lindhorst t. k. essentials of carbohydrate chemistry and biochemistry

MICROREVIEW

DOI: 10.1002/ejoc.201100407

The Bacterial Lectin FimH, a Target for Drug Discovery – CarbohydrateInhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion

Mirja Hartmann*[a] and Thisbe K. Lindhorst*[a]

Keywords: Lectins / Cell adhesion / Carbohydrates / Cluster effect / Inhibitors

Adhesion is a prerequisite for bacteria to colonize cell sur-faces. To accomplish cellular adhesion, many bacteria usecarbohydrate-specific lectins, which are expressed as part ofcapillary protein appendages expanding from their surface,called fimbriae or pili. For bacteria, colonization of cell sur-faces offers advantageous conditions to persist and multiply.For the host, however, bacterial colonization can be affiliatedwith severe health problems such as inflammation. There-fore, to combat bacterial adhesion and inflammatory dis-eases, investigation of the molecular and biophysical detailsof the relevant lectin–carbohydrate interactions is important.Understanding molecular carbohydrate recognition can lead

Contents1. Introduction

2. Early studies on the carbohydrate specificity of type 1 fimbriae

3. Multivalent glycomimetics as inhibitors of type 1 fimbriae-medi-ated bacterial adhesion

4. Assays to test antagonists of type 1 fimbriae-mediated bacterialadhesion

[a] Otto Diels Institute of Organic Chemistry,Christiana Albertina University of KielOtto-Hahn-Platz 3/4, 24098 Kiel, GermanyFax: +49-431-880-7410E-mail: [email protected]

[email protected]

Mirja Hartmann, M.Sc., was born in 1983. She studied Molecular Life Science at the University of Erlangen-Nurembergwith main focus on Drug Discovery, Molecular Biology and Molecular Synthesis. For her master thesis, she developedstrategies for the synthesis of glycoconjugates and their biological testing with Prof. Mikael Elofsson at Umeå Universityin Sweden. In 2007 she received her Master of Science degree from the University of Erlangen-Nuremberg and joined theresearch group of Prof. Thisbe Lindhorst at Christiana Albertina University of Kiel for her Ph.D. studies. Currently, sheis working on biochemical assays and biophysical setups to test type 1 fimbriae-mediated bacterial adhesion.

Thisbe Lindhorst is Full Professor at the Faculty of Mathematics and Natural Science of Christiana Albertina Universityof Kiel since 2000. She studied chemistry at the Universities of Munich and Münster, received her diploma in chemistry/biochemistry in 1988 and her Ph.D. in Organic Chemistry in 1991 at the University of Hamburg in the group of Prof. J.Thiem. After a postdoctoral stay at the University of British Columbia with Prof. S. Withers, she worked on her habilita-tion and became Private Docent in 1998 at the University of Hamburg. In 1997 she was a Visiting Professor at theUniversity of Ottawa in Canada in Prof. R. Roy’s laboratory. Since 2000, she holds a chair in Organic and BiologicalChemistry in Kiel. Her scientific interests are in the field of synthetic organic chemistry and in biological chemistry, andin particular in glycochemistry and glycobiology. Current research is focussed on the study of cell adhesion to glycosylatedsurfaces. She is the author of the textbook “Essentials of Carbohydrate Chemistry and Biochemistry”.

Eur. J. Org. Chem. 2011, 3583–3609 © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim 3583

to the development of high-affinity inhibitors of bacterial lec-tins. That way, interfering with the bacterial attachment tosurfaces proves the vision of an antiadhesion therapy, amongothers, against uropathogenic E. coli (UPEC). One of the mostimportant and best investigated bacterial lectins is the man-nose-specific protein FimH, which is expressed on the tips oftype 1 fimbriae. During the last 30 years, many natural aswell as synthetic mannosidic ligands of FimH have been de-signed and tested for their inhibitory potencies. We reportkey results and comment on key problems and perspectivesof this research.

5. Structure of the type 1 fimbrial lectin FimH

6. Rational design of carbohydrate ligands for the type 1 fimbriallectin FimH

7. Special approaches to the inhibition of mannose-specific bacte-rial adhesion

1. Introduction

There is a class of proteins that reversibly bind carbo-hydrates. These proteins are neither carbohydrate-specificenzymes nor antibodies. Today, the understanding of glyco-scientists is that this type of proteins has evolved to recog-nize specific carbohydrates and select them for binding to

M. Hartmann, T. K. LindhorstMICROREVIEWform carbohydrate–protein complexes. While, apparently,carbohydrate–protein complexation has no obvious conse-quence for the structural integrity of the complexed andsubsequently released saccharide ligand, such molecular re-cognition of carbohydrates can trigger biological responseof different kinds in all types of organisms, such as cell re-cognition, signalling and cell adhesion.

Lectins

The carbohydrate-complexing proteins called “lectins”comprise a remarkable variety of structures, folds, functionsand occurrence. The history of lectins goes back to the endof the 19th century.[1] At that time, work with extracts fromseeds of the castor tree (Ricinus communis) led to the dis-covery that certain plant proteins have the ability to aggluti-nate erythrocytes. The agglutinating protein isolated fromRicinus communis was called “ricin”. Rapidly, many moreproteins with the same feature were discovered in variousplants and were called phytoagglutinins, phytohemaggluti-nins and somewhat later hemagglutinins. Soon they wereemployed in immunological studies, leading to the findingthat specific hemagglutinins react specifically with humanred blood cells of certain blood groups (A, B, or O).[2]

In 1954, the term “lectin” was proposed by Boyd andShapleigh “for these and other antibody-like substances”with blood-group-specific agglutination properties.[3] Theauthors quoted the name to be derived from “the Latinlego, to choose or pick out”; (in fact the Latin verb legeremeans to gather, choose, collect, select, pass through, read,and its perfect passive participle is lectus, meaning selectedand picked out, or read out.) In the 1970s Sharon and Lis[4]

suggested the term lectin as a general name for all proteinsof nonimmune origin that possess the ability to agglutinateerythrocytes and other cell types.

It was discovered very early that the interactions of lec-tins with cells can be inhibited by specific carbohydrates,mono- or oligosaccharides. Consequently, it was concludedthat lectins are specific saccharide-binding proteins. Thiswas first shown for the well-known plant lectin concanava-lin A.[5] As a consequence, the first attempt to classify thefast-growing class of lectins was based on their carbo-hydrate specificity. Today, lectins are being classified on thebasis of their structural features and especially the re-latedness of their carbohydrate-binding sites, which arecalled “carbohydrate recognition domains”, in shortCRDs.[6] It was Drickamer who pointed out the importanceof sequence homologies in the carbohydrate recognition do-main motifs of proteins for their carbohydrate-bindingproperties.[7,8] The carbohydrate specificity of lectin CRDshas remained a key criterion for the assessment of lectinsso far.[9]

Fimbrial Lectins of Bacteria

Early in the history of lectins, it had become evident that,besides numerous plant lectins, most, if not all, organismsexpress this class of proteins, for very different purposes,

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however. Already during the first half of the 20th century,it was discovered that also many bacteria, in particularthose of the Enterobacteriaceae family, have the ability toagglutinate erythrocytes. This hemagglutination activity ofbacteria is almost always associated with the presence ofmultiple filamentous protein appendages projecting fromtheir surface, called fimbriae (from the Latin word for“thread”) or pili (from the Latin word for “hair”) (Fig-ure 1). Fimbriae are adhesive organelles, comprising lectinsubunits, which mediate carbohydrate-specific adhesion tocell surfaces as well as cell agglutination. Thus, bacteria uti-lize the sugar decoration of cells, the glycocalyx, to colonizethe cell surface, wherever cells are in contact with the out-side environment, as for example in the case of epithelialcells.

Figure 1. Transmission electron micrograph of a type 1 fimbriatedE. coli cell. Some hundreds of the adhesive organelles cover thebacterial surface.

Bacterial Adhesion and Diseases

Initial bacterial adhesion to cell surfaces is in turn ampli-fied, leading to the development of a well-organized super-structure, called a biofilm. Biofilm formation is highly ad-vantageous for the colonizing microbes. It facilitates firmand irreversible adhesion to a surface, interlinking bacteriaof different species, which produce a carbohydrate mucusto maintain the biofilm.[10] Through this exopolysaccharidelayer,[11] bacteria can achieve chemical communication andprofit from favourable coordination (quorum sensing).

Biofilm formation can form the basis of a beneficial sym-biosis between a microorganism and its host such as in thegut, where Escherichia coli (E. coli) bacteria produce vita-min K in the large intestine. However, as soon as microor-ganisms invade another habitat or only slightly change theirgenes, disorders such as inflammation, or even apoptosisor uncontrolled cell growth can arise.[12] Typically, bacterialcolonization is accompanied by infectious diseases, and thisconstitutes a major global health problem.[13] In addition,biofilm formation on medical devices and implants fre-quently causes complications[14] and significantly contrib-utes to the pathogenesis of implant-related infections.

Among the most prevalent inflammatory diseases thatare caused by pathogens are urinary tract infections. Thepredominant pathogens in this case are uropathogenic E.

Carbohydrate Inhibitors of Type 1 Fimbriae-Mediated Bacterial Adhesion

coli (UPEC). UPEC can attach to specific niches in the uri-nary tract by virtue of the interaction of their surface fim-briae with carbohydrate receptors on the luminal surfaceof the bladder epithelium, leading to bladder infection andinflammation (cystitis).[15]

Carbohydrate Antagonists and Antiadhesion Therapy

Adhesion to the surface of the host cell is a prerequisitefor colonization and successful reproduction conditions es-pecially for enteric, oral and respiratory bacteria. Adher-ence protects bacteria from being swept away by the normalcleansing mechanism operating on mucosal surfaces suchas urinary flow. Thus, successful adhesion increases theability of the bacteria to colonize epithelial cells, multiplyand eventually also invade the host. As bacterial adhesionis mediated by interactions with cell surface carbohydrates,the intriguing idea to prevent bacterial colonization bycarbohydrate inhibitors of adhesion arises. Thus, carbo-hydrates could be developed as antiadhesive drugs accord-ing to a strategy against pathogens that is an alternative toantibiotics. An antiadhesive therapy[16–18] could help tofight especially those bacteria with multi-antibiotic resis-tance, constituting an increasing problem in medicine.[19] Itcan be considered as rather unlikely that bacteria developresistances against antiadhesive drugs.

Hence, studies on the carbohydrate-specificity of bacte-rial lectins have been largely motivated by the importanceof carbohydrate-mediated bacterial adhesion in infection,and this research has led to the vision to prevent bacterialadhesion by suitable carbohydrate antagonists of naturallectin ligands. There are many different lectins of Gram-negative bacteria known with various carbohydrate specifi-cities. In E. coli, P fimbriae (specific for Galα1,4Gal), S fim-briae (specific for Neu5Acα2,3Gal), and type G fimbriae(specific for GalNAc) are found, as well as the mannose-specific type 1 fimbriae, which are one of the most abun-dant surface structures both in pathogenic and nonpatho-genic Gram-negative bacteria.[20]

Type 1 Fimbriae

Type 1 fimbriae serve as extremely efficient adhesiontools for bacteria inhabiting diverse environments, includingbiotic and abiotic surfaces.[21] They are uniformly distrib-uted on Enterobacteriaceae, commonly between 100 and400 fimbriae per cell. Their length varies between 0.1 to 2micrometer, their width is approximately 7 nm (Figure 1).Fimbriae are present in at least 90% of all known UPECstrains, which are the main cause of urinary tract infectionsin humans, and they have been shown to be important viru-lence and pathogenicity factors.[22] Type 1 fimbriae mediateagglutination of guinea pig red blood cells in an α-man-nose-inhibitable manner and are responsible for bacterialbinding to a wide range of glycoproteins carrying one ormore N-linked high-mannose type oligosaccharide.[23] Type1 fimbriae of the E. coli type have been classified as type

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1E,[24] but are simply referred to as type 1 fimbriae in moststudies, as well as in this account. Because of the impor-tance of type 1 fimbriae for bacterial adhesion, numerousstudies on their carbohydrate specificity were undertaken,in order to design effective inhibitors of bacterial adhesionto mucosa.

2. Early Studies on the Carbohydrate Specificityof Type 1 Fimbriae

Interest in type 1 fimbriae started relatively early. Ap-proximately since the 1970s it was known that hemaggluti-nation mediated by type 1 fimbriae can be inhibited bymannose, methyl α-d-mannoside (MeMan) and mannan.[20]

Then, evidence that type 1 fimbriae are major virulence fac-tors of UPEC motivated a number of more in-depth investi-gations on the carbohydrate-specificity of type 1 fimbriae.Ofek and colleagues showed that the carbohydrate specific-ity of the E. coli type 1 fimbrial lectin can be studied quan-titatively by examination of the inhibitory potency ofmono- and oligosaccharides on the agglutination of yeastcells by bacteria. In this agglutination inhibition assay, theconcentration of MeMan that is required to inhibit 50 % ofyeast agglutination by E. coli 364 (a strain which predomi-nately expresses type 1 fimbriae) was determined to rangebetween 0.15 and 0.40 mm.[25] A number of oligosaccha-rides were tested as inhibitors of yeast agglutination andcompared to the inhibitory potency of MeMan, which wasarbitrarily set to 1 (Figure 2). Mannobiosides Manα1,2Man(1), Manα1,3Man (2) and Manα1,6Man (3), as well as thebranching trimannoside Manα1,6[Manα1,3]Manα1-OMe(4), formed a first group of tested saccharides, of which onlytrisaccharide 4 showed a significantly higher inhibitory po-tency than MeMan [RIP(4)MeMan = 10.5]. In 2006, verysimilar saccharides were investigated as inhibitors of the ad-hesion of type 1 fimbriated E. coli to mannan in an enzyme-linked immunosorbent assay (ELISA).[26] Lindhorst andco-workers employed allyl mannobiosides Manα1,2Manα1-OAll (5), Manα1,3Manα1-OAll (6), Manα1,6Manα1-OAll(7) and Manα1,4Manα1-OAll (8), and the allyl trimanno-side Manα1,6[Manα1,3]Man1α-OAll (9), and largely foundrelative inhibitory potencies similar to those reported byOfek and his colleagues. However, the α1,3-linked disaccha-ride 6 performed significantly better in the ELISA, givinga RIP value of 11 (Figure 2). It should be noted, however,that the standard deviation in this assay was determined as7, which was rather high. Also the allyl trisaccharide 9[RIP(9)MeMan = 20] was a better inhibitor than the analo-gous methyl trisaccharide 4 [RIP(4)MeMan = 10.5], thoughin different assays.

When Ofek and co-workers tested more complex oligo-saccharides, the strict specificity of the type 1 fimbrial lectinfor α-mannosides was confirmed. However, it was alsoshown that the way an α-mannosyl ligand is scaffolded ona particular oligosaccharide is crucial. While for trisaccha-ride 10, Manα1,3Manβ1,4GlcNAc, a RIP value of 21 wasdetermined, its isomer 11, Manα1,6Manβ1,4GlcNAc, had

M. Hartmann, T. K. LindhorstMICROREVIEW

Figure 2. Structures of oligosaccharides tested as inhibitors of mannose-specific adhesion of E. coli together with their relative inhibitorypotencies based on the reference mannoside methyl α-d-mannoside (MeMan). IP: inhibitory potency; RIP: relative inhibitory potency(with IP of MeMan � 1); SD: standard deviation. [a] Values from inhibition of yeast agglutination with E. coli 346;[25] [b] values frominhibition of adhesion of type 1 fimbriated E. coli HB101 pPKL4 to the polysaccharide mannan, measured by ELISA;[26] [c] “Oligoman-nose-9 and -3” are named according to a suggestion made in the literature,[28] where they were tested as ligands for the fimbrial lectinFimH (vide infra).



a RIP of 0.7, thus performing worse than simple MeManas inhibitor of mannose-specific yeast agglutination.Furthermore, while the branched oligomannosides 12 and13 were very good inhibitors, 14 and 15 were significantlyweaker. The data obtained in yeast agglutination assayswere confirmed with guinea pig erythrocytes a little later.[27]

Some of these results, obtained in the 1980s, can be ra-tionalized today on the basis of more modern testing resultswith oligosaccharides 16 and 17, which have been termed“oligomannose-9 and -3”, respectively, by Bouckaert et al.(Figure 2).[28] At the time, however, researchers had no ideaabout the structure of the carbohydrate recognition domainof the type 1 fimbrial lectin and had to rely on models,which were deduced on the basis of the determined testingresults. The model of the type 1 fimbrial carbohydrate com-bining site, which was finally suggested by Ofek andSharon, was therefore based on the findings obtained witha series of oligomannosides and, in addition, based onstructure–activity relationships obtained with a series ofsimple mannosides with varying aglycon moieties (Fig-ure 3).

Figure 3. The nature of the aglycon moiety of simple α-d-mannos-ides is decisive for their potency as inhibitors of type 1 fimbriae-mediated bacterial adhesion.[29] RIP: relative inhibitory potency(with inhibitory potency of MeMan � 1).

Strikingly, mannosides with an aromatic aglycon, such asp-nitrophenyl α-d-mannoside (pNPMan) and 4-methylum-beliferyl α-d-mannoside (MeUmbMan), were shown to ex-ceed the inhibitory potency of MeMan significantly (Fig-ure 3),[29] performing better than all oligosaccharides thathad been examined earlier. In addition, certain substitution


patterns on the aromatic ring showed a favourable effect onthe inhibitory potency of the respective mannnoside. Forp-nitrophenyl-o-chlorophenyl α-d-mannoside (pNoClMan),for example, a RIP value of approximately 720 was deter-mined. As mentioned earlier, this and some other testingresults of the early days can today be decoded on the basisof the crystal structure of the fimbrial lectin (vide infra).

Finally, Sharon and Ofek established a model for thecarbohydrate combining site of the type 1 fimbrial lectinand postulated that it should correspond to the size of atrisaccharide (having three subsites) with a hydrophobicbinding region adjacent to the carbohydrate-binding site,which has a strict specificity for the α-configuration of thebound mannoside. Today, crystallographic studies have par-tially approved Sharon’s model, while the three-subsitemodel could not be confirmed so far. Nevertheless, at pres-ent there seems to be some mystery hidden behind type 1fimbriae-mediated binding of mannosides that has not beenunravelled yet. Therefore, Sharon’ s model might contain arelevance, which is not reflected in the X-ray analysis of thetype 1 fimbrial lectin, called FimH, and thus has not yetbeen appreciated and remains underestimated.

3. Multivalent Glycomimetics as Inhibitors ofType 1 Fimbriae-Mediated Bacterial Adhesion

After the early investigations with natural mannosidesand oligosaccharides, a series of studies has employedmultivalent glycomimetics as inhibitors of type 1 fimbriae-mediated bacterial adhesion. The multivalent presentationof α-d-mannosides was anticipated as a promising approachto high-affinity (avidity, respectively) inhibitors of man-nose-specific bacterial adhesion based on multivalency ef-fects that had been observed in other carbohydrate–lectininteractions. Characteristically, lectins can contain not onlyone but two or more carbohydrate-binding sites, and theirinteraction with carbohydrates is therefore often multi-valent. Multiple lectin–carbohydrate contacts can lead toagglutination on one hand, but on the other hand, theyalso may lead to increased affinities as well as to a higherspecificity of the interaction. When the affinity of a lectinto a carbohydrate ligand is increased by multivalent carbo-hydrate–CRD contacts, the term “avidity” is used to de-scribe the strength of the overall interaction. To achieve anavidity effect by employing multivalent glycomimetics aslectin antagonists is especially attractive, as carbohydratebinding of lectins is typically associated with only moderateaffinity. Thus, the typical stabilities of many lectin–mono-saccharide complexes are low and lie in the millimolar tohigh micromolar range.[30]

Indeed, studies on the inhibition of type 1 fimbriae-medi-ated bacterial adhesion and hemagglutination have revealedthat rather high concentrations of saccharides are requiredfor effective inhibition. In the 1970s, Y. C. Lee and co-workers approved weak binding of galactose and GalNAcbinding to the asialoglycoprotein receptor (ASGPR). How-ever, when multivalent glycomimetics were employed, they

M. Hartmann, T. K. LindhorstMICROREVIEWdiscovered that a linear increase in the number of monosac-charide epitopes, presented in a specific glycomimetic, ledto a logarithmically increased avidity to the ASGPR. Leeand co-workers, at that time, concluded that this findingstrongly suggests that lectins possess more than one carbo-hydrate-binding site, hence are bi- or even multivalent. Theresulting effect in binding multivalent carbohydrates theycalled the “cluster effect”, which today is often referred toas “multivalency effect”.[31] Inspired by Y. C. Lee’s results,chemists have strived after multivalent glycomimetics toachieve high-affinity carbohydrate ligands for lectins. Multi-valent glycomimetics have been designed and synthesizedalong a large variety of architectures, and this field has beenextensively reviewed.[32,33] It has, however, not been easy tosimply reproduce the results Y. C. Lee observed in the caseof carbohydrate binding to ASGPR with other lectins. To-day it can be concluded that multivalency effects, observedin carbohydrate–protein interactions, can result from quitedifferent biological mechanisms and according to quite dif-ferent thermodynamic and kinetic conditions.[34–38] Thus,the effect of variable multivalent glycomimetics in a particu-lar testing system can not necessarily be compared in thesense of quantitative structure–activity relationships(QSARs). Multivalent glycopolymers,[39–42] for example,might interact with a lectin according to fundamentally dif-ferent models than a multivalent glycocluster, which isbased on a carbohydrate core, for instance.[26] Amongmultivalent glycomimetics, glycodendrimers are an espe-cially promising class of molecules, which have been evalu-ated as antiadhesives. Their synthesis and testing results asinhibitors of UPEC has recently been reviewed in detail.[43]

Mannose Glycoclusters with Noncarbohydrate Core

In 1998, the effect of multivalency on the inhibition oftype 1 fimbriae-mediated bacterial adhesion was studied forthe first time,[44] by using a hemagglutination inhibition as-say (vide infra). Branched and hyperbranched amines wereemployed as multivalent scaffold molecules and reactedwith glycosyl isothiocyanates to achieve di-, tri-, tetra-, andhexavalent α-mannose clusters 18 to 21 by thiourea bridg-ing (Table 1). The inhibitory potencies determined withthese glycoclusters and glycodendrimers[45–47] turned out tobe not strictly valency-dependent.[44] Whereas divalent mo-lecule 18 and tetravalent cluster 20 performed 31 and 39times better than the reference inhibitor MeMan, respec-tively, trivalent cluster 19 had a 106-fold higher inhibitorypotential. When the determined inhibitory potencies areconsidered as valency-corrected values (RIPvc), trivalentglycocluster 19 was superior over all other multivalent gly-comimetics tested in this series. In valency correction, theinhibitory potency determined for a specific glycocluster isdivided by the number of clustered mannosyl moieties.Thus, a RIP value of 106, determined for a trivalent gly-cocluster, results in a RIPvc of 35. The hexavalent molecule21, for example, was shown to have the same RIP value asthe trivalent cluster 19, but its valency-corrected inhibitorypotency, RIPvc, is only 18.


Besides the valency of a glycocluster, the structure ofthe aglycon or scaffolding unit was shown to play a crucialrole for lectin-binding properties. Trivalent cluster 22,which, apart from its O-glycosidic linkage, offers greatstructural similarity to thiourea-bridged glycocluster 19[RIP(19)MeMan = 106], performed only 10 times better thanMeMan.[44] Thus, thiourea bridging of mannosyl residueswas especially effective in inhibiting mannose-specific bac-terial adhesion. This was once again confirmed in a studywith tetravalent clusters 23 and 24,[48] which are based ontetraazamacrocycle scaffolds. While cyclen 23 showed a195-fold increased inhibitory potency in a hemagglutinationassay, when compared to MeMan, the more flexible cyclamanalogue 24 exceeded the inhibitory potency of MeMan by780-fold.

These findings showed that tri- and tetravalent glycoclus-ters perform particularly well as inhibitors of type 1 fim-briae-mediated bacterial adhesion, and this led to the devel-opment of a series of further small glycoclusters and clustermannosides (Table 1). A new ELISA (vide infra) was devel-oped to determine the antiadhesive properties of such tri-and tetravalent cluster mannosides, all O-glycosidically at-tached to the respective noncarbohydrate cores.[49] In ad-dition, the idea was born to cluster mannosides via the 6-position of the sugar ring. This concept was considered inorder to combine a favourable aglycon effect with the suc-cess of trivalent clustering of mannosidic ligands. Thus, inthe late 1990s “mannoside donors” (instead of the classical“mannosyl donors”) were designed, each with a function-alized 6-position to achieve a library of analogous clustermannosides with varied aglycon moiety. For clustering pep-tide coupling to branched triacids, or thiourea bridging tobranched triamines was utilized. This approach to high-af-finity ligands for type 1 fimbrial lectin led to cluster glycos-ides such as 25 and 26, which were quite poor inhibitors,however. Nevertheless, direct comparison of two structur-ally similar C-6-clustered inhibitors with aliphatic[RIP(25)MeMan = 0.7] and aromatic aglycon [RIP(26)MeMan

= 8] moieties showed that the aromatic character of the ag-lycon affected binding. On the other hand, the anomericallylinked trivalent cluster mannnoside 27 tested in the sameassay and lacking an aromatic moiety, produced a RIP of90. Overall, the concept of mannoside clustering via the 6-position of the sugar ring cannot be recommended. Anygood testing results with this type of 6-modified clustermannosides must be considered false positive results[49] inlight of the FimH X-ray structure,[50] which indicates thatcomplexation of mannosides within the FimH CRD is notpossible once a scaffolding unit is connected to the 6-posi-tion of the sugar ring (cf. Figure 6).

The first crystal structure of the type 1 fimbrial lectinFimH was published in 1999.[50] It proved that this lectin ismonovalent and possesses just one CRD, thus favourableeffects with multivalent ligands could not necessarily be ex-pected on the basis of this information. In spite of this,many more multivalent cluster mannosides were preparedin the following years up to the present, and they weretested as inhibitors of type 1 fimbriae-mediated bacterial


adhesion and even the isolated lectin FimH. Such researchwas motivated and reasoned by numerous experimental fin-

Table 1. A selection of representative mannose glycoclusters with noncarbohydrate core and their testing results. As many of the structuresare rather complex, α-d-mannosyl residues have been simplified and represented as six-membered heterocycles shaded in grey, here andlikewise in all other following tables.


dings, which showed that especially tri- and tetravalent clus-ter mannosides are indeed potent inhibitors of type 1 fim-

M. Hartmann, T. K. LindhorstMICROREVIEWTable 1. (Continued)



Table 1. (Continued)

[a] SD = standard deviation, included in brackets if literature-reported. [b] RIP = relative inhibitory potency, based on MeMan withIPMeMan � 1. [c] RIPvc = valency-corrected RIP (rounded down). [d] = RIP based on d-mannose with IPMan � 1.


M. Hartmann, T. K. LindhorstMICROREVIEWTable 2. A selection of representative carbohydrate dendrimers and carbohydrate-centred cluster mannosides and their testing results.



Table 2. (Continued)

[a] SD = standard deviation, included in brackets if literature-reported. [b] RIP = relative inhibitory potency, based on MeMan withIPMeMan � 1. [c] RIPvc = valency corrected RIP (rounded down). [d] K abbreviates l-lysine as branching element.

briae-mediated bacterial adhesion and good antagonists ofFimH. Thus, a collection of structurally related tri- andtetravalent cluster mannosides with a C3-aliphatic aglycon,28 to 31, were investigated.[51,52] Here it was again con-firmed that a linear increase in valency can result in a muchmore than linear enhancement in affinity, as observed bycomparison of 28 and 31. However, the attempt to improvebinding to FimH by incorporation of an aromatic moietyadjacent to the clustered mannosides, such as in the case of28 and 29,[51] was not successful. Trivalent cluster manno-side 30 performed approximately 6 times better than its an-alogue 29 with a phenyl ring incorporated at its focal point.On the other hand, the importance of the nature of thescaffolding unit for the affinity of clustered mannosides wasalso revealed. Four clusters with ethylene glycol (EG) spa-


cers were tested (Table 1): trivalent clusters 32 and 33,[53]

both yielding a high RIP of 1063, and tetravalent molecules34 and 35,[54] which had a 100 times lower RIP in the sameassay system. Thus, in spite of the fact that the same typeof linkers were used for clustering in all four cases, theirinhibitory behaviour differed significantly.

In recent years, also additional chemistries such as“click” chemistry,[55] Sonogashira coupling[56] and squaricacid conjugation[57] were employed for the synthesis of clus-ter mannosides, leading, for example, to triazole-linkedclusters 36, 37, 41 and 42, alkyne-linked multivalent man-nosides 38 to 40 and clusters such as 43, linked by squaricacid. With the isolated lectin FimH on hand, such tetra-and hexavalent cluster mannosides were tested in surfaceplasmon resonance (SPR) experiments (vide infra).[58] The

M. Hartmann, T. K. LindhorstMICROREVIEWobtained RIP values vary from 157 for cluster 36 to 41 forcluster 37, and even more drastically, from 4889 for tetrava-lent cluster 38 to 8 for the similar tetravalent 39. This tre-mendous discrepancy points out that the distance betweenthe mannoside ligand and the aromatic moiety as part ofthe spacer is extremely critical for binding efficiency toFimH. An interesting observation was made, when the val-ency of alkyne-linked clusters was changed while the princi-pal architecture of these clusters was maintained. Whentetravalent molecule 39 was expanded to hexavalent struc-ture 40, the relative inhibitory potencies remained the same.The triazole-linked cluster mannosides 41 and 42, on theother hand, led to a significant improvement, such that theRIP value of 41 was increased by a factor of 4.7 when com-pared to that of 36. The synthesis of cluster 42[59] was in-spired by the structure of the highly potent heptyl α-d-man-noside (cf. Table 3) and indeed seems to be a very promisingcandidate for inhibition of bacterial adhesion. Glycocluster42 was shown to exhibit a relative inhibitory potency of2670 (based on mannose) when tested in a hemagglutina-tion inhibition (HAI) assay.

An impressive example for a cluster effect in FimH bind-ing was shown by applying mannosylated lysine-based den-drimers.[60] With growing generations, the relative inhibitorypotencies of the di-, tetra-, octa- and hexadecavalent den-drimers 44, 45, 46 and 47 increased from 455 over 2000 and3571 to 11111, respectively (Table 2). Though the aviditydid not grow logarithmically, it rose to a much greater ex-tent than it would by a linear increase with respect to theincreasing valency.

Scheme 1. Carbohydrate dendrimers such as 50 (Table 2) can be formed in an iterative synthetic sequence employing a glycosyl donor asthe branching element.[61]


Carbohydrate-Centred Cluster Mannosides andCarbohydrate Dendrimers

Carbohydrate dendrimers were obtained by employing aglycosyl donor as the branching element.[61] This synthesisstarts from a 3,6-di-O-allyl mannoside (Scheme 1), whichcan be converted into a respective diol by hydroboration orradical addition of 2-mercaptoethanol. Then, mannosyl-ation with a 3,6-di-O-allylated mannosyl donor delivers thenext generation of carbohydrate dendrimers. When hydro-boration is employed to produce the diol acceptor for glyco-sylation, mannose moieties are linked by a C3 spacer, suchas in the case of 48 and 49 (Table 2). When 2-mercapto-ethanol was added upon the allylic double bonds, moreflexible dendritic structures such as 50 resulted. Interest-ingly, thioethers like 50 could be oxidized to the respectivesulfones (51), to increase the hydrophilicity of the spacers.Adhesion inhibition assays showed that, relative to the firstgeneration precursor 48, a duplication of mannoside resi-dues in 49 led to a fourfold higher inhibitory potency[RIP(48)MeMan = 10; RIP(49)MeMan = 42]. Secondly, againthe importance of spacer properties for protein–ligand in-teractions was apparent. Sulfone-bridged cluster 51 sur-passed the inhibitory potency of MeMan by 25-fold,whereas its less polar analogue 50 was 110 times a betterinhibitor than the reference mannoside.

Finally, carbohydrate-centred cluster mannosides wereintroduced and tested.[26,62] They were designed to closelyresemble the chemical nature of natural oligosaccharides.For steric reasons, C3 spacers were included between the


glucose core and the scaffolded mannoside ligands. It wasanticipated that, once the synthetic sequence to glucose-centred cluster glycosides is established, it can be transfer-red to others, for example, di- and trisaccharide core glyco-sides. Thus, a small library of carbohydrate-centred clusterssuch as 52, 53[26] and 54[62] (Table 2) could be produced,which gave RIP values of 180 for pentavalent cluster 52,230 for octavalent 53 and 190 for dodecavalent mannoside54.[62] Strikingly, these values are all in the same range,pointing out that the enhancement in molecule size and val-ency had no significant effect on inhibition properties ofthe respective cluster mannosides in this case.

Reasoning of Multivalency Effects in Carbohydrate Bindingof Type 1 Fimbriated Bacteria

The multivalency effects observed with many differenttypes of mannoside clusters as inhibitors of type 1 fimbriae-mediated adhesion of E. coli have not been fully under-stood. Generally, a cluster effect can be explained with theclustering of multiple CRDs,[31,34,37,63] but the type 1 fim-brial lectin FimH possesses only one CRD. Interfimbrialclustering of FimH CRDs might occur with large enoughmolecules. In an average glycocluster having about ten C–Csingle bonds between two mannoside residues, the distancebetween these two ligands is in the range of 1.5 nm. As thebacterial fimbriae measure about 7 nm in diameter, bindingof one cluster molecule to more than one fimbrial lectin at atime is very unlikely with cluster mannosides such as thosedepicted in Table 1. Intramolecular binding is a much moresensible explanation of avidity enhancement observed withmultivalent cluster glycosides in this adhesion system.Hence, several putative carbohydrate-binding sites onFimH, in addition to the CRDs known from X-ray studies,were suggested (vide infra).[64] Multiple binding to morethan one carbohydrate-binding site on FimH could accountfor strengthened binding of multivalent ligands.

A most obvious explanation for the observed multiva-lency effects is the elevated effective concentration of man-noside residues in the proximity of the carbohydrate re-cognition domain when a multivalent ligand is employed.Quick rebinding of a mannoside ligand is therefore statistic-ally favoured once a bound ligand is released from theCRD.

Another probable explanation for multivalency effects ininhibition of type 1 fimbriae-mediated adhesion could bethat cooperative effects between ligand and protein play arole. For example, intramolecular preorganization of freeligands could arise when the first ligand of a multivalentglycomimetic is bound to the lectin. This induced spatialorganization could enhance binding of the other ligands(positive cooperativity) or reduce their affinity (negativecooperativity). Moreover, binding of one ligand might in-duce a change in lectin conformation, which enforces thesubsequent binding event of the next mannoside moiety ofa cluster mannoside. This mechanism would correspond toan allosteric activation of the protein achieved by a multi-valent ligand.


Several studies indicated that especially potent clusteredinhibitors of type 1 fimbriae-mediated bacterial adhesionwere tri- or tetravalent. To explain the variant potencies ofthese, the chemical properties of the used linkers are decis-ive. Tuning of the linker chain length and its conforma-tional flexibility makes the exposed carbohydrates more orless easily accessible for lectin binding. Furthermore, theconformational stability of the chosen linker in water (orbuffer), the surrounding solvent in biological systems, is animportant property. Very hydrophilic spacers may dissolvethe carbohydrate cluster so well that binding to a partiallyhydrophobic protein would be disfavoured by enthalpy con-siderations. In contrast, hydrophobic linkers might lead tosupramolecular assemblies of the clusters, in course lower-ing their accessibility for lectin binding. This is a reasonableexplanation of the finding that higher valencies in manycases did not lead to drastically increased inhibitory potenc-ies of cluster mannosides and mannose glycodendrimers.Higher numbers of mannoside residues on a scaffoldingcore molecule of limited size does not necessarily mean bet-ter ligand availability. Individual mannoside moieties mightbe clustered too closely, so that enough space for the lectinto bind multiple copies is not available.

4. Assays to Test Antagonists of Type 1Fimbriae-Mediated Bacterial Adhesion

The development of new drugs is typically based on reli-able estimation of QSAR studies with potential leads. Inaddition to rational ligand design, which is based on theknowledge of the structural properties of the drug target,in vitro methods to screen for potential lead compoundsare indispensable tools. Therefore, any test system has to beparticularly suited for the biological question that is investi-gated. In an in vitro assay, which is typically used for QSARstudies, three major components determine the situationduring testing: the biological receptor or target, the nativeligand and the test compound, which can be an agonist oran antagonist.

If this three-compartment model is translated to thescreening for potent inhibitors of type 1 fimbriae-mediatedbacterial adhesion (Figure 4), the bacterial lectin FimH isthe receptor, the native ligand is approximated by the glyco-sylated surface (e.g. human bladder epithelium cells, ormannan as its mimic), and the test compound is the puta-tive inhibitor of bacterial adhesion, typically an α-d-man-noside derivative. All possible mutual interactions of thethree components have to be considered to influence theoutcome of an assay. In this system, a number of param-eters are important, and they are discussed in the following.(1) Presentation of the lectin: How the lectin is applied inan assay plays a crucial role for the outcome. Three statesof complexity of the receptor system have been employed.The lectin can be applied as recombinantly expressed, iso-lated protein or protein conjugate. Sheared-off bacterialtype 1 fimbriae can be used, or an assay can be performedwith whole bacteria. Even without any knowledge of the


Figure 4. Three different types of interactions play key roles in FimH-mediated bacterial adhesion to mammalian cells. In the developmentof assays to test and optimize putative adhesion inhibitors, all interactions and their mutual interplay have to be considered: the bacteriallectin FimH can either bind to a diversely glycosylated surface, a cell or cell mimetic (interaction I) or to an inhibitor in solution, amultivalent α-d-mannoside in this case (interaction II); additionally, an interplay of the carbohydrates on the surface with those insolution (interaction III) has to be considered. Each change in one of the shown interactions will influence the others, and likewisechanges in the conditions of the binding process have an impact on the whole system. Thus, the results of any FimH binding assay oradhesion inhibition assay are highly dependent on the setup of the applied in vitro test system.

lectin structure, it can easily be understood that the bindingconditions can differ a lot in these three different setups. (2)Glycosylated surfaces: Not only the presentation of the lec-tin FimH but also the glycosylated surface used in the assayinfluences the results. The ligand system can be applied inmany different stages of complexity. Whole cells can be usedto test adhesion at native interfaces. On the other hand, byusing synthetic surfaces it is possible to tune the complexityand structural features of a glycosylation pattern in greatdetail. In this way, structural refinement allows to test forvery specific patterns in a ligand surface, if needed. (3) Pres-entation of the inhibitor: Structural details of the test com-pound that enhance or lower its affinity are the object un-der investigation and can naturally not be varied to tune anassay. But still, they play a role in the way a FimH antago-nist is applied. As mentioned earlier, inhibitors that areclustered can be much more effective than their respectivemonovalent precursors. Immobilized on some surface orother rigid device, the inhibitor’s properties can change andthus lead to an inhibitory effect that differs from the prop-erties of the inhibitor in solution.

In Vitro Assay Systems to Screen for Potent FimHInhibitors

Different types of assays have been developed to test thequality of a potential new inhibitor of type 1 fimbriae-medi-


ated bacterial adhesion. The data that are generated for thesame FimH ligand can vary significantly when obtained indifferent assay systems. To be able to understand some ofthe observed effects and apparent discrepancies, it is usefulto categorize the tests applied according to the conditionsunder which inhibition of adhesion is investigated.

Assays in a Three-Dimensional Setup

The first assays that were performed to screen the inter-actions between type 1 fimbriated bacteria and eukaryoticcells were aggregation assays.[25] In the 1980s Sharon andcolleagues could show that the agglutination of yeast cellsby type 1 fimbriated bacteria, such as, among others, E. coliand some species of Salmonella, can be inhibited by ad-dition of α-mannosides. This agglutination assay using bac-teria and yeast cells is still used today.[65,66] The assay setupis rather close to physiological conditions regarding the gly-cosylated eukaryotic cell as well as in view of the fact thatthe lectin-bearing bacteria can move freely in all three di-mensions. A closely related testing system that has nearlyreplaced the yeast aggregation assay due to its higher sensi-tivity is the hemagglutination inhibition assay.[44,49,58,67,68]

In this assay, an inhibition titre (IT) is determined, whichreflects the concentration of a tested inhibitor that is neededto prevent type 1 fimbriated bacteria from agglutinatingguinea pig erythrocytes. As direct IT values generally differ


much from one screen to the other even if the same bacte-rial strain is used, relative IT values are commonly calcu-lated, which are referenced to a known inhibitor tested inparallel. In most cases, the reference glycoside is methyl α-d-mannoside (MeMan). In 2002, along with hemagglutina-tion inhibition data of type 1 fimbriated E. coli, Lee andco-workers detected the binding event by radioactivity as-says.[60] Mannosides coupled with 125I-labelled human se-rum albumin (HSA) were incubated together with E. colibacteria and different dilutions of the test substance. Aftercentrifugation, the remaining radioactivity of the E. colicells was an inverse measure for the inhibitory effect of thetested carbohydrate ligand. As the tested inhibitors have tocompete with the mannosylated HSA surface for bacterialbinding, half maximal inhibitory concentration (IC50) val-ues can be derived by plotting the % inhibition against theinhibitor concentration on a logarithmic scale to obtain asigmoidal dose response curve. The IC50 value of a test sub-stance reflects the inhibitor concentration at which 50% ofthe binding is inhibited. In dose response curves the IC50

value is the point of inflection. A second 3D approach todetermine inhibitory potencies of FimH ligands was re-ported in 2001.[69] Human cells (neutrophilic granulocytes)and a dilution of potential adhesion inhibitors were addedto a solution of biotinylated type 1 fimbriae. After incu-bation and washing, fluorescein-labelled streptavidin facili-tated the readout by fluorescence-activated cell sorting(FACS). In 2002, it was shown that the binding of man-nose-encapsulated gold nanoparticles (m-AuNP) to type 1fimbriated E. coli cells is inhibited by addition of MeMan.M-AuNPs were thereby introduced as tools for the imagingof FimH mediated bacterial binding in transmission elec-tron microscopy (TEM).[70] Recently, mannosylated nano-particles, which can be used in imaging applications wereintroduced again. In this study, the adhesion of E. coli tomannose-functionalized hematite (iron oxide) nanoparticleswas visualized by TEM.[71]

Detection has been performed with 3H-labelled mannose.After incubation of isolated FimH together with 3H-man-nose and a competing inhibitor, the assay mixture was fil-tered, and the remaining radioactivity could be read out.When using different inhibitor dilutions, Kd values could bederived for the tested substances.[72] In recent work, twoadditional three-dimensional assays were applied to test thebinding between FimH and potential carbohydrate ligands.One of these is the investigation of the binding of fluores-cent-dye-labelled mannosides to isolated FimH in a fluores-cence polarization (FP) assay.[68] In this assay, the fluoro-phore on the FimH ligand is excited by polarized light, soit consequently emits polarized light. The more freely themolecule can move in solution the more disturbed the po-larization of the emitted light. Bound ligands partiallyfreeze and thus move more slowly; therefore, the polariza-tion of the emitted light increases upon binding, which canbe measured. As ligand binding is not competitive in thissetup, the inhibitory effect of a substance is not detecteddirectly. Nevertheless, the binding event per se can be moni-tored precisely. Thus, the characteristic measure for a tested


ligand is not an IT, but the half maximal effective concen-tration, abbreviated EC50. The EC50 can again be deducedfrom a dose response curve, a plot of the gained effectagainst the corresponding ligand concentration applied.

Isothermal titration calorimetry (ITC) is a different andwell-known test method that was first utilized for the detec-tion of FimH binding mannosides by Bouckaert and col-leagues in 2011.[73] Mannosylated ligands can be titratedinto a FimH solution to measure the temperature changesupon saccharide addition and calculate a Kd value to char-acterize the binding of the ligand. Measurement of inhibi-tory effects was not performed, but binding constants forthe ligand–FimH interaction were deduced.

Assays on Surfaces

Besides the three-dimensional test systems, a number ofassay setups have been published that depend on the immo-bilization of one binding partner and subsequent detectionof the binding event to the manipulated surface. As onlyone of the binding partners is in solution and can thusmove freely, the setups can be categorized as two-dimen-sional. The prototype of 2D assays is probably the classicalELISA that depends on the detection of a molecule or pro-tein bound to a predefined surface by incubation with aspecific antibody against the presumably surface-boundcompound. Lindhorst and co-workers used microwell platescoated with mannan (mannose polymer) for ELISA to bindtype 1 fimbriated E. coli cells.[49] These could be detectedwith an antibody against FimA, the predominant proteinof the fimbrial shaft and a peroxidase-labelled secondaryantibody. Upon addition of mannoside solutions in dif-ferent concentrations, dose response curves of the inhibi-tory effect of the tested ligand against the applied concen-tration could be plotted, which led to IC50 values as charac-teristic for each tested ligand. As these ligands competewith the mannan-coated surface for FimH binding, a directinhibitory potency is measured in this case. To be able tocompare two such ELISA measurements with one another,a standard inhibitor has to be tested on the same test plate,so that the poorly comparable IC50 values from differentexperiments can be uniformly referenced. Different ELISAexperiments have been performed by using whole E. colicells,[26,44,48,51–54,61,62,74–76] type 1 fimbriae[77] and the iso-lated FimCH protein complex.[78]

Assays similar to the ELISA method have been devel-oped. They all share the competitive 2D binding situation,but the detection method is nonimmunological. By usingtype 1 fimbriated E. coli cells, two different competitive ad-hesion inhibition assay setups have been introduced.[79]

These assays basically resemble the ELISA and were per-formed on the same mannan-coated test plates, but eitherthe bacteria were biotinylated to be detected with a strepta-vidin–peroxidase conjugate, or directly detectable self-fluo-rescing bacteria expressing the green fluorescent protein(GFP) were used. In addition to mannan-coated plates, co-valently glycosylated assay surfaces were used to screen theaffinity of bacteria to a surface-bound ligand. Both assaysresulted in IC50 values for the tested substances. Covalent

M. Hartmann, T. K. LindhorstMICROREVIEWimmobilization of saccharides on a test surface is alsoneeded for current research and carbohydrate array fabrica-tion. A competitive binding assay on mannosylated chipswas published in 2004.[80] Type 1 fimbriated bacteria werestained with intercalating fluorescent dyes and sub-sequently, together with a reference mannoside solution, ap-plied to a mannoside array to competitively bind to themicrochip. The fluorescence readout was then used toquantify the surface-bound bacteria. A more flexible carbo-hydrate surface can be built up by self-assembled monolayer(SAM) formation on a gold surface. In 2002, an adhesioninhibition assay on mannosylated SAMs in a 96-well for-mate was performed.[81] The adhesion of type 1 fimbriatedfluorescence dye-stained bacteria to the glycosylated SAMwas shown to be inhibitable by addition of a mannosidesolution. As this inhibition again turned out to be depend-ent on inhibitor concentration, IC50 values for the used in-hibitors could be reported. Radioactively labelled bacteriawere also used to detect binding to GP2 (a membrane gly-coprotein) in a competitive manner.[82] To test the inhibi-tory potency of a glycopeptide from soybean, Guo and co-workers[83] used an adhesion inhibition assay, in which theylet E. coli and Salmonella cells adhere to eukaryotic LoVocells (colon carcinoma cells). This adhesion was inhibitedby glycopeptides. To characterize the glycoconjugate GP2as inhibitor, the remaining bacteria cells on the LoVo cellsurface were counted. A different 2D test on human cellswas performed in 2008.[28] A human urothelial cell line wasgrown in 12-well plates. Type 1 fimbriated E. coli were pre-incubated with mannoside solutions and then transferredto the cell surface. After incubation and washing, the blad-der cells were lysed by addition of trypsin and the lysatewas transferred to agar culture plates to determine the re-maining amount of colony-forming units.

Turning the setup upside down, the FimH CRD can alsobe immobilized to microtitre wells, to which the inhibitordilution and a biotinylated polyacrylamide glycopolymerare applied and incubated, generating a competitive bindingsituation. By addition of peroxidase-coupled streptavidin,the amount of bound mannoside-functionalized polymer isdetected. By plotting the corresponding dose responsecurves, IC50 values for the tested substances can be ob-tained.[84]

Assays under Flow

One parameter that plays an important role in physiolog-ical systems is the flow that the binding event has to with-stand. Though in some of the reported assays the samplesare shaken during incubation, the shear stress that the lectinis exposed to is not regulated. This is different in flow-regu-lated SPR measurements. For SPR, two different generalsetups have been applied. In the first SPR measurementswith FimH, either an anti-FimH antibody or a BSA–man-noside conjugate was bound to the Biacore sensor chip.When isolated FimH was coursed over the immobilized an-tibody together with a soluble adhesion inhibitor, only asmall percentage of FimH could be attached to the surface,depending on the binding affinity between FimH and the


added inhibitor.[58,72] Recently, the antibody or BSA conju-gate was replaced by low-molecular-weight mannosides,which were covalently attached to the Biacore chip.[73]

When a mixture of FimH and soluble inhibitors – mannos-ylated fullerenes in this case – passes the glycosylated chipsurface, the lectin can either bind to the Biacore chip andbe detected or stick to the glycosylated fullerenes and bewashed away. Both of these setups for SPR measurementsresulted in Kd values for the FimH–mannoside interaction.

In most cases, noncovalent binding of protein–ligand in-teractions is shorter-lived under shear stress. Such interac-tions are referred to as slip bonds.[85] Nevertheless, for someinteractions of cells with surfaces, it was first proposed in1988[86] that a critical applied tensile force can enhance thebinding strength rather than weakening the adhesion.[85]

FimH was the first bacterial protein for which such catchbonds were discovered.[87] Since 2010, the way bacterial lec-tin FimH tightens the binding to its sugar ligand under ten-sile force is understood in great detail. As long as the FimHprotein is in a relaxed conformation, the N-terminal lectindomain and the C-terminal pilin domain (vide infra) are inclose proximity, and the lectin domain is compressed. Whenshear force is applied, the two domains separate, and thelectin domain is elongated by 11 Å. This elongation processstretches the conformation of the β-sheets of the lectin do-main in such a way that the CRD constricts. This, in turn,tightens the binding of the ligand to the binding site. Thesame mechanism is in effect when a FimH truncate is iso-lated in pure form. As the FimHtr isolates lack the attach-ment to the C-terminal pilin domain, the lectin domain isfrozen in its elongated high-affinity state.[87] This finding isparticularly valuable for the interpretation of the resultsgained in different adhesion or binding assays with type 1fimbriated bacteria under flow. In addition, the affinitystate in which the lectin FimH is applied in different assayshas a great impact on the measured binding strength. Con-sidering these recent findings about the catch bond mecha-nism of lectin, many testing results with the various FimHantagonists might appear in different light and deserve anupdated interpretation.

The evaluation of testing results can be complex and dif-ficult. Even the interpretation of inhibitory potencies withinone assay system can already be challenging. In many cases,it can be very helpful to keep in mind the three-compart-ment model of an assay (Figure 4). To be able to comparethe effect of different compounds, it is essential to test areference compound in the same assay and batch, so that allresults can be referenced reliably. In FimH binding studies,MeMan (Table 3) or pNPMan (Table 4) are mostly used asreference glycosides. In particular, these relative values areneeded to compare the results of different assays. As theconditions under which binding is measured vary from sys-tem to system, the same inhibitor might perform quite dif-ferently in two apparently similar assays. Illustrative exam-ples are the highly diverging values measured for pNPManin many different test systems (Table 3). Usually, the calcu-lated relative values of the inhibitory potencies are in goodagreement, though.


5. Structure of the Type 1 Fimbrial Lectin FimH

Crystal structures of carbohydrate-binding proteins pro-vide valuable information about the modes of interactionsthat mediate carbohydrate recognition.[88,89] It has been fre-quently observed in X-ray diffraction studies with lectinsthat a carbohydrate ligand is complexed by a well-definedarray of hydrogen bonds, involving hydrogen-bond donorand acceptor groups of the ligand and the side chains ofasparagine or glutamine residues, carboxylate groups fromaspartates or glutamates, OH groups in serine side chainsand NH-groups from lysine, tryptophan or histidine resi-dues of the lectin CRD. Water molecules can mediate thesehydrogen bonds, and in some cases divalent metal ions suchas Ca2+ or Mn2+ are additionally involved in carbohydratebinding. Also, sandwiching of the carbohydrate ligand be-tween aromatic amino acid side chains is an importantbinding motif, leading to a relevant contribution of CH–πinteractions in carbohydrate binding.[90,91]

The type 1 fimbrial lectin is called FimH and is as-sembled at the tips of type 1 fimbrial composite structuresaccording to the chaperone/usher pathway.[50,92] Nine fimgenes are required for fimbriae assembly. The tip-fibrillarstructure contains the mannose-specific adhesin FimH,whereas the fimbrial rod is mainly composed of FimA sub-units (Figure 5). Type 1 fimbriae are constructed by a “do-nor strand exchange” (DSE) process, in which the Ig foldof every subunit is completed by an amino-terminal exten-sion from the following subunit.[21]

Figure 5. The type 1 fimbrial rod is assembled from different Fimproteins, which attach to each other by donor strand exchange(DSE). The fimbrial lectin, FimH, terminates the fimbrial fibre atits tip, and the most abundant protein constituent, FimA, attachesthe fimbriae to the bacterial outer membrane.

FimH is a 29 kDa protein with a length of 279 aminoacid residues. Its exact structure has remained unknown forlong, because FimH cannot be crystallized in pure form asit is missing a strand to complete its fold. In addition, it isproteolytically degraded when it is expressed alone. Knightand co-workers reasoned that, according to the two-compo-nent system that is required for fimbriae assembly (accord-ing to the DSE process), the C-terminal part of FimH re-quires interaction with another protein. For fimbriae as-sembly, FimH is complexed with the chaperone FimC, andthis led to the attempt to stabilize FimH by addition ofFimC. This idea delivered the first crystal structure of


FimH in 1999,[50] in a complex with the FimC chaper-one. Cyclohexylbutanoyl-N-hydroxyethyl-d-glucamide (C-HEGA), which had been added for crystallization, wasfound to be bound to the CRD of FimH, where it adoptsa conformation that relatively closely resembles the struc-ture of a mannose pyranoside ring.

Thus, it was revealed that FimH has two domains, a pilusdomain, FimHP, and a lectin domain, FimHL. The N-ter-minal mannose-binding lectin domain FimHL comprisesresidues 1–156, and the C-terminal pilin domain, which isused to anchor the adhesin to the pilus, comprises residues160–279. There is one single CRD to be seen in the FimHcrystal structure, which is located at the tip of the lectindomain. It is capable of accommodating a mannose mono-saccharide in its α-configuration (Figure 6).

Figure 6. A mannosidic ligand is complexed within the FimH CRDsuch that the aglycon of an α-d-configured mannoside pointstowards the entrance of the binding pocket. This allows terminalmannose residues on complex oligosaccharides to be complexed byFimH. β-d-Mannosides cannot be complexed for steric reasons.

In 2002, the crystal structure of the FimC/FimH chap-erone–adhesin complex bound to its physiologically rel-evant ligand α-d-mannose was published, and the aminoacids that are important for mannose complexation wereidentified in great detail.[78] Mannose was found to be bur-ied in a deep and negatively charged pocket. The mannosering makes ten direct hydrogen bonds to the FimH bindingsite, and in addition indirect water-mediated hydrogenbonds are formed. All hydroxy groups of the sugar ring,other than the anomeric position, interact extensively withthe CRDs of FimH, in particular with residues Phe1,Asn46, Asp47, Asp54, Gln133, Asn135, Asp140 andPhe142 (Figure 7). The mannose-binding pocket is sur-rounded by a hydrophobic ridge comprising Ile13, Tyr48,Ile52, Tyr137 and Phe142. The side chains of Tyr48 andTyr137 are positioned such that they form a gate-like struc-ture that has been named the “tyrosine gate”.[21] Thus, man-noside ligands with an aromatic aglycon, such as pNPMan,can establish π–π interactions with the tyrosine gate flank-ing the entrance of the FimH CRD (Figures 7 and 8), lead-


Figure 7. Both graphics show the amino acid residues in an orientation revealed by X-ray diffraction analysis (PDB ID: 1KLF),[78]

depicted in ball-and-stick form. Left: The bottom site of the FimH CRD with docked p-nitrophenyl α-d-mannoside (pNPMan). TheCRD contains the N-terminal Phe1 amino acid of the protein. Prominent hydrogen bonds between the carbohydrate-binding site andthe ligand are depicted as grey dotted lines. Right: The amino acid residues at the entrance of the FimH CRD. The aromatic side chainsof Tyr48 and Tyr137 form π–π interactions with docked pNPMan, leading to an increased affinity of the ligands with an aromaticaglycon.[93]

ing to increased affinities. Thus, the early findings with li-gands such as pNPMan or MeUmbMan (Figure 3) cannow be understood on the basis of the FimH structure.

Figure 8. Structures of the CRD of the bacterial lectin FimH crys-tallized with the tyrosine gate (residues Tyr48 and Tyr137) in anopen or closed conformation. The conformation of the tyrosinegate depends on the complexed ligand. The open gate structure(PDB ID: 1KLF) was obtained with FimH in complex with man-nose (top),[78] while the closed gate conformation (PDB ID:1UWF) arose from complexation of BuMan (bottom).[72] TheFimH receptor structure is depicted as a Connolly surface, and tohighlight the tyrosine gate it is meshed in each case.


In 2005, another crystallographic study was published,which employed two FimH proteins named FimHtr1 andFimHtr2 from two different bacterial strains.[72] TheseFimHtr proteins were truncated to only the FimH lectindomain, built up by residues 1–158. In both cases, althoughno sugar was included in the crystallization setups, it turnedout that butyl α-d-mannoside (BuMan) was bound in theCRD. It was shown that this mannoside originated fromthe LB (Luria-Bertani) medium used to grow the bacteriaduring expression of the protein. The butyl moiety ofbound BuMan extends out of the mannose-binding pockettowards Tyr48 and Tyr137, making van der Waals contactsto both tyrosine rings and Ile52. In case of FimHtr2 theTyr48 and Tyr137 side chains were found in an almost par-allel orientation as in the earlier FimC/FimH struc-tures.[50,78]

In the FimHtr1 structure, on the other hand, the parallelorientation of the Tyr48 ring is prevented and instead it ispacked edge-to-face with Tyr137 (Figure 8). Thus, there isa conformational flexibility of the tyrosine gate at the en-trance of the FimH CRD, which has implications on liganddesign and the interpretation of testing results (vide supra).

In 2008, a truncated version of FimH, FimHtr compris-ing residues 1–158, was finally crystallized in a complexwith a natural ligand, “oligomannose-3” (Figure 2).[28] Itwas shown that the α1,3-linked mannose residue is com-plexed within the CRD and that the Manβ1,4GlcNAcβ1,4GlcNAc portion of “oligomannose-3” interacts with anextended region of the binding site. Interestingly, for thecentral mannose unit, a strong stacking interaction with thearomatic ring of Tyr48 can be observed, whereas Tyr137interacts with the mannose-bound GlcNAc moiety. Thus,the FimH tyrosine gate can be likewise utilized by oligosac-


charide ligands for tighter binding as it has been observedfor synthetic ligands with an aromatic aglycon moiety (Fig-ure 3, Table 4).

6. Rational Design of Carbohydrate Ligands forthe Type 1 Fimbrial Lectin FimH

As the structures of the type 1 fimbrial lectin FimH andits CRD are known today from several crystallographicstudies,[28,50,68,72,78] a rational, computer-aided design ofhigh-affinity ligands to develop inhibitors of bacterial ad-hesion to mucosal surfaces should be greatly facilitated.

Table 3. A selection of representative alkyl α-d-mannosides and their testing results.

[a] SD = standard deviation, included in brackets if literature-reported. [b] RIP = relative inhibitory potency, based on MeMan withIPMeMan � 1.


Only recently, a number of such more rational studies havebeen published, leading to three classes of FimH ligandswith relatively high affinity: (i) long-chain alkyl mannos-ides, (ii) mannosides with variously substituted aromatic ag-lycon moieties, and (iii) mannosides with extended aglyconmoieties.

Long-Chain Alkyl α-D-Mannosides – HydrophobicInteractions with the Tyrosine Gate

In case of the crystallization studies with FimHtr pub-lished in 2005,[72] exogenous BuMan was found to be com-

M. Hartmann, T. K. LindhorstMICROREVIEWplexed within the CRD. As even repeated dialysis could notremove BuMan from the protein, it was concluded that thissimple alkyl mannoside undergoes a stable interaction withthe protein. In fact, van der Waals contacts of the butyl ag-lycon to the phenyl rings of the lectin’s Tyr48 and Tyr137and to the side chain of Ile52 were found. Today, it can bereasoned that in its complexed conformation, BuMan canmimic the hydrophobic face of an oligosaccharide such as“oligomannose-3” (Figure 2).[28] To screen the ligand prop-erties of further mannosides with a more extended hydro-phobic alkyl aglycon, a series of alkyl α-mannosides wassynthesized and tested in SPR experiments and a displace-ment assay using 3H-labelled mannose (vide supra).[72] Asindicated in Table 3, the affinity of the various alkyl α-d-mannosides for FimH increases with the length of the agly-con alkyl chain. The only irregularity in this study was acomparably high RIP value of 440 for heptyl α-d-manno-side (HeptMan), determined in SPR studies. The strongbinding to FimH is assumed to result from interactionswith the heptyl aglycon. These high affinities of HeptManfor FimH were approved in other studies.[68,84] However, thehigh value of the MeMan-based RIP(HeptMan)MeMan =440, determined in the initial SPR study,[72] has not beenconfirmed in any other test system to date (cf. Table 3).

Mannosides with Aromatic Aglycon Moieties – π–πInteractions with the Tyrosine Gate

It has been known since the 1980s that aromatic aglyconmoieties can enhance the affinity of the respective mannos-ides for FimH by a factor of 600 or more.[29] Later, on thebasis of the FimH crystal structures, such findings couldbe rationalized. Thus, π–π stacking interactions of aromaticrings with the amino acid side chains of Tyr48 and Tyr137of the FimH protein improve the affinity of a carbohydrateligand. Also oligosaccharides, the natural ligands for FimH,can interact with this tyrosine gate by means of their hydro-phobic sites.[28]

To further improve the affinity of pNPMan, extensivestudies on variation of the aromatic aglycon of mannosideligands of FimH have been performed, and some of theresults are summarized in Table 4. Variation of the p-sub-stituent of phenyl mannosides had no big effect. It wasshown that neither the reduction of the nitro group inpNPMan (leading to pAPMan), nor its deletion (PMan)changed the compound’s inhibitory potency to a great ex-tent.[67,68] While Sharon and co-workers had reported a dif-ference between PMan [RIP(PMan)MeMan = 40] andpNPMan [RIP(pNPMan)MeMan = 70], somewhat later thisdifference could not be confirmed (cf. Table 4). Only an N-acetylamino substituent in the p-position of phenyl manno-side (pNAcPMan) increased the inhibitory potency by afactor of 4 compared to that of pNPMan. On the otherhand, the favourable effect of o-substitution was confirmed,as introduction of a chlorine substituent in the o-positionof the phenyl ring to yield pNoClPMan increased the affin-ity for FimH significantly by a factor of 200[74] and 720.[29]


Monochloro-substituted mannosides, oClPMan, mClPManand pClPMan did not perform as well as pNoClPManwhen tested in a hemagglutination inhibition assay. Also ina cyano-substituted series of mannosides, oCNPMan,mCNPMan and pCNPMan, it was confirmed thatmannnosides with o-substituted phenyl aglycon performedbest as hemagglutination inhibitors, whereas the p-substi-tuted compounds were the worst inhibitors (Table 4).

The high inhibitory potency of MeUmbMan, which hadbeen reported by Sharon three decades ago, was confirmedin three independent assay systems.[68,72,74] From the X-raystructure of FimH it can be seen that the aromatic systemin MeUmbMan perfectly fits between the phenyl rings ofthe tyrosine gate to build up strong π–π stacking interac-tions. A similar effect could also be achieved with otherfused ring systems, such as naphthalene (for 55). Manno-side 55 has a fivefold higher inhibitory potency thanpNPMan. Interestingly, in the applied assay, mannosideswith an ester-substituted thiophene ring fused to the phenylaglycon (mannoside 56) produced a RIP value of 16 (basedon pNPMan), just as MeUmbMan, though lacking the ex-tended aromatic system of MeUmbMan.

Mannosides with Extended Aromatic Aglycon Moieties –Interactions beyond the Tyrosine Gate?

When MeUmbMan was docked into the FimH bindingsite in its closed-gate conformation,[72] the pyrone part ofthe conjugated system was located exactly between the twophenyl rings of the CRD tyrosine gate. This complexationmode explains the high affinity measured for MeUmbMan(Table 4). Extension of the aromatic system by anotherplanar substituent with the ability to establish additionalor tighter π–π interactions with the tyrosine gate seemed apromising approach towards low-molecular-weight manno-sides with very high affinities for FimH. Thus, a series ofmannosides with varied biphenyl aglycon were synthesizedand tested in hemagglutination experiments,[68] a selectionof which is shown in Table 5. The biphenyl analogue ofpNPMan, 57, and its m-nitro-substituted analogue 58 sup-ported the initial assumption. The inhibitory potencies ofthese biphenyl mannosides exceeded that of pNPMan by afactor of 16 for 57, a value that was also found for MeUmb-Man in the same assay (Table 4). The m-nitro-substitutedbiphenyl mannoside 58 exceeded the inhibitory potency ofpNPMan even by a factor of 62. To identify a substitutionpattern on biphenyl mannosides that is optimal for FimHbinding, the influence of o-, m- and p-cyano-substitution(compounds 59 to 61) was investigated.[68] Other than inthe case of phenyl mannosides (cf. Table 4), the inhibitorypotency could be best enhanced, namely by a factor of 31relative to pNPMan, by the introduction of a cyano groupinto the m-position of the biphenyl system (60). CN resi-dues in the o-position led to a 16-fold inhibitory potency,while the p-substituted product 61 had a RIP value of 4(Table 5). The same inhibitory effect could be achieved,when the biphenyl system was replaced by a benzophenoneaglycon (62).


Table 4. A selection of representative mannosides with aromatic aglycon moieties and their testing results.

[a] SD = standard deviation, included in brackets if literature-reported. [b] RIP = relative inhibitory potency, based on MeMan withIPMeMan � 1. [c] RIP based on pNPMan with IPpNPMan � 1.


M. Hartmann, T. K. LindhorstMICROREVIEWTable 5. A selection of representative mannosides with extended aromatic aglycon moieties and their testing results.

[a] SD = standard deviation, included in brackets if literature-reported. [b] RI = relative inhibitory potency, based on MeMan withIPMeMan � 1. [c] RIP based on pNPMan with IPpNPMan � 1.

When pNPMan was extended by a squaric acid moiety(mannoside 63), also a very high inhibitory potency relativeto pNPMan resulted, with a RIP value of 58 as determinedby ELISA. Introduction of a chloro substituent in the o-position (mannoside 64) led to a RIP value of 223 (Table 5),and this could be rationalized by molecular modelling.[74]

Somewhat different results for 63 and 64 were obtainedlater by employing a competitive binding study.[84]

When the aglycon portion of mannoside 63 was furtherextended by short peptide chains, the affinities decreased[RIP(65)pNPMan = 7 and RIP(66)pNPMan = 14].

It has therefore been argued that the potentially reactivesquaric acid monoamide moiety in 63 might block the bind-ing pocket permanently by covalent attachment to the N-terminus of the lectin, Phe1. This would imply that the af-finity for the corresponding squaric acid diamide 67 should


be lower than the one for 63. Only recently, Lindhorst et al.could show that this is not the case.[93] On the contrary,the squaric acid diamide 67 exceeded the RIP value of itscorresponding monoamide derivative 63 by a factor of 3.

Conformational Flexibility of the Ligand and the Lectin

According to the induced fit model of ligand–receptorbinding, it must be considered that both a carbohydrate li-gand and its lectin receptor change their conformation uponformation of a carbohydrate–lectin complex. Such conforma-tional considerations have, however, not yet been extensivelyinvestigated for carbohydrate binding of FimH. For a fo-cussed library of cluster mannosides and glycodendrimers,molecular dynamic simulations have been performed that


provide an impression of the conformational availability ofthe included mannose moieties.[94,95] Interestingly, predic-tions made in these theoretical studies can be aligned withexperimental results obtained from inhibition adhesion mea-surements. When α-d-mannosides where clustered as multi-valent glycomimetics, the affinities of the respective glyco-clusters and glycodendrimers for type 1 fimbriated bacteria(cf. Tables 1 and 2) showed moderate to good improve-ment.[44,58,60,61] However, when p-nitrophenyl α-d-mannosidemoieties were analogously assembled in order to increase thefavourable affinity of pNPMan, testing results were disap-pointing.[44,96] In fact, clustering of pNPMan hardly led toinhibitory potencies that exceeded that of monovalentpNPMan when tested with type 1 fimbriated E. coli. Thisfinding can be explained by results obtained in molecularmodelling.[94] Molecular dynamic studies with pNPMan gly-coclusters suggest that there is a pronounced intramolecularπ–π stacking of the included phenyl moieties and that theconformational space that is populated by the scaffoldedpNPMan moieties is thus considerably limited. If the sugarmoiety is less available for complexation with FimH inpNPMan glycoclusters than in monovalent pNPMan, conse-quently favourable multivalency effects cannot be expectedin this case, and they have not been measured either.

Thus, the conformational features of lectin ligands cer-tainly influence their receptor binding properties. On theother hand, the fimbrial lectin FimH will also experienceconformational changes upon ligand binding.[97] Most ob-viously, speculations can be made about the meaning of dif-ferent conformations of the tyrosine gate at the entrance ofthe FimH CRD. In crystal structures, the tyrosine gate wasin an either closed[68,72] or open conformation.[78] Conse-quently, the predictions about ligand binding to FimH, de-duced from docking studies, are different depending onwhich FimH conformation was taken as the basis of themodelling.[74] Some mannosides show higher affinity to theclosed gate conformation of FimH, others show the oppo-site preference. It can be speculated that, depending on theligand that is available for FimH binding in vivo, the lectinadopts an appropriate conformation in order to bind astightly as possible. The tyrosine gate in this situation mightadopt any conformation between the two extremes “closed”and “open”. Furthermore, it is intriguing to speculate thatthe conformational flexibility of the entrance domain of theFimH CRD (including the tyrosine gate) might have a func-tion in “selecting” and/or “preorganizing” carbohydratesfor binding. Future research might address such hypotheses.

7. Special Approaches to the Inhibition ofMannose-Specific Bacterial Adhesion

From X-ray diffraction analysis and studies on the as-sembly of fimbriae, it seems to be evident that FimH is thetype 1 fimbrial lectin, located at the type 1 fimbrial terminiand displaying one monovalent carbohydrate recognitiondomain (CRD) in its lectin domain (FimHL). Nevertheless,type 1 fimbriae-mediated bacterial adhesion is a multifac-


eted process, which is not yet understood in all of its details.Consequently, there is also room for novel conceptual ap-proaches both to better understand the mechanisms of bac-terial adhesion as well as to further improve the affinitiesand other advantageous features of inhibitors of bacterialadhesion.

As mentioned earlier, higher-valent inhibitors effect anincreased avidity compared to that of their monovalentcounterparts. These results, obtained with multivalent gly-comimetics as ligands of FimH and as inhibitors of type1 fimbriae-mediated adhesion of E. coli,[32,33,43] are not inaccordance with the monovalent nature of the fimbrial lect-in, though. In addition, clustering of several FimH CRDsby typical multivalent glycomimetics is unlikely, given thesize of fimbriae and their distance dimensions on the bacte-rial surface. In order to take advantage from the reportedmultivalency effects and combine them with the ability ofcarbohydrate conjugates to cluster the bacterial lectins,mannose residues can be coupled to a more extended scaf-fold. A common technique in biochemistry is to display theligand on a protein. Some examples of serum albuminsfunctionalized with 8 to 35 mannose residues have beentested in a competitive binding assay with type 1 fimbriatedE. coli bacteria.[60] Generally, it was shown that the bindingefficiency increased with the degree of mannosylation. Be-sides, as reported for glycoclusters (vide supra), the lengthand the hydrophilic and steric properties of the linker con-necting the mannose moiety and the protein play a crucialrole for the inhibitory potency of the tested compound. Thevalency-corrected relative inhibitory potencies based on thevalues obtained for MeMan were in the range of resultstypically measured for glycoclusters. Most of the tested neo-glycoconjugates had a RIPvc of about 50.[60] Hence, in-creased inhibitory effects, which might arise from lectinclustering or agglutination, were not observed.

Only recently, the idea of particle-bound mannosides wasresumed. Fullerenes were functionalized with twelve man-noside residues, by using two different linker strategies.[73]

It turned out that the valency-corrected inhibitory potenc-ies were unexpectedly low, in the range of 3 (compared tothose of the monovalent counterparts). A considerablemultivalency effect would have increased the avidity by amuch higher factor. On the other hand, it was reported inthis study that the dodecamannosylated fullerenes couldbind up to seven isolated FimH proteins. Clustering of theisolated form of the lectin domain was thus achieved by thisapproach.

An even more extended scaffold molecule was employed,when mannosylated pseudopolyrotaxanes were introducedin 2010.[98] Three, five or ten cucurbit[6]uril-based mannos-ylated “wheel structures” were threaded on polyviologenstrings with approximately eleven viologen units. Thesepseudorotaxane inhibitors were tested in a hemagglutina-tion inhibition assay. The relative inhibitory potencies ofthe threaded mannose wheels were reported to be 180 to300. This is a value typically obtained for a potent mannosecluster. Hence, the desired prominent inhibitory effects ofthese interesting compounds could not be measured,

M. Hartmann, T. K. LindhorstMICROREVIEWthough mannosides were displayed on a voluminous scaf-fold.

Another interesting approach to the development offunctional FimH ligands addresses the topic of multival-ency from a different point of view. It has been speculatedthat multiple binding sites might exist on the FimH ad-hesin.[99] This hypothesis has been encouraged by the find-ing that certain carbohydrate ligands exhibit stimulating al-losteric effects on adhesion,[100] which might be explainedby the existence of allosteric binding sites or by activationof carbohydrate complexation in an analogy to the catchbond.

Although the sequence of the FimH adhesin is highlyconserved, studies by Sokurenko and colleagues[101–103]

have indicated that allelic variation in FimH is correlatedwith different carbohydrate-binding profiles. However, noneof the allelic variations, which gives rise to differences inmannose-binding, occurs within, or even close to the FimHmannose-binding pocket. Again, additional sugar bindingsites on the FimH lectin domain could be an explanationfor this finding. Multiple binding sites on FimH could aidin recognizing large and multivalent carbohydrate receptorson the host cell surface.

Consequently, molecular modelling was performed toidentify putative additional carbohydrate-binding pocketson FimH, and this has led to the suggestion of three loca-tions, which might constitute new potential carbohydrate-binding sites on the surface of the FimH lectin domain, inaddition to the mannose pocket at the tip of the domain.[99]

The hypothesis of multiple carbohydrate binding sites onFimH was tested with a bivalent carbohydrate ligand (Fig-ure 9),[75] which was shaped such that it could concomi-tantly occupy the known FimH CRD and a putative secondcarbohydrate-binding site with a preference for high-man-nose trisaccharides, as suggested by Knight.[99] This manno-side-trimannoside ligand was meant to clamp two carbo-hydrate-binding sites on FimH, but failed as effective inhib-itor of type 1 fimbriae-mediated bacterial adhesion, whentested by ELISA. Thus, the hypothesis of multiple carbo-hydrate-binding sites on FimH remains unapproved to date.

An alternative method to prove multiple binding sites onFimH is photoaffinity labelling of the protein. Accordingto this methodology, mannoside ligands of FimH have tobe equipped with a photolabile functional group that canbe activated for covalent cross-linking to the lectin uponirradiation. Proteolytic degradation and mass spectrometricanalysis can then lead to the identification of receptor loci,which complex carbohydrates. To allow affinity chromatog-raphy with the proteolytic digest of a lectin labelled in thisway and to thus facilitate the analysis, biotin can be incor-porated into a photolabile lectin ligand, to be selected bystreptavidin-based affinity chromatography.

Indeed, photoactive mannoside ligands for FimH havebeen made available and shown to be suited to covalentlylabel FimH.[76] Future studies will reveal whether photoaf-finity labelling is a suitable methodology to reliably identifycarbohydrate-binding sites as well as allosteric sites onFimH and other lectins.


Figure 9. This bivalent glycopeptide ligand was designed to bridgetwo putative carbohydrate-binding sites on FimH and was synthe-sized by squaric acid conjugation of a branched trimannoside anda simple α-mannoside unit.[75]

8. Conclusions

Lectins have been defined as a class of carbohydrate-binding proteins, which are neither antibodies nor enzymes.They have been discovered in all organisms, from plantsand microorganisms to vertebrates including humans. Theyhave been allocated to manifold biochemical processes, andit must be assumed that our understanding of how lectinsfunction is not yet comprehensive.

Bacteria use lectins as part of adhesive organelles, calledfimbriae or pili, to adhere to the glycocalyx of their targetcells. Adherence offers a viability advantage for bacteria, asthe cell surface forms an ideal site for multiplication andpersistency, providing a reliable environment with stable pHand salt conditions and versatile nutrients to feed on. Pre-venting bacterial adhesion by suitable carbohydrate inhibi-tors has been envisaged as a means against bacterial coloni-zation, infection by pathogens, inflammation and other dis-eases and medical problems caused by adhesive bacteria.Antiadhesives might offer an alternative to antibiotic treat-ment, which is threatened by a growing number of multire-sistant bacterial strains.[19] The vision of an antiadhesiontherapy has not yet led to pharmacological applications, butit has greatly motivated current programmes on the devel-opment of high-affinity inhibitors of bacterial lec-tins.[66,68,93,104]

The type 1 fimbrial lectin FimH is crucial for mannose-specific adhesion of most enterobacteria including UPECand thus forms a promising target for carbohydrate drugdevelopment. While at first various mannosides and naturaloligosaccharides were investigated as FimH antagonists,mainly two approaches were taken to develop high-affinityligands of FimH. Since the first crystal structure of FimHwas solved in 1999,[50] FimH ligands have been made (i)according to rational design guided by the crystal structureof the FimH carbohydrate-binding site or (ii) more often,glycoclusters were designed for the multivalent presentationof α-d-mannoside ligands for FimH.

Though the bacterial lectin FimH is a monovalent lectinwith a carbohydrate-binding site that accommodates just oneα-mannosyl residue, frequently favourable effects have been


observed with multivalent cluster mannosides of variousarchitectures. Such multivalency effects have not yet beenfully understood in the case of FimH. For steric reasons, itis unlikely that the rather small glycoclusters that have beenemployed for the inhibition of type 1 fimbriae-mediated bac-terial adhesion can effectively cluster several FimH CRDs. Itappears to be more reasonable to conclude on secondarybinding sites on the lectin surface or interpret the effect ofmultivalent FimH ligands on the basis of statistics such ashigh ligand density in the close proximity of the FimH CRD.

For a sensible interpretation of testing results, it is impor-tant to keep in mind the complexity of most testing systemsand of the in vivo situation of bacterial adhesion to hostcell surfaces. In addition to the lectin–carbohydrate interac-tion, which is “isolated” from the more complex molecularscenario to be used as the basis for rational drug design, anycarbohydrate inhibitor also interacts with the surroundingcarbohydrate environment. Likewise, the fimbrial lectin isembedded into a possibly favourable environment before aninteraction with an added carbohydrate inhibitor occurs.The thermodynamic, enthalpic and entropic, as well as ki-netic circumstances of this multifaceted situation can hardlybe sorted out in all detail. Furthermore, the biophysical pa-rameters of carbohydrate binding will be very differentwhether a hemagglutination assay, an ELISA or SPR mea-surements under flow, among others, are being used to de-termine the inhibitory potency of a particular FimH ligand.Moreover, it was shown that shear forces even influence thestructure of the type 1 fimbrial lectin FimH,[87] leading totighter carbohydrate binding according to a mechanisticprinciple that has been termed “catch bonds”.[85]

All together, it cannot be taken for granted that carbo-hydrate ligands for bacterial lectins can be successfully de-veloped as antiadhesives for an antiadhesion therapy invivo. The multidimensional in vivo scenario of cellular ad-hesion might prevent the success of the rather one-dimen-sional idea of an antiadhesion therapy. In addition, it couldbe a critical limitation for the concept to provide enoughselectivity not to interfere with physiological cell adhesion.Given that most pathogens, bacteria and viruses, possessdifferent kinds of lectins to use several carbohydrates foradhesion, selectivity issues will form a major challenge forany approach to an antiadhesion therapy.

After all, research on lectin ligands is also highly moti-vated by fundamental questions on the mechanism ofcarbohydrate binding. Thus, it will continue to form an im-portant field in the glycosciences and eventually will leadto new applications in life science. The development offunctional glycomimetics and lectin ligands will assist in un-ravelling the secrets of glycobiology and to understand bio-logy beyond genomics and proteomics. The key to under-standing glycobiology will be to identify so far undiscov-ered intrinsic features of carbohydrates, as displayed in thesupramolecular environment of living cells.

Abbreviations

AIBN = azobis(isobutyronitrile), ASGPR = asialoglycoprotein re-ceptor, 9-BBN = 9-borabicyclo[3.3.1]nonane, BuMan = butyl α-d-


mannoside, C-HEGA = cyclohexylbutanoyl-N-hydroxyethyl-d-gluc-amide, CRD = carbohydrate recognition domain, DSE = donorstrand exchange, E. coli = Escherichia coli, EC50 = half maximaleffective concentration, ELISA = enzyme-linked immunosorbentassay, FACS = fluorescence-activated cell sorting, FimH-CRD-Th-His = construct containing the FimH-CRD, thrombin cleavage siteand 6His-tag; FP = fluorescence polarization, Gal = galactose,GalNAc = N-acetylgalactosamine, GFP = green fluorescent pro-tein, HAI = hemagglutination inhibition, HSA = human serumalbumin, IC50 = half maximal inhibitory concentration, IT = inhi-bition titre, ITC = isothermal titration calorimetry, LoVo cells =human colon adenocarcinoma cell line, Man = mannose, MeMan= methyl α-d-mannoside, MeUmbMan = methlyumbelliferyl α-d-mannoside, Neu5Ac = 5-N-acetylneuraminic acid, pNPMan =para-nitrophenyl α-d-mannoside, QSAR = quantitative structure–activity relationship; RIP = relative inhibitory potency, SAM =self-assembled monolayer, SD = standard deviation, SPR = surfaceplasmon resonance, TEM = transmission electron microscopy,TMSOTf = trimethylsilyl trifluoromethanesulfonate, UPEC = uro-pathogenic Escherichia coli.

Acknowledgments

Our own work on carbohydrate binding of type 1 fimbriated bacte-ria was financed in its first period by the Deutsche Forschungsge-meinschaft (DFG) and later by Christiana Albertina University.Most valuable over the years have been the contributions of theLindhorst group members, in particular those of Dr. ChristofferKieburg, Dr. Sven Kötter, Dr. Michael Dubber, Dr. Anupama Pa-tel, Dr. Oliver Sperling, Dr. Christoph Heidecke and for molecularmodelling Dr. Andreas Fuchs and Jörn Schmidt-Lassen. Further-more, we are very thankful to our collaborators in this field of ourresearch, Dr. Ulrike Krallmann-Wenzel, Prof. Dr. Stefan Ehlersand Prof. Dr. Stefan Knight.

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Received: March 24, 2011Published Online: June 15, 2011

lindhorst t. k. essentials of carbohydrate chemistry and biochemistry

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