteraction of differentchromatineffectors with modifiedchroma- compounds.The alkylation takes place viaanN-H insertionreaction selec- tinarrays. tively targeting the non-paired nucleobases in single-stranded DNA and RNA motifs [1]. The useofalkyne-functionalized -diazo carbonyl compounds allows tagging of thenucleic acid substrate withawiderangeof reporter groups such as fluorophores, and affinity tags via ‘click’ chemistry.

[1]K.Tishinov,K.Schmidt, D. Haeussinger, D. G. Gillingham, Angew. Chem. Int. Ed. 2012, 51,12000. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 541

MedicinalChemistryChemical Biology MC089 Medicinal Chemistry Chemical Biology MC090

Does alterationofadenine purinelevelsleadtoaloss-of-fitness AFRETSensor forGABAand Synthetic GABAB Receptor Ligands phenotype in Trypanosoma brucei ? Anastasiya Masharina,Luc Reymond, KeitaroUmezawaand KaiJohnsson Patricia Graven,Margherita Tambalo, LeonardoScapozza,RemoPerozzo Ecole Polytechnique FédéraledeLausanne(EPFL),Route Cantonale, 1015 Pharmaceutical Biochemistry Group, UniversityofGeneva&Lausanne, Lausanne,Switzerland Quai Ernest-Ansermet 30,1211 Geneva,Switzerland γ-Aminobutyric acid (GABA) is the main inhibitoryneurotransmitter in the mammalian nervous system. Despiteits importancethereisnosystem HumanAfrican Trypanosomiasis(HAT),also knownassleepingsickness, available that is able to measure GABA concentrations at the single cell is causedbythe parasites T. brucei rhodesiense and T. brucei gambiense. levelwith good spatio-temporalresolution. The presented workdescribes Availabletreatmentsfor this diseasestill remainunsatisfactory, needing the developmentofaFRETsensor for GABA basedonthe Snifit sensor parentaladministration, are toxic, or parasites have acquiredresistance. design [1]. The GABA-Snifit displaysaGABA-dependent fluorescence Thussafe andpotentnew drugsare needed to treat sleepingsickness. emission spectrum in therange of 500−700 nm that permits sensing mic- Trypanosoma brucei adenosine kinase (TbAK), an enzyme involvedinthe romolar to millimolar GABA concentrations on thesurfaceofliving mam- purinesalvage pathway, wasidentifiedbychemical proteomicstobethe malian cells [2].Additionally,GABA-Snifit can be used to characterizesyn- intracellulartargetof4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]-morpholine thetic GABAB receptor ligands such as agonists, antagonistsand allosteric (compound 1).Thismolecule exhibits very good antitrypanosomal activity modulatorsthatcan be used as therapeutics. with an IC50 of 1 µM, andbiochemical analysis revealed that compound 1is astrongactivator of TbAK1,2. The aimofthe currentproject is to understand themechanism of actionof compound 1. To this endchangesinadenine purinelevelsoftrypanosomes induced by compound 1were measured by meansofanion-pair HPLC/UV method. Several strains of Trypanosoma brucei were tested,e.g.1)wild type strain,2)atetracycline inducible ak overexpression strainW2, hypoth- esizingthatthe presence of compound 1issimilar to overexpression of TbAK), and3)atetracyclineinducible nullmutant ak overexpression strain D299V to provide insightintothe effect of non-physiological high levels of GABA-Snifit. (A)Designprinciple of the GABA-Snifit basedonthe TbAK. GABAB receptor andthe labeling tagsSNAP- andCLIP-tag. (B) Structure of the GABA-Snifit substrate containingaGABAB receptor antagonist. [1] S. Kuettel, A. Zambon, M. Kaiser,R.Brun, L. Scapozza,R.Perozzo,J Med Chem,50(2007)5833. [1] Brun, M. A.; Tan, K. T.; Nakata, E.; Hinner,M.J.; Johnsson, K. J. Am. [2] S. Kuettel, M. Mosimann, P. Maser, M. Kaiser,R.Brun, L. Scapozza,R. Chem.Soc. 2009,131, 5873−5884. Perozzo,PLoSNeglTropDis,3(2009)e506. [2]Masharina,A., Reymond, L.,Maurel, D., Umezawa, K., Johnsson, K. J. Am. Chem. Soc. 2012,134,19026-19034.

MedicinalChemistry Chemical Biology MC091 MedicinalChemistry Chemical Biology MC092

On Homotropic Cooperativity in CYP3A4 Methionine promotes tyrosine oxidation

Christian S. Müller1,2,Tim Knehans3,Patricia L. Bounds1,Ursula von Man- Frédéric Grandjean, Patricia L. Bounds, Thomas Nauser, dach2,Dmitri R. Davydov4,James R. Halpert4,AmedeoCaflisch3,and Wil- Willem H. Koppenol lem H. Koppenol1 Institute of Inorganic Chemistry, Deptartment of Chemistry and Applied 1D-CHAB, ETHZürich, 8093 Zürich, Switzerland1, 2Dept.ofObstetrics, Biosciences, Swiss Federal Institute of Technology, 8093 Zürich University HospitalZürich, 8091 Zürich, 3Dept.ofBiochemistry, University of 4 Zürich, 8057 Zürich, Skaggs School of Pharmacy, UCSD,LaJolla,CA, USA. α-Synuclein, the normal function of which is not yet well understood, may aggregate in response to cellular oxidative stress into Lewy bodies inside Cytochromes P450 (CYP) areasuperfamilythat cancatalyseavari- dopaminergetic neurons, which is ahallmark of Parkinson’s disease[1]. etyofreactions, e.g. the oxidationofnon-activated hydrocarbons.Weinves- We aim to study the oxidation chemistry of α-synuclein in real time using tigatedthe mechanism of interactions of cytochrome P450 3A4 (CYP3A4), the pulse radiolysis technique,which allows the generation of oxidative spe- the principlehuman drug-metabolizing enzyme, with carbamazepine (CBZ). cies, e.g. HO•,insolution within 1µs. We chose to investigate the reaction Amodel of aCYP3A4 complex with CBZwas subjected to molecu- of HO• with asynthetic tyrosine- and methionine-containing peptide corre- lar dynamics simulations in GROMACS [1]tofindpreferred binding loca- sponding to residues 124 –130 near the C-terminus. We observed unex- tions andorientations of the substrate, from whichwechose residues S119, pectedly rapid formation of tyrosyl radicals (Y[O•]): A370, andI369 as appropriatetargets forsite-directed mutagenesis.Mutants 6 –1 –1 AYEMPSE +HO•→AY[O•]EMPSE +HO k=2×10 M s were constructed andscreenedfor CBZ epoxide formation. Wild-type 2 CYP3A4 andI369 mutants exhibited Michaelis-Menten dependenceofthe We also investigated the reaction of HO• with synthetic peptide from β- reaction rate on CBZconcentration. S119 andA370 mutants induceda synuclein, corresponding to residues 118 –131, which contains 3tyrosine change in the kinetics profilestosigmoidalbehavior that suggests homo- and no methionine, where normal rates of tyrosine oxidation were found: 3 –1 –1 tropic cooperativityinthe enzyme-substrate interaction. SYEDPPQEEYQEYE +HO•→products k =5×10 M s Cooperativityinsubstrate interactions with CYP3A4 hasbeen re- We believe that the presence of anearby methionine residue promotes oxi- ported[2,3].Our findingsdemonstrate that the CYP3A4-CBZ interactionis dation of tyrosine, perhaps catalytically. Further experiments are being per- exquisitely sensitive to even modest structuralchangesinthe substrate bind- formed to elucidate the reaction mechanism. ing pocketand mayprovide useful insights regarding mechanismsofcoop- erativesubstrate interactionswithCYP3A4. [1] Spillantini MG, Schmidt ML, Lee VM, TrojanowskiJQ, Jakes R, Goe- dert M(1997) Alpha-synuclein in Lewy bodies. Nature 388 :839–840 [1]BerendsenHJC et al., ComputerPhysics Communications 1995,91: 43. [2]Davydov DR et al., Expert OpinDrug Metab Toxicol 2008,4:1523 542 Chimia 2013, 67, Nr. 7/8 medicinal chemistry and chemical biology

Medicinal Chemistry Chemical Biology MC093 Medicinal ChemistryChemicalBiology MC094

NMRspectroscopic investigationsofporphyrinicphotosensitizers with Neuritogenic Surfaces: Towards Solid Support Mediated nanoparticles as carrier systems Neuronal Organisation

Marianne Hädener, Martina Vermathen PatrickBurch,Fabian Schmid, Karl Gademann

UniversityofBerne,Freiestrasse 3, 3012 Bern, Switzerland University of Basel, Department of Chemistry, St. Johanns-Ring 19 CH-4056 Basel, Switzerland Photodynamic therapy(PDT) is acancer treatment modality, which is based on the selective uptake of aphotosensitizer (PS)bycancertissue. Upon ex- This work aims to provide new solutions for two different challenges: 1) citation with redlight thephotosensitizer mayreact with molecular oxygen implant based neuronal network reconstruction for spinal cord injuries and 3 1 [1,2] ( O2)via energy transfertogeneratesingletoxygen(O2), whichisknown 2) improvement of connections between neurons and electronic sensors. to give rise to oxidative reactions triggering celldeath. Many PS molecules, With our modular and straightforward method, we were able to coat glass however, arehydrophobicand have astrongtendency to aggregateinaque- slides with structurally diverse neuritogenic natural product analogues ous solution decreasing their photosensitizing andbiological effectiveness. (NNPA) to induce neuronal differentiation in rat pheochromocytoma cells.[3] The use of nanoparticlesasPScarrierspresents apromising approach for The robustness of this approach is underlined by the fact that reuse of al- enhancingPDT efficacy [1]. ready in vitro applied slides is possible with maintained activity. If anon- Aprevious studywas aimedatinvestigating theaggregationbehavior of coated slide is placed in close proximity to aslide coated with NNPA within severalporphyrinic compounds, which arepromising PS in PDT, by NMR the same cell media, no enhanced cell differentiation activity could be de- spectroscopic methods.Throughobservation of NMR resonancebroadening tected on its surface. This opens the possibility of neuronal organization on andporphyrin ringcurrent inducedshifts,the extent of aggregationinphos- solid support. phate buffered saline could successfullybeassessed [2]. Our presentworkis concentrated on the search for an appropriate nanocarrier system for selected porphyrins. Carrier systems such as poly(DL-lactide-co-glycolide) (PLGA),polyvinylpyrrolidone (PVP)orβ-cyclodextrin arecurrentlyprobed by NMR spectroscopic methods for their abilitytoforminclusion complexes with porphyrinic compounds. Cell uptake is probedusing cultured HeLa cells andfluorescencebased methods. Since aminoacidresidues have been found to enhancethe biologicaleffects of porphyriniccompounds [3], our focus is on aminoacidconjugates of the dihydroporphyrin chlorin e6. Coated Control

[1]Bechet D. et al, Trends Biotechnol. 2008, 26,612. [1] R. van den Brand, et al, Science 2012, 336,1182. [2]Vermathen M. et al, papersubmitted forpublication. [2] B. Eversmann, et al, Solid-State Circuits 2003, 38,2306. [3]Jinadasa,R.G.W.etal, J. Med. Chem. 2011, 54,7464. [3] P. Burch et al, Chem. Eur. J. 2013, 19,2589.

MedicinalChemistry Chemical Biology MC095 MedicinalChemistry Chemical Biology MC096

ABioorthogonalInVivoLigationReaction and its Application for Non- Convergent preparation of unsaturated acyclicnucleosidephosphonate Invasive Bioluminescent Imaging of Protease Activity in Living Mice prodrugs

AurélienGodinat, Hyo Min Park, StephenC.Miller, Ke Chen, Douglas UgoPradère,VincentRoy,AurélienMontagu, JanBalzarini, Robert Hanahan, Laura E. Sanman, Matthew Bgyo, Allen Yu, Gennady F. Nikitin, Snoeck,Graciela Andrei, LuigiAgrofoglio Andreas Stahl, Elena A. Dubikovskaya* Institut de Chimie Organique et Analytique,UMR CNRS 7311, Université Swiss Federal Institute of Technology of Lausanne, CH-1015 Lausanne, d’Orléans, BP 6759 45067Orléans, France and Rega Institutefor Medical Switzerland Research,K.U.Leuven, 3000 Leuven,Belgium

Thediscovery of biocompatiblereactions had atremendous impacton Acyclicnucleoside phosphonates (ANPs) is akey class of antiviralagent chemical biology, allowing the study of numerous biologicalprocesses di- with compoundsofprimary importanceagainst both DNAand retrovirus rectlyincomplexsystems. Here we report that D-cysteineand 2- infections.Because of theioniccharacter of their phosphonate moiety, the cyanobenzothiazolescan selectively react with each other in vivo to gener- ANPs have to be functionalizedasprodrugsbyinsertion of biolabilepro- ate aluciferin substrate forfirefly luciferase. First, the production of alucif- tecting groups. Our grouprecentlyreportedanewtypeofunsaturated ANPs erinsubstrate can be visualized in alivemouse by bioluminescence imaging bearinga(E)-4-phosphono-but-2’-en-1’-yl sugarlikebackbone.Suchmodi- andfurthermore allowsinterrogation of targetedtissues using a″caged″ fiedmoietyproved to be well mimickingthe conformation of theC1-O4- luciferinapproach. We therefore appliedthis reaction to the real-timenon- C4-C5atomsfromnatural substrate by X-raystudies with thymidylateki- invasive imaging of apoptosis associated with caspase 3/7. Caspase- nase.Direct conversion of nucleoside phosphonates intotheir prodrug form dependent release of free D-cysteinefrom the caspase 3/7 peptide substrate is particularly cumbersome andoftenlow-yielding.Inordertoopenaneasy Asp-Glu-Val-Asp-d-Cys allowed selective reactionwith 6-amino-2- accesstoprodrugsand assessthe antiviralpotency of our ANPs,wedevel- cyanobenzothiazole in vivo to form 6-amino-D-luciferin with subsequent opedconvergentsynthetic strategies to allowsimultaneous insertion of the lightemissionfrom luciferase. Moreover, the split luciferin approach ena- phosphonate moietyand the biolabileprotectinggroups.Cross metathesis bles themodular construction of bioluminogenic sensors, where either or (CM) andMitsunobu reaction (MR)wereemployed for the preparation of a both reactionpartners could be caged to report on multiple biological small library of unsaturated ANPprodrugsbearingcarbonyloxymethyl events,suggestingfurther applications forbothbioluminescence andspecif- (POM, POC) andalkoxyalkyl (HDP) biolabile protecting groups [1,2]. ic molecular targetinginvivo.

[1]Agrofoglio,L.; Roy, V.; Pradère, U. PCT WO2010146127A1 [2]Agrofoglio,L.; Roy, V.; Pradère, U.; Balzarini, J.; Snoeck,R.; An- drei, G. PCT WO2012034719A1. medicinal chemistry and chemical biology Chimia 2013, 67, Nr. 7/8 543

MedicinalChemistry Chemical Biology MC097 MedicinalChemistry &ChemicalBiology MC098

Efficient conversion of nucleosideanalogs to 5’-methylene-phosphonate Newtherapeuticoptions for thetreatment of life-threatening prodrug derivatives parasiticdiseases

UgoPradère,FranckAmblard, StevenJ.Coats, Raymond F. Schinazi T. Küster,‡ N. Lense,† F. Barna,‡ A. Hemphill,‡ M. K. Kindermann,† J. W. Heinicke,† C. A. Vock† Center for AIDS Research,LaboratoryofBiochemical Pharmacology, DepartmentofPediatrics, EmoryUniversitySchoolofMedicine and † Institute of Biochemistry –InorganicChemistry, Felix-Hausdorff-Str. 4, VeteransAffairs Medical Center,Decatur, Georgia30033, D-17487 Greifswald,Germany; United States, andRFS Pharma,LLC,1860 Montreal Road,Tucker, Georgia30084, United States ‡ InstituteofParasitology, VetsuisseFaculty,University of Berne, Länggass-Str. 122, CH-3012 Berne,Switzerland Nucleoside phosphonates that contain aphosphonatebioisostereofanatural 5’-phosphate group have received considerable synthetic andmedicinal Echinococcusmultilocularis (fox tapeworm)isconsidered to be themost chemistryattention overthe years. However,the ioniccharacter of these dangerous human pathogenicparasiteoccurring in CentralEurope.Infec- phosphonicacidderivativesisgenerally an obstacle for cellular penetration, tions with theparasiteare quite rare (0.5 –2.5 cases/100.000 inhabitants); whichoftentranslatestoalack of biological activity for these nucleoside therapyofchoice is surgical removalofinfectedtissues.However,alterna- analogs. In order to maskthe negative charges, phosphonate prodrugsbear- tivetreatmentsfor inoperablepatients areurgently needed,because thereare ing biolabileprotectinggroup suchaspivaloyloxymethylgroups (POM) onlyveryfew parasitostatic andnoparasitocidal chemotherapeutic options have been developed. Because the preparationofsuchphosphonatepro- available for infected persons.Progressionofthe untreated diseaseleadsto drugsisgenerally tedious andlow yielding, we developedageneraland almostcertain deathwithin approx. 15 –20yearsafter infection. efficient method to prepare nucleoside 5’-methylenebis(POM)-phosphonate During thelastapprox. 25 years, ruthenium complexes have attractedin- prodrugsthrough the condensation of tetra(POM)-bisphosphonate with pro- creasing interestaspotential therapeutics for thetreatment of cancer.[1,2] tected5’-aldehydenucleosides via amodifiedHorner-Wadsworth-Emmons We will present our recentpromisingdata,[3] demonstratinghydrolytically reaction. Ourstrategy wasapplied to the 2’-deoxy-2’--fluoro-2’--C- stable ruthenium complexes to be apossibletherapeutic optionalsofor the methyl nucleoside series whose most advanced member, PSI-7977 (GS- treatmentofalveolarechinococcosis,which is thenameofthe diseaseori- 7977) is currentlyinphaseIII clinicaltrials as an anti-HCV agent. ginating from fox tapeworm infections.Inaddition, some newresults from our research on antiparasitic agents will be shown.

[1]W.H.Ang, P. J. Dyson, Eur. J. Inorg. Chem. 2006,4003. [2]A.Bergamo,C.Gaiddon, J. H. M. Schellens, J. H. Beijnen, G. Sava, J. Inorg. Biochem. 2012, 106,90. [3]T.Küster, N. Lense,F.Barna,A.Hemphill, M. K. Kindermann, J. W. Heinicke, C. A. Vock, J. Med. Chem. 2012, 55,4178.

MedicinalChemistryChemical Biology MC099 MedicinalChemistryChemical Biology MC100

RNA-Metal ions interactionsbysmFRET SPRasatool forthe detection of newRNA bindingmotifs

MokraneKhier,Sebastian L.B. König, DannyKowerko, Roland K.O. Sigel Moritz Stoltz1,JulianZagalak1,HarryTobwin1,Erich Michel2,Frédéric Allain2, Jonathan Hall1 Institute of InorganicChemistry, UniversityofZurich, Winterthurerstrasse 190, CH-8057, Zurich,Switzerland 1Institute of Pharmaceutical Sciences,Department of Chemistryand Ap- pliedBiosciences,ETH Zurich,Zurich, Switzerland. RNAfolding andactivitydepends vastlyonthe nature of metalions 2Institute forMolecularBiology andBiophysics, ETH Zurich,Zurich, Swit- presentinsolution. ThepotentialofRNA for medical andbiotechnological zerland. applications growsdaily,and understanding RNA-metal ions interactions andhow thelater maytune theactivityofthe former become thus of great RNAs play important rolesinmanybiologicalprocesses, includingdisease interest. In our study, we apply"Single Molecule FRET" to characterize the mechanisms.Amajorchallenge in RNAbiology is understanding theirreg- interactionofanRNA hairpinloop, knownas"EBS1"(Exon Binding Site ulationbyRNA binding proteins (RBPs).Therefore it is important to detect 1),with its RNAorDNA cognate. Ourresults reveal that thesetwo interac- newinteractions andnew potentialtargets. tions differ slightlyinconformation, whichisingood agreementwith NMR The RBFOXfamilyisanimportant alternativesplicingfactor in humans, studiesperformedonthe same system[1].Inaddition, kinetic analysis responsiblefor thehighdiversityofprotein isoforms. Previously, the showsthatthe EBS1-RNA interactiondisplays kinetic heterogeneityinthe SELEXmethod (Systematic EvolutionofLigands by ExponentialEnrich- presence of Mg++,While theEBS1-DNAinteractionwas homogenous in the ment)revealedthatRBFOXbinds to astrictconsensus sequenceGCAUG presence of K+ andMg++.The origin of theheterogeneityseenwith EBS1- [1][2],which is highly enriched in proximitytoalternativesplicingsites.[3] RNAinteractioninthe presence of Mg++ couldbeexplained by aspecific requirementfor divalent metalions, knowingthatprevious works hadpoint We have developedanewSPR-basedmethod to identifyRNA sequences out twopotentialbinding sitesfor divalent metalion in EBS1 hairpinloop recognizedbyRBPsusing libraries of RNAs.Duringthe investigation, we [2]. Ourresults supportthe involvement of one of thetwo specificmetal identifiednew RNAbinding motifsfor RBFOXwith high nM affinities. ions in determiningthe correctphosphodiesterbondcleaved by thegroup II These newmotifswereshowntobefunctionalincellularpull-down assays intron, an activeRNA molecule to whichbelongEBS1hairpin[3]. whichindicated anew functionofRBFOX independent of alternativesplic- ing. Financialsupportbythe SwissNationalScienceFoundationand theUniversityof Zurichand aregratefullyacknowledged. [1]Jin,Y.etal.,EMBO. 2000, 22, 905-12. [2]Ponthier, J. L. et al., J. Biol.Chem. 2006, 281, 12468-74. [1]Skilandat, M.,and Sigel, R.K.O., submittedfor publication. [3]Yeo GW et al., PLoS Genet. 2007, 3, e85. [2]Kruschel, D.,Sigel,R.K.O., 2008, J. Inorg. Biochem.102: 2147-2154. [3]Su, L.J.,Qin,P.Z., Michels, W.J.,and Pyle,A.M., 2001, J. Mol. Biol. 306: 655-68.