François Diederich, born in1952 in the Grand Duchy ofLuxembourg, received hisPh.D. degree in 1979 from theUniversity of Heidelberg. Afterpostdoctoral studies at theUniversity of California at LosAngeles (UCLA) andHabilitation at the Max-Planck-Institut fürmedizinische Forschung inHeidelberg, he joined in 1985the Faculty of the Departmentof Chemistry andBiochemistry at UCLA wherehe became full Professor in

1989. In 1992, he moved to the ETH Zürich, where he currentlyserves as the head of the new Department of Chemistry andApplied Biosciences. His research interests span from medicinal

Medicinal chemistry inacademia: molecularrecognition with biologicalreceptorsFraser Hof and François Diederich*

Laboratorium für Organische Chemie, ETH Hönggerberg, HCI, CH-8093 Zürich, Switzerland. E-mail:diederich@org.chem.ethz.ch

Why carry out medicinal chemistry at a university, when it means competing withthe billion-dollar research efforts of the pharmaceutical industry? In academicresearch, the race to get a drug to market is not the prime motivation. Instead,university-based medicinal chemistry is driven by the search for new knowledgeand the opportunity to educate a new generation of chemists. Furthermore,academia can complement commercial efforts by addressing diseases neglected byprivate industry.

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Fraser Hof was born inMedicine Hat, Canada. Heobtained a B.Sc. in chemistryat the University of Alberta in1998, and a Ph.D. at theScripps Research Institute in2003 under the direction ofProfessor Julius Rebek, Jr. Heis currently a NovartisStiftung post-doctoral fellowin the laboratory of ProfessorDiederich at ETH Zürich. Hisresearch centres oninhibiting the malarialprotease plasmepsin II.

chemistry, with a focus on molecular recognition studies, toadvanced fullerene and acetylene-based materials with noveloptoelectronic properties.

Pharmaceutical research hastraditionally been considered the realm ofindustry, but academic laboratories havealways played a role in developing newideas and methods. No matter what theresearch setting, medicinal chemistry is afertile field for learning about molecularbehaviour. Medicinal chemistry is asignificant activity in numerouslaboratories around the world, whereverthere is expertise in chemical synthesis andan interest in biological phenomena. Herewe present three case histories from ourown laboratory that demonstrate why andhow medicinal chemistry is carried out inthe academic setting.

Thrombin inhibitors fromstructure-based designIn the active site of a rigid enzyme(thrombin), we were able to carry outsome fundamental molecular recognitioninvestigations. This approach turns the“receptor design” paradigm of traditionalmolecular recognition chemistry insideout—here, one must design appropriateguests to engage the receptor from withinits concave binding pockets. The detailedexamination of ligand interactions withinprotein environments teaches valuablelessons in molecular recognition.

In the blood coagulation cascade,thrombin catalyses clot formation via the

cleavage of the soluble blood proteinfibrinogen into insoluble fibrin. Thrombinhas long been a prime pharmaceuticaltarget because clotting disorders areamong the leading causes of death in thedeveloped world.1 Moreover, the X-raystructure of thrombin shows that its activesite has limited flexibility.2,3 These factorshave driven various structure-basedinhibitor design programs.4,5

Owing to the rigidity of the active site,related ligands with functional groupmutations form complexes of similargeometry, allowing direct correlation ofmeasured changes in the complexation freeenthalpy with the contributions of

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individual ligand substituents. Our ownefforts at thrombin inhibition began with arigid tricyclic core6 which can directvectors into the hydrophobic bindingpockets P and D while introducing aphenylamidinium group to occupy theselectivity pocket S1 (Fig. 1). The crystalstructure of the best first-generationinhibitor ((±)-1) complexed with thrombinconfirmed the predicted binding mode.7

Stepwise mutation of the inhibitor thenallowed exploration of the molecularrecognition properties of discrete regionswithin the thrombin active site.

One of the inhibitor’s imide carbonyloxygens accepts a hydrogen bond from thebackbone NH group of Gly216 (Fig. 1).8

When the hydrogen bond is removed, byreplacing the carbonyl with a methylenegroup, the enzyme-inhibitor complex isdestabilised by 3.4 kJ mol21. Thisprovides a good estimate of theincremental free enthalpy arising from asingle hydrogen bond in a biologicalsetting.

The P pocket of thrombin is a smallhydrophobic cavity lined with aromaticresidues (Tyr60A and Trp60D). In thefirst-generation inhibitors, modelling andcrystallography showed that an imidecarbonyl occupies this sub-site. Theseinhibitors demonstrate only modest (lessthan tenfold) selectivity for thrombin overtrypsin. Since the P pocket is not presentin related proteases, such as trypsin, weproposed that replacing this carbonyl witha more hydrophobic residue wouldincrease both the affinity for thrombin and

selectivity over trypsin.The imide oxygen in question was

replaced with a variety of alkyl groups toyield inhibitors (±)-2–(±)-6 (Fig. 1).8

Substitution with an isopropyl group ((+)-4) gives an impressive combination ofincreased affinity (7 nM) and selectivityover trypsin (740-fold). The crystalstructure of (+)-4 complexed withthrombin shows that, as predicted, theisopropyl group is nestled snugly againstthe aromatic residues that line the Ppocket.8

Unlike the cosy P pocket, the D pocketis spacious enough to accommodate cyclicbinding elements such ascyclohexylmethyl ((±)-7) and benzyl ((±)-8) groups (Fig. 2). According to

modelling, both substituents adopt anedge-to-face orientation with respect toTrp215. Their similar affinity for thrombinsuggests that specific electrostatic CH–pinteractions need not be invoked for thebenzyl complex, supporting earlierproposals that edge-to-face aromaticinteractions are mainly dispersive innature.9

Fluorinated organic compounds are wellrepresented among current therapeuticagents, yet the nature of noncovalentinteractions experienced by organicfluorine is still an issue for discussion.10

We therefore treated the D pocket ofthrombin to a fluorine scan,11,12 anapproach that might not have beenfavoured in an industrial setting, but onewhich yielded results of interest toresearchers in academia and industry alike.

Compounds (±)-9–(±)-14 (Fig. 2) eachinhibited thrombin with similar efficiency,with one notable exception. The 4-fluoroderivative (±)-11 shows fivefold enhancedactivity (DDG = 4.4 kJ mol21) relative tothe parent compound (±)-8. The crystalstructure of the enzyme-inhibitor complexreveals that the carbon-bound fluorineatom is in close contact with the enzyme.The fluorine atom is only 3.1 Å away fromthe a-carbon of Asn98, forming a dipolarC–H…F–C contact. It is also only 3.5 Åaway from the carbonyl carbon of thesame residue. Further exploration of thelatter unusual interaction led to thediscovery within the Cambridge StructuralDatabase13 of 43 examples of sub-van derWaals (d(F…C) < 3.09 Å) contactsbetween organic fluorine atoms andcarbonyl carbons. The fluorine atomsclearly prefer a position close to thepseudo-trigonal axis of the carbonyl unit.At short distances, the F…CNO angletends toward 90°; at longer separation theangle dependence is weaker. Thisattraction may arise from the interaction ofthe partially negative fluorine with thepartially positive carbonyl carbon. Protein

Fig. 1 De novo designed thrombin inhibitors that explore the binding affinity and selectivityavailable within the small, hydrophobic P pocket. The carbonyl group that hydrogen bonds toGly216 is highlighted in red. a) Ki (trypsin)/Ki (thrombin).

Fig. 2 Exploration of the thrombin D pocket, including a series of compounds that constitute afluorine scan. On the right are depicted the unusually short fluorine contacts (Å) observed in thecomplex of thrombin with the active enantiomer of (±)-11.

data base mining subsequently showed thatthe fluorine atoms of ligand CF3 groupsmay also be able to form short dipolarC–F…C(O) contacts. Using this newinformation, structure-based designsshould be able to use rational fluorinesubstitution to improve ligand affinity.

Novel design of bisubstrateinhibitors of catechol O-methyltransferaseMuch can be learned about molecularbehaviour through the detailed scrutiny ofissues such as linker effects and enzymekinetics. The enzyme catechol O-methyltransferase (COMT) uses thecofactor S-adenosylmethionine (SAM) anda Mg2+ ion to methylate biologicalcatechols, including L-dopa and dopamine,which can hinder treatment of Parkinson’sdisease by L-dopa. Concomitant treatmentwith COMT inhibitors decreases the rateof L-dopa metabolism, increasing theamount of the drug delivered to thebrain.14 Nitrocatechol derivatives thatmimic the natural substrates have beendeveloped as COMT inhibitors, includingtwo currently marketed drugs (Tasmar®and Comptan®).15,16 In a novel approachto COMT inhibition, we created inhibitorsoccupying both the SAM and catecholbinding sites of the enzyme.17 Theseinvolve an inhibitor design where acatechol derivative is attached via avariable linker to the 5A position ofadenosine. The methionine element ofSAM is omitted on the understanding thatits complementary polar binding pocketcould be hydrated without great cost infree enthalpy. The success of the resultinginhibitors justifies this simplification,providing an important lesson in inhibitordesign.

A small family of inhibitors wassynthesised in which only the structure ofthe linker between adenosine and catecholwas varied (Fig. 3).17–20 The effect of thelinker on inhibitory strength is striking;within this family, the inhibitoryconcentrations (IC50 = inhibitorconcentration at which 50% Vmax isobserved) span five orders of magnitude!Evidence for the bisubstrate action of thebest inhibitor 18 was provided by enzymekinetics and X-ray crystallography.18

Structural analysis of the ternary complexof COMT, Mg2+ and 18 shows the adenineand ribose moieties contacting theexpected residues within the SAM pocket,while the nitrocatechol moiety occupiesthe catechol binding pocket as predicted(Fig. 3).

What gives rise to the variableperformance of inhibitors 15–20? Linkersthat are too long (15 and 16) or too short(20) provide only micromolar-rangeinhibition of COMT. Comparing inhibitors17 and 18 reveals a more subtle effect.

Inhibitor 18 has a fully flexible linker,allowing hydrophobic collapse of the twoaromatic binding elements: catechol andadenine. Introduction of one double bondin the linker of 18 removes one degree offreedom, and the effect of this structuralrigidification is significant: 18 is 20 timesmore potent than 17.

Drugs for a developingworld—plasmepsin II, a newantimalarial targetAnother major role for academic medicinalresearch is to tackle diseases of thedeveloping world which are oftenneglected by the pharmaceutical industry.Ideally, these publicly produced leadcompounds will be further developed intoviable drugs in collaboration withcommercial organizations. Malariaremains one of the most significantinfectious diseases, affecting up to 500million people annually.21 The increasingprevalence of Plasmodium strains resistantto existing treatments demands thedevelopment of new antimalarial drugswith novel modes of action.22

Plasmepsin II is an aspartic proteaseused by Plasmodium parasites for theproteolysis of human haemoglobin toprovide nutrients during their rapid growthand multiplication.23 The related humanaspartic protease, renin, undergoes a largeconformational change that exposes a newhydrophobic flap pocket upon bindingappropriate inhibitors.24–26 Owing to thehomology between renin and plasmepsinII, we hypothesised that a similarconformational shift may also occur inplasmepsin II. In the absence of a

plasmepsin II structure in the flap-openconformation,27 we initiated a structure-based design project for plasmepsin IIinhibition using a homology model thatsuperimposes the conformational shiftsobserved for renin onto plasmepsin II.28

Using computer modelling, weidentified a suitable scaffold for inhibitingplasmepsin II. The structure engages thecatalytic aspartate dyad with a bicyclicammonium ion while directing a largearomatic substituent into each of thehydrophobic S1/S3 cleft and the flappocket (Fig. 4).28–30 In the modelledstructure of plasmepsin II the S1/S3 cleftis shallow and solvent-exposed, suggestingthat little specificity can be gained in thisarea. But the addition of a single chlorineatom (as in (±)-24) to fill space at the rearof the hypothetical flap pocket provides afourfold boost in activity.28 Omission ofeither of the two aromatic bindingelements (structures not shown) results intenfold decreased activity, suggesting thatboth the S1/S3 cleft and the hypothesisedflap pocket must be filled for efficientligand binding.

The biological results obtained withthese first-generation inhibitors suggestthat the opening of the flap pocket ispossible for plasmepsin II. This hypothesishas now been validated with aconceptually similar ligand. This work,carried out at Roche, provided the firstcrystal structure of plasmepin II in theflap-open conformation.31 The goodagreement between the experimentalstructure and the homology modelconfirms the accuracy of the computer-based structure prediction.

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Fig. 3 Linker effects within a family of bisubstrate inhibitors of COMT, and the crystal structureshowing the elongated binding conformation of the best inhibitor, 18. The water-coordinatedmagnesium ion is shown in green.

In conclusionWe are well aware that there are otherimportant motives for academic medicinalchemistry that are not addressed within thelimited space of this article. Notableexamples are the searches for practicalsolutions for interfering withprotein–protein binding and for binding toflat protein surfaces.32,33 Such advanceswould make available a plethora of newtargets for disease treatment. Anotherimportant objective for academicmedicinal chemistry is to contribute to thedevelopment of efficient in silicoscreening methods of virtual libraries.

Along with new lessons in molecularrecognition, design strategies and leads forneglected diseases, medicinal research inacademia provides another significantbenefit. It is the most important, and oftenoverlooked, reason for carrying outresearch in an academic environment: theintroduction of students and postdoctoralchemists to the fascinating problems ofbiological recognition within the contextof chemical synthesis. In this way,interdisciplinary research in academia actsas an ‘incubator’—training newresearchers, many of whom go on to makevaluable contributions to thepharmaceutical industry.

Notes and references1 M. F. Scully, in Essays in Biochemistry,

ed. K. F. Tipton, Portland Press, London,1996, p. 17.

2 W. Bode, I. Mayr, U. Baumann, R. Huber,S. R. Stone and J. Hofsteenge, EMBO J.,1989, 8, 3467.

3 D. W. Banner and P. Hadvary, J. Biol.Chem., 1991, 266, 20085.

4 P. E. J. Sanderson and A. M. Naylor-Olsen,Curr. Med. Chem., 1998, 5, 289.

5 K. Menear, Curr. Med. Chem., 1998, 5,457.

6 R. Grigg and S. Thianpatanagul, J. Chem.Soc., Chem. Commun., 1984, 180.

7 U. Obst, V. Gramlich, F. Diederich, L.Weber and D. W. Banner, Angew. Chem.,Int. Ed. Engl., 1995, 34, 1739.

8 U. Obst, D. W. Banner, L. Weber and F.Diederich, Chem. Biol., 1997, 4, 287.

9 S. Paliwal, S. Geib and C. S. Wilcox, J.Am. Chem. Soc., 1994, 116, 4497.

10 J. D. Dunitz and R. Taylor, Chem. Eur. J.,1997, 3, 89.

11 J. A. Olsen, D. W. Banner, P. Seiler, U.Obst Sander, A. D’Arcy, M. Stihle, K.Müller and F. Diederich, Angew. Chem.,Int. Ed., 2003, 42, 2507.

12 C.-Y. Kim, J. S. Chang, J. B. Doyon, J. T.A. Baird, C. A. Fierke, A. Jain and D. W.Christianson, J. Am. Chem. Soc., 2000,122, 12125.

13 CSD version 9.24, November 2001.14 P. T. Mannistö, I. Ulmanen, K. Lundström,

J. Taskinen, J. Tenhunen, C. Tilgmann andS. K. Kaakkola, Prog. Drug Res., 1992,39, 291.

15 J. Borgulya, M. D. Prada, R. Dingemanse,B. Scherschlicht, B. Schläppi and G.Zuercher, Drugs Future, 1991, 16, 719.

16 E. Nissinen, I. B. Linden, E. Schultz and P.Pohto, Arch. Pharmacol., 1992, 346, 262.

17 B. Masjost, P. Ballmer, E. Borroni, G.Zuercher, F. K. Winkler, R. Jakob-Roetneand F. Diederich, Chem. Eur. J., 2000, 6,971.

18 C. Lerner, A. Ruf, V. Gramlich, B.Masjost, G. Zuercher, R. Jakob-Roetne, E.Borroni and F. Diederich, Angew. Chem.,Int. Ed., 2001, 40, 4040.

19 C. Lerner, R. Siegrist, E. Schweizer, F.Diederich, V. Gramlich, R. Jakob-Roetne,G. Zuercher and E. Borroni, Helv. Chim.Acta, 2003, 86, 1045.

20 C. Lerner, B. Masjost, A. Ruf, V.Gramlich, R. Jakob-Roetne, G. Zuercher,E. Borroni and F. Diederich, Org. Biomol.Chem., 2003, 1, 42.

21 M. Nacher, Trends Parasitol., 2001, 17,563.

22 C. H. Sibley, J. E. Hyde, P. F. G. Sims, C.V. Plowe, J. G. Kublin, E. K. Mberu, A. F.Cowman, P. A. Winstanley, W. M. Watkinsand A. M. Nzila, Trends Parasitol., 2001,17 , 582.

23 C. Boss, S. Richard-Bildstein, T. Weller,W. Fischli, S. Meyer and C. Binkert, Curr.Med. Chem., 2003, 10, 883.

24 C. Oefner, A. Binggeli, V. Breu, D. Bur, J.P. Clozel, A. D’Arcy, A. Dorn, W. Fischli,F. Gruninger, R. Guller, G. Hirth, H. P.Marki, S. Mathews, M. Muller, R. G.Ridley, H. Stadler, E. Vieira, M. Wilhelm,F. K. Winkler and W. Wostl, Chem. Biol.,1999, 6, 127.

25 M. G. Bursavich and D. H. Rich, Org.Lett., 2001, 3, 2625.

26 M. G. Bursavich, C. W. West and D. H.Rich, Org. Lett., 2001, 3, 2317.

27 O. A. Asojo, S. V. Gulnik, E. Afonina, B.Yu, J. A. Ellman, T. S. Haque and A. M.Silva, J. Mol. Biol., 2003, 327, 173.

28 D. A. Carcache, S. R. Hörtner, A. Bertogg,C. Binkert, D. Bur, H. P. Märki, A. Dornand F. Diederich, ChemBioChem, 2002, 3,1137.

29 D. A. Carcache, S. R. Hörtner, A. Bertogg,F. Diederich, A. Dorn, H. P. Märki, C.Binkert and D. Bur, Helv. Chim. Acta,2003, 86, 2192.

30 D. A. Carcache, S. R. Hörtner, P. Seiler, F.Diederich, A. Dorn, H. P. Märki, C.Binkert and D. Bur, Helv. Chim. Acta,2003, 86, 2173.

31 J. Yuvaniyama, A. D’Arcy, C. Oefner, H.Loetscher, D. Bur, G. E. Dale, H. P. Märki,F. K. Winkler, R. P. Moon and R. G.Ridley, Symposium on “Parasite Proteasesas Drug Targets”, University of Glasgow,UK, 2000.

32 H. S. Park, Q. Lin and A. D. Hamilton,Proc. Natl. Acad. Sci. U. S. A., 2002, 99,5105.

33 L. T. Vassilev, B. T. Vu, B. Graves, D.Carvajal, F. Podlaski, Z. Filipovic, N.Kong, U. Kammlott, C. Lukacs, C. Klein,N. Fotouhi and E. A. Liu, Science, 2004,in press.

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Fig. 4 First-generation inhibitors of plasmepsin II, and their proposed occupation of the active site.The Trp41 residue that swings aside to open the flap pocket is highlighted in red. The design ofthese inhibitors was based on an enzyme structure constructed by homology modelling.