ANALYTICAL CHEMISTRY/MEDICINAL CHEMISTRY557676 CHIMIA 2008, 62,No. 7/8

Analytical Chemistry 73 Analytical Chemistry 74

IndicatorDisplacement Assays as MolecularTimers Real-time, on-linemonitoring of organic chemical reactions using ex- tractive electrosprayionization tandem mass spectroscopy FriederikeZaubitzer,Alexei Pozdnoukhov, Kay Severin* Liang Zhu1;Gerardo Gamez1;Huanwen Chen2;Haoxi Huang1;Konstantin 1 1 *Institut desSciences et Ingénierie Chimiques, EcolePolytechnique Chingin ;Renato Zenobi Fédérale de Lausanne (EPFL), CH-1015 Lausanne,Switzerland 1ETHZurich, Zurich,Switzerland; 2College of Chemistry, JilinUniversity, Indicatordisplacementsassays (IDAs) have emerged as powerful analytical Changchun, China

tools. They arebased on dyes which compete with analytes for binding to 1 synthetic receptors.Sofar, IDAshave primarilybeen used to determine the Extractive electrospray ionization mass spectrometry (EESI-MS) for identity and/orthe quantity of certain analytes. real-time, on-line monitoring of organicchemicalreactions wasdemon- We show that amulticomponent IDAcan also be employed to obtain infor- stratedfor awell established pharmaceutical process reaction (one-step mation about the history of chemical inputs. Asimple mixture of three Michaeladdition reactionwith phenylethylamine (PEA) and acrylonitrile in ethanol)2 andawidely used acetylation reactioninthe presence of anuc- commercially available dyes and the organometallic complex [(Cp*RhCl2)2] 3 is employed to timethe addition of ADP and ATP with good resolution [1]. leophilic catalyst, 4-DMAP (multiple-step acetylation reaction of benzyl alcohol with acetic anhydride catalyzed by 4-DMAP in dichloromethane). EESI-MS,with acommerical quadrupole-time-of-flight (Q-TOF) mass spectrometer,provides real-timeinformation that allows determining the optimumtimefor terminating the reaction based on the relative intensities of the precursors and products. In addition, analysis viaEESI-MS permits on-line validation of proposedreaction transients,which appears during the catalytic pathway of 4-DMAP,relying on tandem MS. The relatively simple setup allows this method to be implemented on anytype of MS instrument with ESI /APCI interface. The EESI-MS features an instant response (<0.2s) and does not require sample pre-treatment, making it apowerful and con- venient toolfor on-line characterization and full controlofchemicaland Fig.1.: The ADP and ATP addition timesdeterminedbythe molecular timer in pharmaceutical reactions, resulting in maximized product yield and mini- comparison with the real addition times for 12 test samples. The predictions are mized environmental costs. shown as filled symbols (5 measurements each) and the real addition timed are [1]H.W.Chen, A. Venter and R. G. Cooks, Chem.Commun., 2006, indicatedbyempty symbols. 2042-2044 [2]R.Clinton, C. S. Creaser and D. Bryant, Anal.Chim. Acta., 2005,539, The signal of the timer is read by UV/Vis spectroscopy and the data is ana- 133-140. lyzedvia amultivariate analysis. [3]G.Hofle,W.Steglich and H. Vorbruggen, Angew.Chem., Int. Ed. Engl., 1978,17, 569-583. [1]A.Buryak, F. Zaubitzer,A.Pozdnoukhov, K. Severin, submitted.

Analytical Chemistry 75 Medicinal Chemistry 76

Calcilytics –ANew Treatment for Established Osteoporosis Total Synthesis of the Resorcylic Lactone-based Leo Widler KinaseInhibitorL-783277 Novartis InstitutesofBioMedicalResearch, WKL-136.3.93, CH-4002Basel, Switzerland Tatjana Hofmann, Karl-Heinz Altmann Osteoporosis is characterized by low bone mass andmicro-architectural deteriorationofbone tissue thatleads to fragility and increased risk of fractures. InstituteofPharmaceutical Sciences,ETH Zurich, Wolfgang-Pauli-Str. 10, Traditional therapiesfor osteoporosis inhibit bone resorption andprevent further 8093 Zurich, Switzerland bone loss.However,asmany osteoporosis patients have alreadylost asubstantial amount of bone at the time of diagnosis, thereisaneed for agents that stimulate Kinaseshave emerged as important drug targets in cancer and inflammatory newboneformation. The only anabolic treatment forosteoporosis, approvedfor diseaseand several low-molecular-weight kinase inhibitors have now been the US andEUmarkets, is Forteo®/Forsteo® (Teriparatide, the 1-34 fragment of introduced into clinicalpractice.[1] The natural product L-783277 (1)be- parathyroidhormone (PTH))which causes asignificant increase in bone mass and longs to the family of resorcylic acid lactones (RALs),which includes com- reduces vertebral fracture risk substantially. This peptide must be administered by pounds such as zearalenone, C292 (LL-Z1640-2), hypothemycin, or radici- daily subcutaneousinjection and the therapy is costly. An orally active, low col, and which exhibit adiverserangeofbiological activities.[2] L-783277 molecularweight compound with thesameefficacywould be ahighly attractive (1)isapotent inhibitor of the Ser/Thr kinase MEK.[3]Wehave accom- alternative for the patient. plished the first totalsynthesis of macrolactone 1,which is based on the Insteadofapplying exogenous PTH, mobilization of endogenous stores of the consecutiveassembly of the key fragments A, B,and C.[4] The develop- ment of an efficient enantioselective synthesis of 1 and amore detailed hormone can be envisaged. PTH is storedinrelatively large amounts in parathyroid characterizationofits biological effects are the primarygoals of this re- cells and its secretion is controlled by acalcium-sensing receptor (PCaR) located search project. This presentation will discuss the details of the synthesis of 1 on the cell surface. Antagonists of PCaR (calcilytics) mimic astate of and the preparation of anumber of analogs. Preliminary data on the in vitro hypocalcemiaand stimulate PTH release to the blood stream. biologically activity of these compounds will also be presented. The starting point for the Novartis calcilyticsproject was aproprietary structure

found in aHTS screenusing afunctionalassay in the FLIPR format based on OTBS OTBS recombinant human PCaR. Optimization of the series resulted in an increase in in OH O OH O OH O vitro potencybyafactor of >100. First oral applicationsinrats with these highly C OR + O OR O potent calcilytics were rather disappointing with regard to PK/PD parameters. The OH presentation will focus in thesecond part on how these limitations were overcome. O Br O OH O O O O O O OH PK/PD datainrats anddogs will be shown that mark thebestofour derivatives A B D 1 attractive fordevelopment as oral calcilytics.There is excellent correlation of drug R=CH2CH2Si(CH3)3 L-783277 exposure andPTH release. High levels of PTH are reached in plasma within minutesinboth species after p.o.. application whichrevert to baseline in about 1-2 [1] Krause, D. S. and Van Etten, R. H. N. Engl. J. Med. 2005,353, 172-187. hours. This profile is aprerequisite for bone anabolic action, sinceitiswellknown [2] Winssinger, N. and Barluenga, S. Chem.Commun. 2007, 1,22-36. that persistently elevated levels of PTH stimulatenot only osteoblasts (bone [3] Zhao, A.; Lee, S. H.; Mojena, M.; Jenkins, R. G.; Patrick, D. R.; Huber, forming cells)but also osteoclasts, theboneresorbing cells. The net result is H. E.; Goetz, M. A.; Hensens, O.D.; Zink, D. L.; Vilella, D.; Dom- increased bone turnover rather than thedesired gain in bone mineral density browski, A. W.;Lingham, R. B.; Huang, L. J. Antibiot. 1999,52(12), (BMD). 1086-1094 [4] Hofmann,T.and Altmann, K.-H. Synlett 2008 in press MEDICINAL CHEMISTRY557777

doi:10.2533/chimia.2008.577 CHIMIA 2008, 62,No. 7/8

Medicinal Chemistry 77 Medicinal Chemistry 78 NMR Derived Binding Epitopes of Myelin-Associated-Glycoprotein TheFirst Nonpeptidic, SmallMolecule, Highly Selective Somatostatin 5 Antagonists Receptor Antagonists: AChemogenomics Approach

BrianCutting1,DanielStrasser1,Sachin Shelke1,GanpanGao1,Oliver Rainer E. Martin,Luke G. Green, Wolfgang Guba,NicoleKratochwiland Schwardt1,Soerge Kelm2 andBeatErnst1,* AndreasChrist

1UniversityofBasel, Klingelbergstrasse50, CH-4056Basel, Switzerland Medicinal Chemistry III, Discovery Chemistry, F. Hoffmann-La RocheLtd, 2University of Bremen, Leobenerstr. NW2B2, D-28359 Bremen, Germany 4070 Basel, Switzerland

Following injury to the adult CNS, regeneration is inhibited by asignal Somatostatin (SST) or somatotropin release-inhibiting factor (SRIF) is a transductionpathway initiatedbythree proteins, Nogo-A, Omgp andMAG, peptidehormonedistributed throughout the body exhibiting multiple bio- interacting with the Nogo receptor.Ithas been shown that blockingthe logical functions, including antisecretagogue, antiproliferative and neuro- bindingsite of MAG diminishes theinhibition of regeneration [1]. transmitteractivities. SST acts via five distinct G-proteincoupled receptors (SST1-5), which all have been cloned and characterized. Particularly, SST Herein, we provide an interpretation of the mode of binding of related MAG acting via SST5 receptors has beenfound to activate and up-regulate antagonists. This interpretation is achieved through asynergistic use of STD NMDA receptor functionand to control hormonal secretions (e.g.,growth NMR [2] and antagonist affinities. Themethod allows thecharacterization hormone). So far no small-molecule, selective SST5 receptor antagonists of the mode of binding, i.e. the contribution of differentparts of the anta- have been described. Therefore, receptorspecific antagonists are of high gonists to the overall affinity.The bindingwas determined through three interest as toolstostudy the diverse pharmacology of SST. In order to iden- methods, surface plasmon resonance, Hapten inhibition assays and competi- tify chemicalentry points achemogenomics strategy based on screening tive NMR binding experiments. The potential of the method to complement knownGPCRligandsrelatedtoSSTR5(via sequence similarity analysis of medicinal chemistry SAR analyseswill be described. One major advantage the transmembrane consensusdrugbinding site) wasemployed. From vari- is that the structure of thereceptor is not required. ous hits, thewell-known H1 ligand Astemizole was chosen as the most promising starting point and successfully transformed into the first small [1]A.A.Vyas, O. Blixt, J.C. Paulson, R.L. Schnaar, J. Bio. Chem. 2005, molecule SST5 receptorantagonists showing nanomolar binding affinity, 280,16305. high selectivity and promising physicochemical properties.[1] [2] M. Mayer, B. Meyer, Angew. Chem. Int. Ed. 1999, 38,1784.

H hSST5 Ki =13nM O N N N hSST1 Ki =1.75 µM

O S hSST2 Ki >10µM O H N 2 O O hSST3 Ki >10µM NH 2 hH1 Ki =2.64 µM

[1] R. E. Martin et al., J. Med. Chem. 2007, 50,6291.

Medicinal Chemistry 79 Medicinal Chemistry 80 Defining PET-like properties to support radiotracer discovery and their Efficient ligand affinity calculationsusing use in drug development novel computational methods

Emmanuelle Briard,Yves P. Auberson Holmfridur B. Thorsteinsdottir,Michael Podvinec, Torsten Schwede, Markus Meuwly Novartis Institutes forBioMedicalResearch, Global Discovery Chemistry, CH-4002Basel, Switzerland University of Basel, Klingelbergstrasse 50/70, CH-4056 Basel, Switzerland

The development of radiotracers for in vivo Positron EmissionTomography (PET)isquite expensive and still essentially progressing by trial and errors. Theidentification of anumber of factors that can predictthe potential of a In recent yearsvirtual screening tools are becoming increasingly important radiotracer candidate before radiosynthesis would therefore provide substan- for drug discovery.However,the scoring functions many of these methods tialsavings in timeand efforts. We have surveyed the literature, compiled use often lack predicting power. Additionally the poses generated by the andcompleted aset of properties describing clinically used radiotracers as dockingalgorithmsare often sub-optimal.Previously aMM-GBSA method well as unsuccessful candidates using assays and methods routinely used in to calculatebinding free energies has been validated using the well characte- drug discoveryprograms. Among others, we will discuss the impact on PET rized HIV-1protease and16known ligandsasatest system [1].Here, we imagingofphysicochemical and permeation properties,pharmacokinetics extend this method to distinguish correct from incorrectposes and secondly and nonspecific binding. to improve the correct poses generatedfrom virtual screening methods by molecular dynamicssimulations. Furthermore MM-GBSA is used as a Finally some of the main applications of PET imaging in drug development more advanced scoringtechnique to obtain amore reliable rankingofthe will be exemplified: 1) The use of aspecific radiotracer to optimize the the- ligands. rapeutic dose and achieve efficacy, while minimizing the side effects. 2) The use of radiolabeled drug to confirm brain distribution and to identify One of the main obstacles in the routine application of molecular dynamics relationship among dose, plasma concentration and brain concentration. 3) simulations in large-scale virtualscreening projects is the calculation time The investigation of pharmacodynamic effects using PET imaging, correlat- thatisrequired. GRID computingisapossible solution to this problem. ing receptorbinding and regional effects on physiological parameters. This work aimstodevelop amethod forparallelizationand showthat paral- lelized molecular dynamics is able to reproduce bindingfree energies in a comparable way to classical molecular dynamicssimulations and can there- fore be applied to virtual screening projects.

[1] HB. Thorsteinsdottir, T. Schwede, V. Zoete, M. Meuwly, Proteins. 2006,65, 407 MEDICINAL CHEMISTRY557878 CHIMIA 2008, 62,No. 7/8

Medicinal Chemistry 81 MedicinalChemistry 82

Histamine-3 Receptor Inverse Agonists for the Treatment of Obesity: Identification and Characterization of HighlyPotent Activators of the Validation of the Target and Identification of Novel Series NR4A2 Pathway -aNovel Approach to TreatParkinson’s Disease? Jean-MarcPlancher,Pascale David Pierson, Christian Freichel, Silvia Gatti, S. Hintermann 1,M.Chiesi 1,M.Tintelnot-Blomley 1,U.von Krosigk1,B. Cornelia Hertel, Jörg Huwyler, Peter Mohr, Toshito Nakagawa, Matthias Hengerer 1,W.Le2,S.Ametamey 3,M.Honer 3 Nettekoven, Susanne Raab, Hans Richter, Olivier Roche, Rosa María Rodríguez Sarmiento, Monique Schmitt,Franz Schuler, Tadakatsu Takaha- 1 Novartis Institutes for BioMedical Research,GDC,4002 Basel, shi, Sven Taylor, Christoph Ullmer, Ruby Wiegand Switzerland; 2 BaylorCollege of Medicine, Houston, Texas77030, USA; 3 Centerfor Radiopharmaceutical Science of ETH,PSI and USZ, 8093 F. Hoffmann-La Roche Ltd; Pharmaceuticals Division; B092/4.18C; CH- Zürich, Switzerland 4070 Basel; Switzerland The nuclear receptor NR4A2 (Nurr1), amemberofthe family of orphan Obesity is amajor risk factor in the development of conditions such as nuclearhormone receptors, plays acritical role in thedifferentiation and hypertension, hyperglycemia, dyslipidemia, coronary artery disease and survival of midbrain neurons. Nurr1–/– mice have no differentiated dopami- cancer.Several pieces of evidence, including data in primates, have hig- nergic neurons in the substantia nigra and striataldopamine(DA)levels are hlightedthe beneficial effects of H Rinverse agonists in the regulation of 3 reduced by 98% leadingtodeath shortly after birth. In heterozygous Nurr1- food intake and body weight. deficient mice, striatal DA levels and locomotor activity progressively de- Apharmacophore model, based on selected published H Rligands, and va- 3 creasewith age and the vulnerability of midbrain DA neurons to neurotox- lidatedbyprevious investigations[1],was used to identify several scaffolds. ins is increased [1,2]. Nurr1 was shown to be involved in theregulation of Among those,the indole-2-carboxamide appearedtobeofgreat interest as a genes coding for e.g. aromatic aminoacid decarboxylase, tyrosine hydrox- novel seriesofHRinverse agonists. Extensive structure activity relation- 3 ylaseand the DA transporter[3]. These observations triggered the hypothe- ship investigations, rewarded by the identification of several compounds sis thatactivating the remaining Nurr1 proteinindegenerating DA neurons reversing (R)-α-methyl-histamine-induced water intake increase, and reduc- might delay the onset of Parkinsonian symptoms. ing food intake in rats, are presented. Biochemical, pharmacokinetic and Recently, highly potent, brain penetrable activators of the Nurr1 signaling pharmacodynamic characteristics are discussed. pathway were disclosed [4]. The hit identification and SAR studies leading to these isoxazolo-pyridinones as well as their profiling in vitro and in vivo O O R1 in differentmodels will be discussed. N NR3 [1]Zetterström RH, Solomin L, Jansson Letal. Science 1997, 276,248. R2 [2]LeWD, Conneely OM,HeYetal. J. Neurochem. 1999, 73,2218. [1] Roche, O.; Rodriguez Sarmiento, R. M. Bio. Med. Chem. Lett. 2007, 17,3670-3675; Netteko- [3] Hermanson E, Joseph B, Castro Detal. Exp. Cell Res. 2003, 288,324. ven, M.; Roche, O.; Rodriguez Sarmiento R. M. in press [4] Hintermann S, Chiesi M, vonKrosigk U, et al. Bioorg, Med. Chem. Lett. 2007, 17,193.

Medicinal Chemistry 83 Medicinal Chemistry 84

Identification and Characterization of High-affinityMyelin-associated Inspirationfrom nature_EMD! Glycoprotein (MAG) Antagonists Michel Dard1,Stephanie Lemoult2,Aart Molenberg1,CorinnaMauth1, Ruzica Ranevski1* XiaohuaJiang,Stefanie Mesch, Oliver Schwardt, Beat Ernst 1 Institut Straumann AG, Peter Merian Weg 12, Basel, Switzerland Insitute of Molecular Pharmacy, University of Basel, Klingerbergst. 50, 2 BIORA AB,Acompany of the Straumann Group, Medeon Science Park, 4050 Basel, Switzerland Malmö, Sweden

Enamel Matrix Derivative (EMD) is the active ingredient in Emdogain® Followinganinjury of neurons in the central nervous system (CNS) of adult (BIORA AB, Straumann, Malmö, Sweden), acommercially used product mammalians, functional repair is not possible. The two main obstaclesto for regeneration of periodontal tissue. The main component of the derivative regeneration are inhibitor proteins located in the myelin sheaths and the is amelogenin, which is secreted by ameloblasts into the enamel formation of glial scars. MAG, amember of the Siglecs family (sialic acid- compartment. Extraction of EMD for the Emdogain® preparation is carried binding immunoglobulin-likelectins) is one of these inhibitor proteins [1].It out from the molar teeth of 6-month-old pigs. At this time, amelogenin was shown that blocking the inhibitoryactivity of MAG leadstoneuron corresponds to approximately 90% of total tissue protein, and the stages of regeneration [2].Inprevious studies, by employingthe Fragment-based In secretion and early maturation in enamel formation have been reached. Situ Combinatorial Approach, we have successfully identified the highaf- The effects of EMD in the periodontal area on cementum, periodontal finity antagonist 1 [3]. ligament, bone formation, and wound healing have been studied quite Herein,wereport the optimization of lead 1 and the evaluation of the new extensively over the years [1-23]. Recently, researchers have started to look antagonists by surface plasmon resonance experiments. more at the bioactive parts of EMD (sometimes also referred to as EMP; Enamel Matrix Proteins) [20-22]. Even though EMD mainly consists of amelogenin (20 kDa) [11,16,24-26], it cannot be ruled out that other proteins besides amelogenin have biological activity. Therefore, in order to NH OH understand the overall biological activity of EMD, it is essential to COONa NO2 O OH investigate its components. AcHN O O N HO 1 N N N H

[1] Filbin, M. T. Nat. Rev. Neurosci. 2003, 4,703. [2] Vyas, A. A., Blixt, O., Paulson, J. C., and Schnaar, R. L. J. Bio. Chem., 2005, 280,16305. [3] Ernst, B., Cutting, B.,Shelke, S. PCT WO 2007/105094 A1. MEDICINAL CHEMISTRY557979 CHIMIA 2008, 62,No. 7/8

Medicinal Chemistry 85 Medicinal Chemistry 86

DiscoveryofSelective5-HT5A Antagonists Inhibition and Dispersion of Pseudomonas Aeurginosa Biofilms by Glycopeptide Dendrimers Sabine Kolczewski,Rodolfo Gasser, NicoleKratochwil, Lucinda Steward, Claus Riemer,Olivier Roche, Jürgen Wichmann, Thomas Woltering Emma Johansson1, Shanika Crusz2,Steve Diggle2,Miguel Camara2,Paul Williams2,Tamis Darbre1,Jean-Louis Reymond1* F. Hoffmann-La Roche Ltd PharmaceuticalsDivision 1Department of Chemistry and Biochemistry, University of Berne, Frei- DiscoveryChemistry estrasse 3, 3012 Berne, Switzerland, 2Institute of Infection, Immunity and CH-4070 Basel Inflamation, UniversityofNottingham,Nottingham NG7 2RD, UK Switzerland

Selective quinoline5-HT5A antagonists based on 2-aminoquinoline deriva- tives were discovered with the help of site directed mutagenesis and recep- tor pharmacophore modeling of the 5-HT5A bindingpocket. It was predicted that the addition of groups in position 5would potentially lead to improved potency and selectivity. This was confirmed when the non-selective 5-HT5A antagonists initially interactedwith only afew of the bindingpocket amino acids, whereas when anew exit vector in position5ofthe quinolines was introduced, this led as predicted to the interaction with other amino acids within thebinding pocketand an increase in potencyand selectivity.Quino- The antibiotic-resistant pathogenic Pseudomonas aeruginosa (PA)bacte- rium causes lethal respiratory tractinfections in cystic fibrosis patients by line 1will be presented as aselective5-HT5A antagonist with good pharma- cokineticproperties. means of biofilms, whose formation depends on several factors including lectins. [1] We recently discovered high-affinity ligands for one of these lec- F F tins, LecB, based on themultivalentdisplay of C-fucosides on apeptide dendrimer framework.[2].Here we report in vitro studies showingfor the first timepotent biofilm formation inhibition and biofilm dispersion of clin- O S ical PA strainsvia specific inhibition of LecB. The approach might lead to a HN O new therapeutic strategy againstPAinfections.

O [1] D. Tielker, S. Hacker, R. Loris, M. Strathmann, J. Wingender, S. N N Wilhelm, F. Rosenau, K.-E. Jaeger, Microbiol. 2005, 151,1313-1323. H [2] E. Kolomiets, E. M. V. Johansson, O. Renaudet,T.Darbre, J.-L. Reymond, Org. Lett. 2007, 9,1465-1468. 1

Medicinal Chemistry 87 Medicinal Chemistry 88

AzanorbornanesasPotent Inhibitors of Plasmepsins Rational Design of T315I BCR-Abl Inhibitors for the Treatment of Drug-Resistant Chronic MyelogenousLeukemia(CML): BGG463 Martina Zürchera),Luzi Baranduna),Pascal Wyssa),Solange Meyerb), DanielBurb),François Diedericha) Paul W. Manley,Josef Brüggen, Andreas Floersheimer, Pascal Furet, Michael R. Jensen, Jürgen Mestan, Carole Pissot, Sandra Cowan-Jacob a) ETH Zürich, Wolfgang-Pauli-Str.10, 8093 Zürich, Switzerland b) Actelion, Hegenheimermattweg 91, 4123 Allschwil, Switzerland Novartis Institutes for BioMedical Research, CH-4002, Basel, Switzerland The aspartic proteases plasmepsins of Plasmodiumfalciparum areinterest- ingantimalarialtargets [1]. We found that azanorbornanes of type 1 inhibit CML is caused by the dysregulated Abl tyrosine kinase activity of the st these enzymeswith IC50 values down to the nanomolar range [2,3]. The BCR-Abl oncoprotein.Although well managed with 1 -line imatinib and compounds are designed to occupy both the flap as well as the S1/S3 pocket 2nd-line nilotinib or dasatinib therapy, some patients relapse due to the with vectorsthat can be attached to the scaffold.Inorder to access awide emergence of clones expressingdrug-resistant mutant forms of BCR-Abl. variety of molecules, amodular,facile synthesis was developed. Of these, T315I BCR-Abl is the most resistant, where mutation of the gatekeeper Thr residue to Ile abrogates the activity of the inhibitors through Pro43 loss of abinding interaction to the side-chain hydroxyl, coupled with HN Asp121 flap N unfavourable steric interactions. Based upon our understanding of the O Val105 requirements for BCR-Abl inhibition and the crystal structure of sorafenib f l a p H O Val82 in complex with RAF, we postulated that adiarylurea might provide agood Met75 Tyr115 HO Thr114 pharmacophore element to bind to BCR-Abl, without having any Phe111 S Phe120 interaction with Thr315. This was confirmed from the structure of AFG210 S1/S3 in complex with the kinase domain of wild-type Abl. Although neither Trp41 N H S1/S3 vector sorafenib nor AFG210 showed activity in transfected Ba/F3 cells, structural O2S S N modifications led to BBT594 which inhibited T315I BCR-Abl O NH H N O Met15 autophosphorylation (IC 44 nM)and proliferation (GI 117 nM) in Ba/F3 H 50 50 H HN O O – cellsexpressing thismutant. Further optimisation gave BGG463 possessing O O Asp214 Asp34 good oral efficacy at well-tolerated doses in amurine model of CML.

BBT594 O N H [1] S. E. Francis, D. J. Sullivan, Jr.,D.E.Goldberg, Annu. Rev. Microbiol. N CF H 3 O N N CH 1997, 51,97–123. AFG210 N 3 N O H C 3 N N [2] F. Hof, A. Schütz, C. Fäh, S. Meyer, D. Bur, J. Liu, D. E. Goldberg, F. O

O CF3 Diederich, Angew. Chem., Int. Ed. 2006, 45,2138–2141. N O BGG463 H H N N H H [3] M. Zürcher, T. Gottschalk, S. Meyer, D. Bur, F. Diederich, N N O

N CF3 H3C ChemMedChem 2008, 3,237–240. O N NCH3 MEDICINAL CHEMISTRY558080 CHIMIA 2008, 62,No. 7/8

Medicinal Chemistry 89 MedicinalChemistry 90

Full Automation for Microwave Synthesis Workflows GlycomimeticsasPotential Anti-Cancer Agents Claudia Bello,Pierre Vogel AmiraAbou-Hamdan,Ph.D. Laboratory of Glycochemistry and Asymmetric Synthesis, Swiss Federal Chemspeed Technologies AG, Rheinstrasse 32, Institute of Technology (EPFL), CH-1015 Lausanne, Switzerland CH-4302 Augst(BL), Switzerland During the past decade, research into the causes of human cancer, the molecular Since microwaves have been introduced as an alternative source of heating in basisofmalignant transformation and gene-environment interactions that organic chemistry laboratories in the late 90’s, the number of publications on contribute to individualcancer risks, has made significant progress.Unfortunately, Microwave Assisted Organic Synthesis (MAOS) has been growing exponentially. cancer remains amajor disease burden worldwide. Further efforts have to be done in order to improve our knowledge aboutthe mechanisms of carcinogenesis and One of the most eye catching advantages of MAOS compared to classical methods tumour development with the aim to find new therapies and to develop new are reaction times significantly being reduced from typically several hours to just anticancer drugs. minutes. HO Alibrary of sugar mimetics that are analogues of swainsonine OH Although, this achievementhas resolved avery important bottleneck in the has been developed by Vogel and co-workers. These new α- H discoveryresearch, other tedious steps of the workflownow became the “rate N mannosidase inhibitors(e.g. 1)have shown ahigh affinity for N OH determining step”: H atarget enzyme involved in cancer progressioni.e.: Golgi α- -dispensing of solid and liquid chemicals 1 mannosidase II. 1,2 Inhibitionofthis keyenzymeofprotein -automated capping and crimping glycosilationpathway has beenproposed to be auseful -transfer of vials, from and to the Microwave syntheziser approach to cancer treatment -sampling and work-up of the reaction mixture HO OH Modification of thearomaticmoiety of compound 1 Experimental Methods H has led to aseries of products that show inhibitory Fully unattended, automated synthesis via Wittig reaction was done on afully OH N activities toward α-mannosidase (from jack bean) automated microwave workstation, the SWAVE. N 3 H R similar to that of 1.These new inhibitors (e.g. 2-4) Results and Discussions weretested on different cancer cell lines and were Based on selected applications examples from worldwide renowned “CombiChem” R=4'-(trifluoromethyl)biphenyl(2) biphenyl (3) found to inhibitcancer cellgrowth (e.g. 2 has IC50 = labs we will show in this presentation, how the Chemspeed SWAVE Microwave allyloxy (4) 48M on SKBR3). Compound 3 and,toalesser synthesis platform, not only accelerates the reaction step,but also dramatically extent, 4 alsoexhibited growth inhibitory activities. Another series of analogues increases the productivity for all pre- and postsynthetic steps of atypical containing the same dihydroxypyrrolidine moiety, but with more differentiated Microwave chemistry workflow. substituents was synthesized and tested againstseveral cancer cell lines. These Conclusion show even better anti cancer activity than 1-4.More extensive studies are now in Microwave assisted syntheses were successfully automated and results were in progressinorder to elucidate the mechanism of action of these compounds. Data accordance with literature and theory [1]. suggest that the α-mannosidaseinhibitory activity is not the main cause of cancer [1] www.biotagepathfinder.com -Wittig Olefin Synthesis cell growth inhibitory activity.

Medicinal Chemistry 91 Medicinal Chemistry 92 Targeting Glycopeptide Dendrimers-Cytotoxic Agent Conjugates AnalysisofGDB and ZINCDatabases using Color-Coded Towards TumorCells Self-OrganizingMaps

RasomoyBiswas,Tamis Darbre, Jean-LouisReymond* Lorenz C. Blum,Tobias Fink, Ruud vanDeursen, KongT.Nguyen, and Jean-Louis Reymond Department of Chemistry and Biochemistry, University of Berne Freistrasse 3, 3012 Berne (Switzerland) DepartmentofChemistry and Biochemistry, UniversityofBerne, Freiestrasse 3, CH-3012Berne, Switzerland Dendrimers are being investigated by various groups as selective drug deli- very vehicles for cancer cells [1].Weshowed recently that covalent glyco- The classification and contentanalysis of large databasesofmolecules peptide dendrimer conjugatesofcolchicine exhibitselective cytotoxicity is intrinsically complex. Here we usedself-organizingmaps (SOMs) to towards HeLa cells compared to MEF cells, presumably by means of recep- analyze the contents of the chemical universe database GDB1 (26.4 M tor-mediatedendocytosis and activation by metabolic degradation in the structures) and theZINC database2 (2 Mdrug-likemolecules), using [2] dividing tumor cells .Here we present the improvement of the systemsby a200x200 and 100x100 set of neurons, respectively.The SOM based modifyingthe glycopeptide dendrimer structures systematically using posi- analysis automatically organizesthe databasesaccordingtostructural tive,neutral andnegativeamino acid residues. features suchasmolecular weight, rotatable bond count, ring count, or

400 polar surfacearea. Amultidimensional color-coding scheme wasused to colortheseSOMs according to the average descriptor values perneu-

300 ron of as themain color (Hue value), the standard deviation as agrey scale (saturation value), and the neuronoccupancy as thecolor intensity 9.69 90.3 200 (lightness value). The color-code results in sharp and intensecolors for well populatedneurons withwell defined values only,while scattered val- 100 ues or poorly occupied neuronsappear greyorwhite respectively.This

0 allows astraightforward and detailed insightintothe databases. 100 101 102 103 104 FL2-H [1] T. Fink, J.-L. Reymond, J. Chem.Inf. Model., 2007, 47, Figure 1Figure2 342-353. Figure1: Schematic structure of aconjugate. Figure 2: Representative exam- [2]J.J.Irwin and B. K. Shoichet, J. Chem.Inf. Model., 2005, pleofahistogram from flow cytometry experiment, showing percentage of 45,177-182. viable cell (9.69) at the left of themarker, non-viable at right (90.3).

[1]E.R.Gillies, J. M. J. Fréchet, Drug Discovery Today, 2005,10, 35-43 [2]D.Lagnoux, T. Darbre, M. L. Schmitz, J.-L. Reymond, Chem. Eur. J., 2005, 11,3941-3950. MEDICINAL CHEMISTRY 581 CHIMIA 2008, 62,No. 7/8

Medicinal Chemistry 93 Medicinal Chemistry 94

Which Residues Dictate Src-Specific Conformational Plasticity? Synthesis of Novel Cationic NSAID Derivatives to Facilitate Electrically-assisted Transdermal Delivery Ralitza Boubeva, Andrea Cristiani, Loris Moretti, Oscar Vadas, Remo Majdeline El Mahrab Robert,Leonardo Scapozza, Yogeshvar N. Kalia Perozzo, Leonardo Scapozza University of Geneva, School of Pharmaceutical Sciences Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, Quai Ernest-Ansermet 30, 1211 Genève 4–Switzerland University of Geneva, University of Lausanne, Quai Ernest-Ansermet 30, NSAIDs(Nonsteroidal Anti-Inflammatory Drugs) are commonly used for the CH-1211 Geneva 4, Switzerland treatment of inflammation and pain; however, their oral administration is associated with potentiallyfatal gastrointestinal side effects.[1] Improved Tyrosine kinaseswere among the first to be discovered as human transdermal delivery would increase theirlocal bioavailability and expand the oncoproteins. Src tyrosine kinases are key signaling protein tyrosine kinases rangeofindications for targeted, topical therapy. The aim of this study was to that are of considerable interest as drug targets in cancer and many other synthesise novel cationic ester NSAID derivatives optimised for transdermal administration by anodal iontophoresis; the skin has an isoelectric point (pI) of ~4- diseases. The catalytic domains of different tyrosine kinases adopt strikingly [2] similar structures when they are active. By contrast, crystal structures of 4.5, and is permselective to cations. Our hypothesis is that the cationic prodrugs will electromigrate into the skin under the influence of the applied iontophoretic inactive kinases have revealed aremarkable plasticity that allows the current. When they reach the viable epidermis and dermis they will be subject to adoption of different conformations in response to interactions with specific hydrolysis by endogenous esterases and hence release the active molecule.[3] regulatory domains or proteins. Therefore, investigating on the conformational states of Src tyrosine kinase might short cut the drug design The chargedesters (1a, 1b)were synthesised by acarbodiimide condensation using [4,5] for both effective and highly specific compounds. dicyclohexylcarbodiimide (DCC). The main challengeencountered during the Acomparative structural analysis has been performed using molecular synthesis was the removal of the reaction by-product dicyclohexylurea (DCU). modeling. The results of this analysis depicted apool of residues at the Esterification of the carboxylic acid groups in diclofenac and indomethacin with choline, acharged aminoalcohol, introduced apH-independent positive charge into interface of the two lobes that appears to be important for the protein the molecule through the quaternary ammonium.Inthe next part of the project we conformation and motion. With the aim of assessing the findings of the will investigatethe iontophoretic delivery kinetics of the newly synthesised comparative analysis biochemical and biophysical studies of the molecules. Cl conformational plasticity of the c-Src kinase domain have been performed. O

CH H C The results of these studies together with the comparative structural analysis NH 3 3 O + CH Cl O O N 3 will be presented. CH 3 CH3 O N

CH3 + N O Cl CH3 H3C

1a:Diclofenac-choline ester 1b:Indomethacin- choline ester

Medicinal Chemistry 95 Medicinal Chemistry 96

Investigation of the Ribose binding motif in Catechol-O- Description and ComparisonofClassical Cyclic Voltammetry with HT- Methyltransferase (COMT) Cyclic Voltammetryfor the determination of Redox Potentials

ManuelEllermann[a],Ralph Paulini[a],Edilio Borroni[b],Gerhard Zürcher[b], Anabel Felix, Holger Fischer°, Manfred Kansy°, Matthias Hamburger* Roland Jakob-Roetne[b],François Diederich*[a] *University of Basel, Klingelbergstrasse 50, CH-4055 Basel, Switzerland [a]Laboratorium für Organische Chemie, ETH Zürich, Wolfgang- °Fa. Hoffmann-La Roche AG, Grenzacherstrasse 124, CH-4009 Basel, Pauli-Str. 10, 8093 Zürich Switzerland [b]PharmaDivision, Präklinische Forschung, F. Hoffman-La Roche AG, Grenzacherstr. 124, 4070 Basel Electrochemistry is comprised of several aspects of molecular sciences [1- 4]. Besides the movement of charges in an electrical field, electron-transfer Catechol-O-Methyltransferase (COMT)isone of the key enzymes involved induced processes are an importantissue in chemistry and life-sciences [1- in catecholamine catabolism.Therefore it is one of the main targets forthe 4]. Among other electroanalytical methods such as differential pulse vol- treatment of CNS disorders such as Parkinson’s disease [1]. Highly potent tammetry or paleography, cyclic voltammetry is one of the techniques with bisubstrateInhibitors have been developedbyde novo design [2], but the its largest scope as it provides information of thermodynamic data, kinetic exactbindingmotif at the ribose moiety still requires further explanation parameters or stability issues. [3]. In our ongoing work we investigate theseinteractionsand compare this Standard cyclic voltammetric measurements are usually performed with ubiquitousmotif to other proteins. macro electrodes in asingle electrochemical cell. An external collaboration with Gatlik (Gatlik Ltd., Basel/CH) gave rise to the Electroactive Pharmaceutical Screening System (EPSS), anovel HT- cyclic voltammetric screening/profiling system which allows electrochemi- cal determinations under physiological conditions in the 96-well format. Therefore, the focus of the current study was the development of anew ap- plication based upon the EPSS system with asubsequent technical modifi- cation to measure molecules under physiological-like conditions, and a comparison of classical and HT- cyclic voltammetry.

[1] P. T. Männistö, S. Kaakola, Pharmacol. Rev. 1999,51, 593-628. [1] Heinze, J., Angewandte Chemie, 1984. 23,831-918. [2]C.Lerner, A. Ruf,V.Gramlich,B.Masjost,G.Zürcher, R. Jakob- [2] Brett, C.M.A. and A.M. Brett Oliveira, Electrochemistry: Principles, Roetne,E.Borroni,F.Diederich, Ang. Chem.Int. Ed. 2001,40, 4040- Methods and Applications.5th ed. 2002,Coimbra, Portugal:Oxford 4042. SciencePublications [3]R.Paulini, C. Trindler, C. Lerner,L.Brändli, W. B. Schweizer,R.Ja- [3] Atkins, P. and J. de Paula, Physical Chemistry.7th ed. 2002,New York: kob-Roetne, G. Zürcher, E. Borroni, F. Diederich, ChemMedChem [4]GosserJr.,D.K., Cyclic Voltammetry:Simulation and Analysis of 2006,1,340-357. Reaction Mechanisms.1st ed. 1993,New York: VCH. MEDICINAL CHEMISTRY558282 CHIMIA 2008, 62,No. 7/8

Medicinal Chemistry 97 Medicinal Chemistry 98

MolecularModeling Of Glycopeptide Dendrimers LigandsOfThe Fu- Design andSynthesis of Strongly Binding Trna-Guanine cose BindingLectinPA-IIL From Pseudomonas aeruginosa Transglycosylase (TGT) Inhibitors Undergoing Charge-Assisted Hydrogen-Bonding Rameshwar U. Kadam,Tamis Darbre and Jean-LouisReymond* Philipp C. Kohler1,Tina Ritschel2,Simone R. Hörtner1,Bernhard Stengl2,Björn Wagner3,Manfred Kansy3,Gerhard Klebe2,François Diederich1 Department of Chemistry and Biochemistry, University of Berne, Freies- 1 trasse3,CH-3012,Berne, Switzerland P. C. Kohler, Dr. S. R. Hörtner, Prof.Dr. F. Diederich, Laboratory of Organic Chemistry, ETH Zürich, Hönggerberg, HCI, 8093 Zürich, Switzerland. The fucose-specific lectin PA-IIL of the opportunisticpathogen Pseudomo- 2 T. Ritschel, B. Stengl, Prof. Dr. G. Klebe, Institute of Pharmaceutical Chemistry, nasaeruginosa is implicated in tissue bindingand colony formation leading Philipps-University Marburg, Marbacher Weg 6, to lethal airways infections in cystic fibrosis patients.Inview of developing 35032 Marburg, Germany. novelanti-infectivePA-IIL inhibitors,recently our laboratory developed 3 B. Wagner, Dr. M. Kansy, Pharmaceuticals Division, Discovery Chemistry,F. multivalent ligands to this target [1-2]. To study the importance of multiva- Hoffmann-La Roche AG, 4070 Basel, Switzerland. lency for ligand binding, molecular docking and molecular dynamicsatna- nosecond timescale were carried outwith the protein-dendrimer complex to Bacillary dysentery,orshigellosis, is adiarrheal diseaseoccurring worldwide, but further explore the binding mode of the dendrimer to the lectin (Figure 1). mainly in developing countries. The emergence of multi-resistant bacterial strains makes the development of new specific antibiotics an urgent issue. The bacterial enzyme tRNA-guanine transglycosylase (TGT) has been identified as aviable target for new drugs [1].

Figure 1. Molecular model of the peptide In collaboration with the group of Prof. G. Klebe aclass of inhibitors with Ki dendrimer binding to the fucose-specific values down to the single-digit nanomolar range has been developed [2,3]. These lectinPA-IIL from Pseudomonas aerugi- compound are based on a lin-benzoguanine scaffold that is substituted at the 2- nosa position. The rational drug design is based on computational modeling done with the program MOLOC (F. Hoffmann-La Roche)[4]. Introduction of an amino substituent at the 2-position has increased the binding affinity by afactor of 50–70 as compared to the unsubstituted scaffold [2,5]. This findingcan be rationalized by theestablishment of an additional hydrogen bond [1] E. Kolomiets, E. M. V. Johansson,O.Renaudet,T.Darbre, J.-L. Rey- and, more importantly, an increase in basicity of the imidazole moiety. The mond, Org. Lett 2007, 9,1465. corresponding pKa value is raised from 5.2 (unsubstituted lin-benzoguanine) to substantiallyhigher values of 5.9–6.7, depending on the substituent. We therefore [2] E. M. V. Johansson, E. Kolomiets, F. Rosenau, K.-E. Jaeger,T. Darbre, assume protonation of the J.-L. Reymond, New. J. Chem. 2007, 31,1291-1299.

Medicinal Chemistry 99 Medicinal Chemistry 100 InteractionsofPolymyxin MwithBacterialLipopolysaccharides Synthesis and conformational analysisofphenyl-a-CH2 and CF2- galactosides. Interactions with theplant lectin viscumin.

MariaKolympadi,a MarcoFontanella,b Chiara Venturi,b Sowmini Kumaran,JiriMares,OliverZerbe* Jesús Jiménez-Barberob*and Pierre Vogela* University of Zurich,Winterthurerstr.190, CH –8057 Switzerland a Swiss Federal Institute of Technology (EPFL), Batochime, CH-1015Lau- sanne, Switzerland b Centro de InvestigacionesBiológicas, CSIC,Ramiro Polymyxins present agroup of cyclic polycationic polypeptides isolated de Maeztu 9, 28040, Madrid, Spain from various strains of Bacilluspolymyxa and related species. They are ca- pableofinhibiting the growth of awidevariety of Gram-negative bacteria The ongoing study of protein-carbohydrate interactions has created the need and also possess anti-endotoxin activity. LPS, astructural component of the for the preparation of non-native saccharides in order to blockortoinhibit outermembrane of Gram-negative bacteria, elicit awide range of toxic ef- certainbiological events.[1] C-glycosides, where the anomeric oxygen has fects in avariety of organism with the human among the most susceptible been replacedbyamethylene unit, areinterestingmimetics due to their sta- species(1).Polymyxins are peptides that can effectivelykill bacteriaaswell bility.Nevertheless, the absence of theanomeric effect results in an in- as neutralize free LPS (2). The precise mode of its binding to LPS and the creased flexibility of thesepseudosaccharides, giving rise to thequestion structural features involved therein are unknown. whether the entropic excess can be compensated by the binding with are- ceptor protein. We are attempting to answer this question by examiningdif- An understanding of the structural aspects of its binding with LPS in a ferent substitution on the methylene linker. Substitution by two fluorine membrane-mimicking environment would be very useful for the rational atomsisexpected to alter significantly the conformational behaviourofthe developmentofcompounds with anti-endotoxin activity. Accordingly we molecule relative to thehydrogen substitution, as well as to the nativelin- chose to characterise interactions of Polymyxin M(Mattacin) produced by [2] kage. Therefore, two simple phenyl galactosides, 1 and 2,have been pre- Paenobacillus kobensis Mwith Re LPS (obtained from the D31me4 strain pared and their conformer distribution in solution has been evaluated using a of E. Coli)inthe presence of phospholipid (DPC) micelles. In our study, we combination of forcefield calculations and NMRstudies. Interactions with have purified Re LPS and PolymyxinM,and studied the interaction be- theplant lectinviscumin, which is agalactoside-specific enzyme, are also tween them by variousNMR techniques. These studies include aspects of described. how PMX andLPS are oriented on the phospholipid micelles. HOOH HOOH O O HO HO HO HO [1] S. Bhattacharjya, Biopolymers. 41 (1997), 251-265. F [2] J. Howe, Biophys. J. 92 (2007), 2796 –2805. F 1 2

[1] C. R. Bertozzi, L. L. Kiessling, Science. 2001, 291,2357. [2] J. Jiménez-Barbero, R. Demange, K. Schenk,P.Vogel, J. Org. Chem. 2001, 66,5132. MEDICINAL CHEMISTRY558383 CHIMIA 2008, 62,No. 7/8

Medicinal Chemistry 101 Medicinal Chemistry 102

StructureActivity Studies for Hypermodified Epothilone Analogs with The non-globular domaininP. falciparum enoyl-ACP reductase Potent in vitro AntitumorActivity (PfFABI) andits role in substrate specificity andturnover

Karl-Heinz Altmann, Pascal Wyss, Christian N. Kuzniewski Lauciello, Leonardo,Lack, Gabriela, Scapozza, Leonardo, Pe- rozzo, Remo DepartmentofChemistry and AppliedBiosciences, ETH Zürich, Wolfgang- Pauli-Str. 10, CH-8093 Zürich, Switzerland Pharmaceutical Biochemistry Group, School of Pharmaceutical Sciences, UniversityofGeneva,UniversityofLausanne, Quai Ernest-Ansermet 30, Epothilones are microtubule-stabilizing agents with potent in vitro and in CH-1211Geneva, Switzerland vivo antitumoractivity. Although the SAR of thishighly promising com- pound class has been extensively investigated, specific aspects still remain The analysis of complete genomes hasshown that Intrinsically Unstructured unaddressed[1].Inasynthetic endeavor to deviate more and more strongly Proteins (IUPs) are common among organisms. Thanks to their structural from theoriginalnatural productleads we discoveredthatthe hypermodi- flexibility, non-globular domains are involved in several cellular activities fied trans cyclopropylepothiloneanalogs 1 and 2 inhibithuman cancer cell carried out by protein-protein, protein-DNAand protein-RNA interactions. growth in vitro with low- to sub-nM IC50 values. Most remarkably their ac- Genes of Plasmodiumfalciparum areknown to be characterized by long tivity is significantly more pronouncedfor the multidrug resistant KB-8511 insertions called Low Complexity Regions (LCRs), which often give rise to cell line than forthe correspondingdrug-sensitive KB-31line. With this regions lacking awell-defined 3D structure. [1] These regions were recently potentially highly active macrocycle in hand the aim is to further probe the shown to be important for proteinfunctions in this parasite. [2] effects of side chain modifications for thisnew chemotype. With an efficient This work aims at elucidating the role plaid at amolecularrecognition level synthesis for structures of type 1-4 available[2],aconvergent new synthetic by thenon-globular domain present in P. falciparum enoyl-ACP reductase strategyfor the generation of macrolidescaffold 5 was developed. The (PfFabI), an enzymeinvolved in the type-II Fatty Acid Synthesis (FAS-II) omission of thenaturally occurringmethylgroup at C16has been shown not pathway. For this purpose, amatureform deletion mutant lacking the PfFabI to be connectedwith major activity lossesfor natural epothilonesand is thus 43-resuidues longLCR wascreated, and kinetic parameters were compared expected to producehighly active analogs in combination with the newly to thoseofthe mature, wild type PfFabI, using crotonoyl-CoA and enoyl- designed macrolidestructureand various heterocycles as thearomatic side ACPs. Structuraland stability studies were also performedinpresence and chain. absence of the cofactor NADH.

N X The results of this study show that the non-globular domain of PfFbaI is not R Y X important in maintainingthe overall structure of the enzyme, but directly X N N HO O O HO O O HO O O influences the affinity of PfFabI forits artificial and naturalsubstratesand the catalytic efficiency of the enzyme. Moreover, the deletion mutant 1:X-Y=CH2-CH2 3:R=Me, X=S 5 2:X-Y=(E)-CH=CH 4:R=H,X=CH=CH X=heterocycle O O O showed to be more susceptible to NADH stabilization.

[1] F. Cachoux et al., Angew. Chem.Int. Ed. 2005, 44,7469. [1] Brocchieri, L., Genome Res.,2001. 11(2): p. 195-197. [2]C.N.Kuzniewski et al., Org. Lett. 2008, 10,1183. [2] Jean, L., et al., Mol. Biochem. Parasit.,2005. 144(2): p. 187-197.

Medicinal Chemistry 103 Medicinal Chemistry 104 Control of abnormal metal-protein interactions in neurodegenerative Thermodynamic and Kinetic Binding PropertiesofMAG-Antagonists disorders by metallothionein-3 Stefanie Mesch,MorenaSpreafico,AngeloVedani, Oliver Schwardt, GabrieleMeloni,Milan Vašák BeatErnst

University of Zürich, Winterthurerstr.190, CH-8057Zürich, Switzerland Institute of MolecularPharmacy, University of Basel, Klingelbergstr. 50, 4056 Basel, Switzerland In the brain, the zinc and copper homeostasis is regulated by asmall metal- loprotein, metallothionein-3 (Zn7MT-3),which is down-regulated in neuro- The injuredadult mammalian central nervous systemisaninhibitory envi- degenerative disorders such as Alzheimer(AD), Creutzfeldt-Jacob and Par- ronment for axon regeneration due to specific inhibitoryproteins. One of kinson. These diseases share common pathological hallmarks including mis- theseneurite outgrowth inhibitors [1] is themyelin-associated glycoprotein folding of amyloid-β (Aβ), prion proteinand α-synuclein, the formation of (MAG)[2]. It belongs to the siglec family (sialic-acid binding immunoglo- protein aggregates, abnormal metal-proteininteractionsand oxidative stress. bulin-like lectin). In previousstudies, we identified high affinity antagonists In AD, Cu(II) and Zn(II) are involvedinthe disease progression by mod- [3,4]. However, they showed unsatisfactory kinetic binding properties, i.e. ulatingthe formation and toxicity of soluble and insoluble oligomers and fast off-ratesand therefore shortresidence times. aggregates of the Aβ peptide. Whereas the copper-induced Aβ aggregation We therefore investigatedthe influence of various modifications on the in- is relatedtothe ROS production and neurotoxicity, the zinc-induced Aβ sufficient kinetic properties of these MAG antagonists. For this purpose, aggregation is neuroprotective. Theprotective effect of extracellular different substitutionswere introduced to the 4position of the neuraminic Zn7MT-3 from Aβ toxicity in neuronal cell cultures is notunderstood. We acid derivative 1. show that Zn7MT-3 notonlyscavenges free Cu(II) ions [1], but also removes Cl Cu(II) bound to Aβ [2]. AmetalswapbetweenZn7MT-3 and soluble and aggregatedAβ-Cu(II) is the underlyingmechanism by whichthe ROS pro- H OH N OH COONa ductionand relatedcellular toxicity is abolished. In this process, copper is R1 O H O reduced by the protein thiolates forming Cu(I) Zn MT-3, in which an air N O 4 4 R2 stable Cu(I)4-thiolatecluster and two disulfide bonds are present [2]. To O F examine whether the discovered effect represents ageneral protective role in 1 F othermetal-linked neurodegenerativepathologies, similar studiesusing The thermodynamic and kinetic properties of the new antagonists were ana- prion peptides in complexwith Cu(II) were conducted. We show that lyzedbysurface plasmon resonance studies. Zn7MT-3 by asimilar metal swap reaction removes abnormally bound Cu(II) from the prion protein, impeding the ROS production. This finding [1] Crocker, R. H., Curr. Opin. Struct. Biol. 2002, 12,609. signifies asofar unrecognized protective role of this protein in the brain. [2] Quarles, R. H., J. Neurochem. 2007, 100,1431. [3] Shelke, S., Gao, G-P., Mesch, S., Gäthje, H., Kelm,S., Schwardt, O., [1] G. Meloni,P.Faller, M. Vašák, J. Biol. Chem., 2007, 282,16068-16078 Ernst, B., Bioorg. Med. Chem, 2007, 15,4951. [4] Method for the identification of new leads for drug candidates, PCT/WO2007/105094 A1, priority: March14, 2006. MEDICINAL CHEMISTRY558484 CHIMIA 2008, 62,No. 7/8

Medicinal Chemistry 105 Medicinal Chemistry 106

Discovery of NMDA-Glycine Site Inhibitors from the ChemicalUn- Natural Product-Like Furo[3,4-c]pyranones: Lead Structures for iverse Database GDB NovelAnticancer Agents

a b a a Kong ThongNguyen,Salahuddin Syed, Stephan Urwyler, Sonia Bertrand, Alina L. Nussbaumer ,Stephan Ruetz ,Cyril A. Fuhrer ,Michel Jordi , Daniel Bertrand,Jean-Louis Reymond Florian Garoa and Robert Hänera

Department of Chemistry and Biochemistry, University of Berne, aDepartment of Chemistry, University of Bern, CH-3012 Bern, Switzerland Freiestrasse 3., 3012 Bern, Switzerland bNovartis Institutes of Biomedical Research, CH-4002 Basel, Switzerland

Natural products have played an eminent role in the discovery and Onepossibility to discover new small molecule drugs would be to search developmentofnew drugs.1 exhaustively through the entire chemical space using virtual screening tools We recently reported the synthesis of different natural product-like furo[3,4- andidentify promising ligands for synthesisand testing. Herein we report c]pyranones.2 The furopyranone scaffold 1 contains the cis-stilbene motif, as the firstexample of such an approach for ligands of the NMDA-receptor we can find it in stilbenoids,includingresveratroland combretastatin A-4. glycine site, an important drug target implicated in synaptic plasticity, neu- The cis-stilbene motif was identified as the pharmacophoreinthe ronal development, learning and memory. Starting with the Chemical Un- compounds, which exhibited interesting anticancer properties in different iverse Database GDB [1], whichcontains all organic molecules of C, N, O, humancancer cell lines.3 To conduct amore detailed structure-activity Fupto11atomspossible under considerationofsimple chemical stability relationship(SAR) study, strategies were carriedout to synthesize and synthetic feasability rules, aBayesian classifier trainedwith61known symmetric and asymmetric methoxy substituted α-diketonestointroduce NMDA-receptorligands was used to identify 15'061 virtualhits, which were new substitution patterns in the furopyranones scaffold. expanded to 69'367 stereoisomers and docked into the glycine site of the In addition, increased antiproliferativeactivity in HEK293 and SJSA1 NMDA-receptor[2] (pdb: 1PB7). From the 712 ligands docking stronger cancer cellswas observedwith abenzylester substituent at thephenyl ring. than glycine, 23 compounds were selected and either purchased or synthe- Theinfluence of new substitution patternsatthe phenyl ringtorefineour sized. Activity screeningusing NMDA receptor radioligandbinding assays structure-activity relationships(SAR) studies, is also investigated. gave 5hits not previously reported to interact with this receptor, two of which were expanded to 8furtheractiveanalogs, suggesting that anew lead R´ structure has been found. Electrophysiological data show that the com- R´´´ O pounds act as glycine antagonistatthe glycine site. H H (+/-) 1

[1] a) T. Fink, H. Bruggesser, J.-L. Reymond, Angew. Chem. Int. Ed. 2005, R´´ H O 44,1504-1508; b) T. Fink, J.-L. Reymond, J. Chem. Inf. Model. 2007, O 47,342-353. [2] H. Furukawa, E. Gouaux, EMBO J. 2003, 22,2873-2885 [1]R.Messer, C. A. Fuhrer,R.Häner, Curr. Opin. Chem. Biol. 2005, 9,259-265. [2]C.A.Fuhrer, R. Messer, R. Häner, TetrahedronLett. 2004, 45,4297-4300. [3]C.A.Fuhrer, E. Grüter, S. Ruetz, R. Häner, ChemMedChem 2007, 2,441-444.

Medicinal Chemistry 1071Medicinal Chemistry 08

Structure-Activity RelationshipsofC12-C13-Oxazoline Derivatives Modulation of Antibacterial Activity by SubstitutedSiderophores of Epothilone A Bernhard Pfeiffer,Kurt Hauenstein, Philipp Merz, Jürg Gertsch, Karl-Heinz V. Bitsch, E. Desarbre, B. Hofer, C. Müller,M.G.P.Page, F. Richalet Altmann* BasileaPharmaceutica InternationalAG, Grenzacherstr. 487, CH-4005 Derpartment of Chemistry and Applied Biosciences, ETH Zürich, Basel,Switzerland Wolfgang-Pauli-Strasse 10, CH-8093 Zürich The inhibition of cancer cell growth by epothilones (Epo) (1), natural Gram-negative bacteria (GNB) are becomingincreasingly resistanttowards products isolated from the myxobacterial strain Sorangium celluosum,is β-lactam antibiotics. Reduced permeability of the outer membrane due to basedonthe stabilization of cellularmicrotubules, i.e. theyexhibit a“taxol- mutations in porins is oneofthe factors leadingtoresistance. Recently,it like” mechanism of action. The widespread interest in the chemistry and was demonstrated that BAL0019764 (PTX2416, fig. 1, R1 =OH, R2 =H, biology of these macrolide-based microtubule-stabilizing agents, expedited [1]) displays enhanced antibacterial activity, especially against P. aerugino- by their potent in vitro and in vivo antiproliferative activity, has resulted in sa species compared to aztreonam, amonobactam antibiotic commonly used an advanced understanding of the structure-activity relationships for for the treatment of GNB infections.The presence of siderophore moiety epothilones. The advances in the synthesis and semi-synthesis of hydroxypyridone is considered to enhance the penetration through the epothilones lead to numerous active analogues of which at least seven have membrane. The new antibiotic possibly formsacomplex with iron (Fe3+), entered clinical evaluation as potential anticancer drugs and one of them has which is recognized and takenupinto bacteria by specific transport systems recently obtained FDA approval for the treatment of breast cancer [1]. [2]. One such epothilone analogue, trans-epothilone A(2), was shownto O OR exhibits antiproliferative activity in asimilar concentration-range as the O 2 R =OH, OMe, Me,NH Aztreonam 1 2 natural epothilone A(1a)itself [2]. As afurther extension of our previous OH N R =H,Me O 2 O R work on trans-epoxide and trans-cyclopropane-based analogs of Epo Awe N N 1 H H now present the synthesis and biological evaluation of aseries of epothilone N N N N H N H N derivativesthat are characterized by the presence of a2-substituted trans- 2 2 S O N S O N O SO H O SO H fused C12-C13-oxazoline ring (3). Some of these derivatives show 3 3

antiproliferative activities and tubulin-polymerizing potenciesthat are Fig. 1 comparable with those of the parent compound Epo A. Aclear structure- It was observed thatmodifications of substituents R1 and R2 presentonthe activity relationship can be delineated for these analogs with respect to the siderophore moietydramatically influencedthe antibacterial activity, espe- nature of the 2-substituent on the oxazoline ring. Possible interaction modes cially in P. aeruginosa strains. Preparation of those compounds as well as a of these new epothilone derivatives with tubulin have been investigated by more detailedtable of activitieswill be presented. molecular-modeling. [1]GillA.E,Taylor K, Lewendon A, UnemiN,Uji T, Nishida K, SalamaS, Singh R, Micetich RG. ICAAC 2003 Sep 14-17; 43: abstract no. F-552. [2] Mushtaq S, Livermore D.M. ICAAC 2003 Sep14-17; 43: abstract no.F- 554. MEDICINAL CHEMISTRY558585 CHIMIA 2008, 62,No. 7/8

Medicinal Chemistry 109 Medicinal Chemistry 110

The Cation–π Interactioninthe S4 Pocket of Factor Xa Computer Simulation of PleD Dimerization

Laura Salonena,Kaspar Schärera,David W. Bannerb,François Diedericha Franziska F.-F. Schmid,Markus Meuwly

a ETHZürich, HCI, Wolfgang-Pauli-Strasse 10, 8093 Zürich, Switzerland Department of Chemistry,University of Basel, Klingelbergstrasse 80, CH- b F.Hoffmann-La Roche Ltd., PRBD-E, Molecular Structure, Bldg 65/312, 4056 Basel, Switzerland 4070 Basel, Switzerland The bacterialsecond messenger cyclic di-guanosine monophosphate (c- Thrombin was atargetinmolecular-recognitionstudies in the Diederich diGMP) is implicated in pathogenesis of many bacteria and its cellular level group. Inhibitors 1 and 2 were synthesized to investigatethe cation–π inte- is tightly controlled by diguanylate cyclases (DGCs) and phosphodiesterases raction in the Dpocket of thrombin. Surprisingly, the onium ion inhibitor 1 (PDEs). DGC activity to synthesize the symmetric signaling molecule is did not show much activity against thrombinbut was found to be ahighly controlled by protein allostery and dimerization.[1,2] Bound c-diGMP in an active factor Xa inhibitor. Thecorresponding noncationic inhibitor 2,how- allosteric binding site prevents product formation, presumably by altering ever, showed reversed activity. Presumably, the cation–πinteraction be- the protein dynamics, i.e. less flexibility in inhibition- and active-site.[3] tween Trp215 in the Dpocket of thrombin and the onium ion is not strong Based on microbiology experiments on PleD of {\it C. crescentus}, DGC enough to compensate forthe desolvationenergylost during binding. In the dimerization is afurther level of activity control. S4 pocket of factor Xa,the aromatic box formed by the threearomatic resi- PleD activation with ${\rm BeF_3 ^-}$ shows structural rearrangements in dues Phe174, Tyr99, and Trp215 is able to compensate for the loss in desol- the domains D1/D2 to improve the dimerization interface and possibly vation energy. The cation–πinteraction was quantified by comparingthe facilitating dimer formation.[4] binding affinitiesof1and 2.Asecond, different ligandseries will also be reported[1]. The dimerization process is investigated for truncated PleD (D1D2 domains) in its activated and inactivated state using molecular dynamics (MD) simulations. Residual contribution to the interaction energy is studied to identify key features. In addition the role of the preserved residue Tyr26 in the dimerization process is probed.

{[1]} &B.Christen {\it et al.}, \emph{J. Biol. Chem.}, \textbf{2006}, \emph{281}, 32015. \\ {[2]} &R.Paul {\it et al.}, \emph{J. Biol. Chem.}, \textbf{2007}, \emph{282}, 29170. \\ {[3]} &F.F.-F. Schmid {\it et al.}, \emph{J. Mol. Biol.}, \textbf{2007}, \emph{374}, 1270. \\ {[4]} &P.Wassmann {\it et al.}, \emph{Structure}, \textbf{2007}, \emph{15}, 1. \\

[1] K. Schärer,M.Morgenthaler, R. Paulini, U. Obst-Sander, D. W. Banner, D. Schlatter, J. Benz,M.Stihle, F. Diederich, Angew. Chem., Int. Ed. 2005, 44,4400.

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Computational Study of Ligand-induced EffectsinDengue HPLC Microfractionation in theSearch for Antimalarial Compounds: Methyltransferase an Application of the Haematin Polymerization Assay

a a a b Claudia Simões-Pires ,Sandra Vargas ,Andrew Marston ,Marçal Paulo , Tobias SCHMIDT, Torsten SCHWEDE, Markus MEUWLY Jean-Robert Ioseta andKurt Hostettmann a aLaboratory of Phytochemistryand Pharmacognosy, University of Geneva, 30 Quai DepartmentofChemistry &Biozentrum, UniversityofBasel, Ernest Ansermet, CH 1211 Geneva 4, Switzerland. Klingelbergstrasse 50/80, CH-4056 Basel, Switzerland bLaboratory of Chemistry of Natural Products, Department of Chemistry,Federal University of Paraíba, Brazil. Dengue fever is amosquito-borne viral infectious disease predominantly prevalentintropical regions with annually 50–100 million cases and Ascreening assay basedonthe inhibition of synthetic hemozoin(beta-haematin) formation [1] to identify novel antimalarial substances was adapted to the around 25000 death worldwide. No vaccinations or specificdrug treat- screening of complexmixtures. ments are available.[1] The Dengue virus is an enveloped virus with a Initially, the test was applied to plantextracts and fractions. This includeda single-stranded, positivesense RNA genome, bearing a5’cap 1structure. specific sample preparation followed by SPE pre-purification in order to improve The viral RNA capping machinery is essential for RNA stabilityand for solubilisationofthe samples and avoid interferencewith unwanted materialsuch as viral replication, making it an attractivetarget for drug design.[2] Apre- tannins. Astandardoperating protocol hasbeen developed for routine screening in vious study focusedonthe inhibition of the activityofthe methyltrans- both eppendorf tubes and 96-well plates. In ordertoobtain fast results for abioguidedisolation, amicrofractionation ferase(NS5MTase),[3] which catalyzes thetransferofamethyl group procedurewas established for the aqueous extract of Syzygium cumini (L.) Skeels fromS-adenosyl-L-methionine (SAM) to the immatureviral RNAcap (Myrtaceae). structure. The NS5MTase enzyme posses two adjacentbinding sites, one S. cumini is atropicaltree which has beenextensively used in traditionalmedicine, for SAM and one for the RNAcap structure. mostly forantidiabetic purposes, in countrieslike India, thePhilippinesand Brazil In this work, the molecular mechanism of ligand binding to the NS5MTase [2]. An antiparasitic activity has already beendemonstrated in the genus Syzygium is investigated at an atomisticlevel, using computer simulations.We against Plasmodium falciparum [3]. The preliminary screeningofthe aqueous extractobtained from the stembarkofS. focus on ligand-induced effects on thestructure and flexibilityofone cumini demonstrated amarkedinhibition of beta-haematin formation. binding site whenaligand binds to the adjacentcavity. Suchinforma- An HPLC-UV-MS analysis of this extractwas performed, followedbythe tion is highly valuable both for amore thorough understanding of the evaluation of thecollected fractionsinthe beta-haematin polymerizationassay. biological system as well as for further computer-aideddrug development The work described here provides aone-stepmethod which enablesadirect efforts where the incorporation of protein flexibilityiscrucial to improve correlation between activity with specific knownand unknown chemical entities. computationalpredictions.[4] [1]Ncokazi K.K.,Egan, T.J. Anal Biochem. 2005,338,306-319. [2]Villasenor, I.M. andLamadrid, M.R.A. JEthnopharmacol. 2006,104, 129-131. [1] D.J. Gubler, Novartis Found Symp, 2006, 277,3. [3]Braga,F.G., Bouzada, M. L. M.,Fabri, R.L.,Matos, M.O.,Moreira,F.O., Scio, E. andCoimbra, E.S. JEthnopharmacol. 2007,111, 396-402. [2] M.P.Egloff, D. Benarroch,B.Selisko, J.L.Romette, B. Canard, EMBO Journal, 2002, 21,2757. [3] M. Podvinec,T.Schwede, personal communication, 2008 [4]M.K. Gilson, H.X.Zhou, Annu. Rev. Biophys. Biomol. Struct, 2007, 36,21. MEDICINAL CHEMISTRY 586 CHIMIA 2008, 62,No. 7/8

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Bifunctional Glycodendrimers as Ligands for LectinsofPathogenic Structural basis forsilver-mediated killingofthe PseudomonasAeruginosa human pathogen Vibrio cholerae Magdalena A. Swiderska,Tamis Darbre, Jean-Louis Reymond* MinliTao1,Joachim Diez2,GünterFritz3,Julia Steuber1 Department of Chemistry and Biochemistry, University of Berne, Freiestrasse 3, 3012 Berne, Switzerland 1) Department of Biochemistry, University of Zurich, 8057 Zurich,Switzer- Pseudomonas aeruginosa,animportant opportunisticpathogen asso- land; 2) Swiss Light Source at Paul Scherrer Institut, 5232 Villigen PSI, ciated with lethal infections in cystic fibrosis patients,synthesizes two lec- Switzerland; 3) Fachbereich Biologie, University of Konstanz, tins, PA-IL, specific for D-galactose, and PA-IIL, specific for L-fucose, 78457 Konstanz, Germany which are responsible for the biofilm formation by these bacteria.[1,2] One of the latest developments in this areaisthe study on neoglycoconjugatesbear- ing multivalent carbohydrates in their structure.[3] Recently we reported the Silver compounds are widely used as microcidals in the clinic andfor public discoveryoftetravalent C-fucosyl peptidedendrimerswhich exhibit high health hygiene.Weshow that Ag+ targetsarespiratory, Na+ -translocating affinity for the PA-IILprotein.[4] Heterofunctionality of these macromole- NADH:quinoneoxidoreductase (Na+ -NQR) of Vibriocholerae,the causa- + 2+ cules should enable simultaneousinhibitionofbothPA-IL and PA-IIL. A tive agentofthe diarrheal disease cholera. Both Ag (Ki =8nM) and Hg combinatoriallibrary of second-generation heterofunctional dendrimers, (Ki =90nM) acted as competitiveinhibitors with respecttoNADH oxida- bearingboth fucose andgalactose, was constructed using split-and-mix pro- tion by the holo-complex and its individual FADdomain.Crystals of the tocol. Theligands with thehighest affinity to the lectins were identifiedby FAD domain diffracted to 1.8 Å, and phases were determined by MAD of a on-bead screening of the library and resynthesized. The dendrimers’ inhibi- crystalsoakedwith K Pt(NO ) .The structureofthe FAD domain with tion properties and their bifunctionality potentialwill be presented. 2 2 4 Hg2+ revealed aheavy metal binding site which blocks access of NAD(H) to the FAD cofactor. Pseudomonas sp. (causing woundinfections) and Legio- + [1] Tielker, D.; Hacker,S.; Loris,R.; Strathmann, M.;Wingender, J.; nella sp.(contaminants of drinking water) contain Na -NQRs which are Wilhelm, S.; Rosenau, F.; Jäger, K.-E. Microbiol. 2005, 151,13131- highly related to the V. cholerae enzymestudied here. Thenovel structure of 1323 the FAD domain opensnew strategies to combatthese human pathogens. [2] S. P. Diggle, R. E. Stacey, C. Dodd, M. Camara, P. Williams, K. Winzer, Environmental Microbiol. 2006, 8(6), 1095-1104 [3] I. Deguise, D. Lagnoux, R. Roy, NewJ.Chem. 2007, 31,1321-1331 [4] E. Kolomiets, E. M. V. Johansson,O.Renaudet, T. Darbre, J.-L. Reymond, Org. Lett. 2007, 9,1465-1468

Medicinal Chemistry 115 Medicinal Chemistry 116

Structural Analysis of Serum Albumin usingArtificial Sensor Anions Navigating in Tanimoto Space

Toshiaki Taura,Ken Suyama Ruud vanDeursen and Jean-LouisReymond

UniversityofAichi Prefecture, Nagakute, 480-1198 Aichi, Japan Universit¨at Bern, Freiestrasse 3, 3012 Bern,Schweiz Virtual chemical space exploration has gained interest for drug discovery.1 Recently we reported Chemical SpaceTravel as atoolfor generating ana- Serumalbumin is essential to the transportofmetal ions, fatty acids and other log libraries.2 Herein we report the analysis and docking study of such small molecules or ions in the blood, comprising 60% of the plasmaproteins librariesspanning chemical space between AMPAand CNQX,two lig- [1]. Only humanserum albumin, the crystalstructure was determined and ands of the AMPA-R,animportantneurologicaldrug target. otheralbumintypes thanhuman wasnot analyzed,probably due to the im- perfectcrystallization. In this study, usingstable metal complex anions as a sensor,local structuresofsomeserumalbuminsand binding structureimages of thesensoranionsaround abindingsite were clarified. The sensor anions are assumed to bind the proteins in the area defined as Site Iinsubdomain IIA of serum albumin [2]. The binding mode was examined through calorimetric experiments and docking studies by computational methods.

The binding constantsbetween serum albumins and sensor anions were de- termined in aqueous buffer solutions(pH=7.1, 7.4) by the calorimetric me- thod. The bindingenergies were estimated by molecular mechanical calcu- lationsafter docking simulations of the sensor anionwith structuraloptimi- zation of protein side chains (without that of backbone structures). Albumin structuresofbovine, sheep and pig are obtainedfrom human structures (PDB structures)bychanges of the amino acid sequence. Then, we comparedthe binding free energy of the sensor anions with these types of serum albumins to the binding constantsinthe samesystem.Anewtype of structure-activity relationshipsstudy will be presented. Fig. 1:Binding Energiesofthe 13’146analog library compounds as function of their Tanimoto similaritycoefficientstoAMPAand CNQX.

[1] T. Peters,Jr., Allabout Albumin, 1996,Academic Press. [1] T. Fink and J.-L. Reymond, Angew. Chem. Int. Ed 2005, [2]D.C.Carter, J. X. Ho, Adv. Protein Chem., 1994, 45,153-204. 44,1504. T. Fink and J.-L. Reymond, J. Chem. Inf. Model. 2007, 47,342. [2] R. vanDeursen and J.-L.Reymond, ChemMedChem 2007, 2, 636. MEDICINAL CHEMISTRY558787 CHIMIA 2008, 62,No. 7/8

Medicinal Chemistry 117 Medicinal Chemistry 118 Aggregation of Chlorin-based Potential Photosensitizers in Aqueous Grafting of extracellular loops of Yreceptors onto a β-barrelscaffold Solution and theirInteraction with Phospholipid Vesicles Reto Walser,Oliver Zerbe Martina Vermathen,Peter Bigler University of Zurich, Winterthurerstr. 190, CH-8057 Zurich, Switzerland Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, 3012 Bern, Switzerland G-protein coupled receptors (GPCRs)belong to the class of seven- Chlorins are porphyrinic compounds with one of the pyrrole ring double transmembrane proteins and play apivotal role in signal transduction proc- bonds hydrated leading to ared-shift of the electronic absorption maxima. esses. The GPCRs shareacommon structural motif of an extracellularN- This makes chlorinsinteresting candidatesaspotentialphotosensitizers in terminal segment, linked to abundle of seven membrane-spanning helices Photodynamic therapy (PDT) [1]. However, chlorins tend to self-associate that areconnectedthrough three loops on the intra- and extracellular sides, in aqueous solutions thereby reducing theirwater solubility and membrane followed by an intracellular C-terminal segment1,2. incorporation efficiency,both crucial factors in PDT. In the presentstudy, derivativesofthe naturallyderived compound chlorin e6 (I) bearing various Unfortunately,wild-type GPCRs are very difficult to produce recombi- substituents at theringperiphery (R1,R2)are characterized NMR spectros- nantely. In this work we aim at developing asmall, preferably soluble pro- copically and compared with respect to their aggregation behavior and tein,which possessesastablecore determining the global fold, and com- membrane interaction. Aggregate formation, previously shown for (III) [2], prises surface-exposed loopsthat canbemodified without compromising is studied by analysis of 1HNMR spectroscopic signalbroadening and cha- folding. In such asystem the extracellular portions of aGPCRcould be racteristic ring-current induced shifts of the chlorinsignals. Moreover, asso- transferred onto that core resultinginachimeric protein that couldbecon- ciation with dioleoyl-phosphatidyl-choline (DOPC) vesiclesinsolutionis sidered as a“minireceptor”. Such aprotein contains all the parts of the re- monitoredbyspectral perturbation mapping of the DOPC 1HNMR signals. ceptor hypothesized to be importantfor ligand binding(hence the term “re- Thedata indicate that the formation of large and stable aggregates strongly ceptor”),but at the sametimedisplays the favourable characteristicsof reducesmembrane incorporation efficiency. On the other hand, oligomer small, soluble proteins (hence the term“mini”). formation still enables the chlorins to efficiently distribute into thevesicular Our efforts are concentrated on the constructionand characterization of such phase. pH and concentration effects arediscussed in this context. aminireceptor for the so-called Y-receptor3 family of GPCRs, that is tar-

R 2 (I) R1 =CH2COOH R2 =CH3 geted by peptides of the neuropeptide Y(NPY) family. The scaffold is de-

(II) R =CH R =CH NH N 1 3 2 3 rivedfrom β-barrelproteins, and NMR techniques areused to assess to NH which extentthe global fold is retained in the loop-modified mutants. N HN (III) R1 = CO2H R2=CH3

O CO2H CO H R 2 [1] Palczewski, Science, 2000,289, 739 HO C 1 (IV) R =CHCOOH R =CHO 2 1 2 2 [2] Rasmussen, Nature, 2007,450, 383 [1] A. Castano et al., Photodiag. Photodynam.Ther. 2004, 1,279. [3] Grundemar, Neuropeptide Yand Drug Development, Academic Press, [2] S. Gomietal., Heterocycles 1998, 48,2231. San Diego, 1997

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Selective DNA-binding of metal complexes embedded within aprotein Studies of theMechanismfor Activation of Caspase-8 by NMR host:anovel technologyfor improved anticancer therapy Nadine Keller1,JiriMares2,Oliver Zerbe2 andMarkus Grütter1 Creus,M., WohlschlagerT.,Zimbron, J.M., Sardo A., Ward T.R. 1Depatment of Biochemistryand 2Institute of OrganicChemistry, University Department of Chemistry, University of Basel, Spitalstrasse 51, CH-4056 of Zurich,Winterthurerstr. 190,CH-8057 Zurich,Switzerland Basel,Switzerland Apoptosis is aspecificformofprogrammedcelldeaththatplays avitalrole Bioinorganic drugs,such as theplatinum-drug oxaliplatin, are important in multicellularorganisms.Two different pathways areknown fortriggering anticancer compounds. However, they are highly toxicand causeserious apoptosis:The extrinsic(extracellular) andthe intrinsic(intracellular) path- side-effects, due to lack of selectivitytotheir DNAtarget. way[1].Inthe extrinsicpathway theFas ligandbinds to theFas receptor resultinginreceptoroligomerizationfollowedbythe recruitment of FADD. We hypothesized that theincorporation of awell-defined protein environ- FADD can then bind to avariety of otherproteins, such as theinitiator ment around aDNA-binding complex would increaseselectivity toward a caspases-8and -10. Activated caspase-8 is released fromthe DISC andac- specificDNA targetthrough second coordination interactions. Here we de- tivates theexecutionercaspases-3and -7. scribe achemogenetic screen to identifyvariants of metal complexes em- GenerationofanactiveCaspase-8requireslimitedproteolysis.Duringthis bedded within aprotein scaffold exhibiting increased DNA-binding activity in vitro. activationprocessCaspase-8 is autoproteolytically cleaved at twoAsp sites. Theactivesite of theenzymecontains an catalytically important Cysresi- We used streptavidin as a‘targeting host-protein’ and abiotinylated metal due.Caspase activationisbelievedtobeinduced by dimerization(thein- complex as a‘non-selective drug’. We foundthat the nature of the complex duced proximity model) [3]. as well as definedsinglepoint-mutations around the ‘activesite’ of the pro- In this workwehavedetermined thesolutionstructure of a266 residue mu- tein-host influenced target-recognitionand binding to cancer-specificDNA- tant of Caspase-8, in whichthe catalytic Cyswas replaced by Ala.Thismu- sequences. tant cannot undergoautoproteolysis, andremains monomeric.Inparticular, thepositionofalong loop, whichcontains thecleavagesites, couldbede- Theseproof-of-principle studies represent, to our knowledge, the first ex- finedinthe structure. Processing of Procaspase-8 wasfollowedinthe NMR ample of increased selectivity of bioinorganic drugs toward cancer-specific tube by adding smallamountsofactiveCaspase-8.Moreover, propertiesof DNAsequences usingaproteincarrier. We believe that this novel technolo- mutants, in which thecleavagesite Aspresiduesare replaced by Ala while gy maylead to improved anticancer (bio)chemotherapy. preserving theactive-site Cysare presented. Finally,amutant that contains theAsp andCys residues, but in which dimerizationisimpairedcould showntobesimilarinstructure.

[1]Danial, N.N. andS.J.Korsmeyer,Cell 2004,116, 205. [2]Wajant, H.,Science 2002.296,1635. [3]Salvesen, G.S.,Dixit, V.M., PNAS USA 1999,96, 10964.