Drug Discovery Today: Technologies
Vol. 6, No. 1–4 2009
Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Non Protein Therapeutics
Toward multivalent carbohydrate drugs Roland J. Pieters Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands
Proteins that bind and/or convert carbohydrate structures represent a vast potential for therapeutic development. The use of multivalency is nature’s solution to
Section Editor: Peter Timmerman – Pepscan Therapeutics B.V., PO Box 2098, 8203 AB Lelystad, The Netherlands
overcome the limited affinities of carbohydrate ligands and has also proven to be a successful strategy for inhibitor development. Suitable protein targets are those with closely spaced binding sites such as the cholera toxin B-subunit. However, also protein targets of unknown tertiary and quaternary structure, such as the adhesion protein of the pig pathogen Streptococcus suis, have experimentally proven to be highly suitable. To enhance the search process for new multivalent ligands for new targets, a glycodendrimer microarray technology is developed which rapidly identifies multivalency effects using little precious protein and ligand material.
Introduction Considering the involvement of carbohydrates in many biological processes, interference with its recognition processes is a natural route for the development of new therapeutics. There are numerous protein targets that are involved in disease whose natural ligands are carbohydrate structures [1]. It is a fact that most of these targets represent a vast but unharnessed potential for drug development [2]. This is due to several challenges that are linked to the natural carbohydrate ligands or inhibitors of these types of proteins. E-mail address: R.J. Pieters ([email protected]) 1740-6749/$ ß 2010 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2009.12.001
First of all, carbohydrates are very polar and exhibit poor oral availability. Furthermore, when in circulation they are subject to rapid renal clearing. Besides these pharmacokinetic issues, the affinities of individual carbohydrate ligands for their binding sites are typically too low for use as therapeutics. Several solutions for the mentioned issues have been found in selected cases or are challenges to be tackled in research. To prolong the circulation time, the binding of a carbohydrate to plasma proteins can be beneficial. This is taking place for compound 1 (ArixtraTM [3]), which functions in the blood as an anticoagulant and is protected from elimination since it is bound to the AT-III protein. Oral availability can also be improved by modifying the carbohydrate so it becomes a glycomimetic, which still can make all the intermolecular contacts of the original carbohydrate ligand, but all unneeded polar hydroxyls have been removed, and additional hydrophobic interactions have been added. A prominent example for this is the prodrug compound 2 (TamifluTM) whose free carboxylic acid is an inhibitor of viral neuraminidase [4]. In comparison to its functional relative 3 (RelenzaTM [5]), the structure of 2 was modified and was made more drug-like, resulting in oral availability. Compound 4, the Gaucher disease drug ZavescaTM [6], is also orally active and this lipophilic iminosugar is even able to pass the blood– brain barrier [7]. For certain applications, no oral availability is required, such as application in the intestines, in the lungs or on the skin. To tackle the other major drawback of carbohydrates as therapeutics, their typical low affinity, a strategy e27
Drug Discovery Today: Technologies | Non Protein Therapeutics
Vol. 6, No. 1–4 2009
Figure 1. Examples of carbohydrate (derived) drugs on the market.
that nature itself frequently uses in carbohydrate–protein interactions, that is multivalency, can be used. Multivalent ligands can bind much more strongly to their targets than their monovalent counterparts [8–12]. Especially when chelation is possible (Fig. 2), affinities can be orders of magnitude increased due to multivalency. In those cases the linking units between the subligands have the appropriate length to effectively bridge between adjacent carbohydrate binding sites of a multisite target protein. Despite all the challenges faced by scientists working on carbohydrate-derived therapeutics, several drugs have made it to the market or are in clinical trials. In Fig. 1, several examples are shown. Strikingly, the size of these compounds varies from a relatively lipophilic monosaccharide to a highly polar and charged pentasaccharide. As mentioned, different solutions for the pharmacokinetic challenges were found in each case. Besides these purely carbohydrate (derived) examples, numerous examples exist of (potential) therapeutics that contain one or more carbohydrate moieties, in addition
to a noncarbohydrate part, that can play an important role in the functioning of the compound. Examples include the glycopeptide antibiotics such as for example vancomycin [13] and also the DNA binding abilities of topoisomerase I inhibitor rebeccamycin are affected by the sugar [14].
Glycodendrimers One major problem that is likely to become far worse is the increase in the occurrence of antibiotic-resistant pathogens [15]. This problem represents a multifaceted challenge for medicinal chemistry researchers to develop new therapeutics or even new methods to deal with the bacterial threat. One such new method that holds promise is to interfere with the adhesion process of the bacteria [16–18]. For bacteria the adhesion is often required to establish an infection or to proceed to invasion. Bacteria have evolved adhesion proteins or adhesins for this purpose that are specific for carbohydrate structures present on the specific host tissues to be infected. To inhibit the adhesins would mean to inhibit the bacterial
Figure 2. Schematic representation of a monovalent receptor–ligand interaction (a) contrasted to a divalent interaction (b), based on a chelation-type mechanism that allows simultaneous binding of the two subligands.
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Vol. 6, No. 1–4 2009
Drug Discovery Today: Technologies | Non Protein Therapeutics
Figure 3. Structures of multivalent inhibitors that exhibited greatly valency-dependent inhibitory potencies. Compounds 5–7a are inhibitors of the adhesion of bacterial pathogen Streptococcus suis (6a being 167-fold more potent than a monovalent ligand). Compounds 5–7b containing the GM1oligosaccharide (GM1os) inhibit the cholera toxin B-subunit (CTB5), with an up to 47,500-fold potency increase. Compounds 5–7c are simplified or partly glycomimetic CTB5 inhibitors with potencies for 6c and 7c that rival that of the endogenous GM1os-based ligand.
infection, without actually killing the pathogen, and consequently with a reduced likelihood of resistance build-up. The compounds 5–7a as well as related structures [19] are inhibitors of the pathogen Streptococcus suis, which usually infects pigs and represents a major cost burden for the pig industry (Fig. 3). However, the pathogen can also cause meningitis in humans. It binds to the galabiose sequence (Gala1,4Gal). In an assay reporting on the inhibition of bacterial binding it was shown that the tetravalent 6a was a ca. 167-fold more potent inhibitor than a monovalent galabiose derivative [20]. Cholera bacteria that cause the cholera disease are producers of a toxin, the cholera toxin (CT). This toxin consists of a single disease-causing A-subunit that is surrounded by five lectin-like B-subunits. The B-subunits are responsible for attaching the toxin to the intestinal surface by binding to the exposed GM1-oligosaccharide (GM1os) moieties [21]. The five B-subunits represent a well-defined multivalent protein ˚ target with binding sites for the GM1os units spaced ca. 30 A apart. The advent of ‘click’ chemistry in combination with chemoenzymatic synthesis of the complex carbohydrate allowed us to assemble multivalent versions of the GM1os into glycodendrimers 5–7b [22]. In the evaluation of these compounds the divalent 5b was a dramatically more potent inhibitor with a close to 10,000-fold potency increase relative to a monovalent GM1os derivative. With an additional increase observed for 6b, the octavalent 7b was 380,000-fold more potent (IC50 = 50 pM), which after division by the eight ligands, that is correction for valency, still remains a strong 47,500-fold. The galactose dendrimers 5–7c represent a simplified or a partly glycomimetic version of the multivalent GM1 compounds 5–7b. The pentasaccharide of GM1os was reduced to
a simple monosaccharide while the rest of the hexose rings were replaced by PEG modules. The inhibitory potency did suffer because of this transformation because the relatively large binding site of the CT remains partly unoccupied. Nevertheless, the multivalency effects were still very strong and the compounds 6–7c were competitive with the natural GM1os ligand regarding their inhibitory potency [23]. Carbohydrate binding sites are not usually as large as the CT site and it can be expected that complicated carbohydrate structures can be reduced to a mere mono- or disaccharide without the loss of potency. Presenting these mono- or disaccharides on a dendrimer in a multivalent fashion can subsequently lead to vastly increased potency for certain target proteins.
Glycodendrimer chips Carbohydrates have been displayed on microarray chips in many cases. They are being screened by large series of carbohydrate binding proteins, antibodies and also pathogenic organisms [24–27]. These studies yield a wealth of information for the design of new therapeutics without using a lot of material. In short, these studies are very efficient. Such an increase in efficiency is also needed for the study of multivalent carbohydrate–protein interactions. One step forward is the display of glycodendrimers on a microarray surface. As can be seen in Fig. 4, glycodendrimers can be displayed on a microarray surface in such a way that the number of the carbohydrates on each spot is the same and only the valency differs from spot to spot [28]. Evaluation of these spots with fluorescent lectins showed that in the case where a multivalency effect was expected because of the presence of multiple closely spaced binding sites, this could indeed be observed in a single experiment, using only minute quantities of www.drugdiscoverytoday.com
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Drug Discovery Today: Technologies | Non Protein Therapeutics
Vol. 6, No. 1–4 2009
Figure 4. On the left, a cartoon depiction of the glycodendrimer array surface is shown, with ever increasing valency yet with a carbohydrate content that is the same in all cases. On the right, the actual structures of the mannose dendrimers are shown.
synthetic and biological materials. The mannose structures were incubated with fluorescent mannose binding proteins, the lectins ConA and GNA. In the case of ConA strong signals were seen but no major differentiation with respect to valency. By contrast, the very weakly binding GNA showed a much stronger binding to the higher valent mannosides on the chip and seemed to benefit much more from the multivalent presentation than ConA. This result correlated well with the possibility for chelation, which is unlikely to occur for ConA. The distance between binding sites is too large, ˚ . We whereas in GNA binding sites are spaced as close as 14 A believe that further developments along these lines will lead to the identification of many instances of a strong multivalent binding, and thus will yield novel lead compounds.
Conclusions Proteins that bind and/or convert carbohydrate structures represent a vast potential of target structures for the development of therapeutics. Whether such therapeutics will emerge depends on our ability to overcome the challenges ahead. Aspects such as bioavailability or clearance can be overcome either by creating glycomimetics or by using tricks involving transporter or plasma proteins while the issue of low affinity can be tackled by multivalency. Multivalent presentation has a great potential in the modulation of protein carbohydrate interactions, because nature itself has evolved carbohydrates to act in a multivalent fashion to reach biologically relevant potency levels. Glycodendrimer technology has proven to be an excellent platform to further explore these interactions. In selected cases very high potency increases have been observed with these dendrimers. The modular synthesis allows convenient variation of e30
www.drugdiscoverytoday.com
valency and also of spacer length, a crucial factor in potency optimization. To become of interest to the drug development community, still more efficiency is needed in both the synthesis and the evaluation of multivalent systems. To this end glycodendrimer microarray technology may play an important role. It allows the rapid evaluation of several multivalency effects without using large quantities of synthetic and biological materials. While ultimately all challenges for the development of glyco(mimetic) therapeutics need to be solved, multivalency studies with glycodendrimers promise to greatly facilitate the needed affinity increase.
References 1 Werz, D.B. and Seeberger, P.H. (2005) Carbohydrates as the next frontier in pharmaceutical research. Chem. Eur. J. 11, 3194–3206 2 Ernst, B. and Magnani, J.L. (2009) From carbohydrate leads to glycomimetic drugs. Nat. Rev. Drug Dis. 8, 661–677 3 Petitou, M. and van Boeckel, C.A.A. (2004) A synthetic antithrombin III binding pentasaccharide is now a drug! What comes next? Angew. Chem. Int. Ed. 43, 3118–3133 4 Kim, C.U. et al. (1997) Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity. J. Am. Chem. Soc. 119, 681–690 5 von Itzstein, M. et al. (1993) Rational design of potent sialidase-based inhibitors of influenza-virus replication. Nature 363, 418–423 6 Platt, F.M. et al. (1994) N-Butyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis. J. Biol. Chem. 269, 8362–8365 7 Aerts, J. et al. (2006) Substrate reduction therapy of glycosphingolipid storage disorders. J. Inherit. Metab. Dis. 29, 449–456 8 Mammen, M. et al. (1998) Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Ed. 37, 2755–2794 9 Lundquist, J.J. and Toone, E.J. (2002) The cluster glycoside effect. Chem. Rev. 102, 555–578 10 Pieters, R.J. (2009) Maximising multivalency effects in protein– carbohydrate interactions. Org. Biomol. Chem. 7, 2013–2025
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Lee, R.T. et al. (1989) Binding characteristics of N-acetylglucosaminespecific lectin of the isolated chicken hepatocytes: similarities to mammalian hepatic galactose/N-acetylgalactosamine-specific lectin. Biochemistry 28, 8351–8358 Kamitakahara, H. et al. (1998) A lysoganglioside poly-L-glutamic acid conjugate as a picomolar inhibitor of influenza hemagglutinin. Angew. Chem. Int. Ed. 37, 1524–1528 Gerhard, U. et al. (1993) The role of the sugar and chlorine substituents in the dimerization of vancomycin antibiotics. J. Am. Chem. Soc. 115, 232–237 Bailly, C. et al. (1999) Calories from carbohydrates: energetic contribution of the carbohydrate moiety of rebeccamycin to DNA binding and the effect of its orientation on topoisomerase I inhibition. Chem. Biol. 6, 277–286 Alekshun, M.N. and Levy, S.B. (2007) Molecular mechanisms of antibacterial multidrug resistance. Cell 128, 1037–1050 Imberty, A. et al. (2008) Glycomimetics and glycodendrimers as high affinity microbial anti-adhesins. Chem. Eur. J. 14, 7490–7499 Sharon, N. and Ofek, I. (2000) Safe as mother’s milk: carbohydrates as future anti-adhesion drugs for bacterial diseases. Glycoconj. J. 17, 659– 664 Pieters, R.J. (2007) Intervention with bacterial adhesion by multivalent carbohydrates. Med. Res. Rev. 27, 796–816
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Joosten, J.A.F. et al. (2004) Inhibition of Streptococcus suis adhesion by dendritic galabiose compounds at low nanomolar concentration. J. Med. Chem. 47, 6499–6508 Branderhorst, H.M. et al. (2008) Synthesis of multivalent Streptococcus suis adhesion inhibitors by enzymatic cleavage of polygalacturonic acid and ‘click’ conjugation. Org. Biomol. Chem. 6, 1425–1434 Fan, E.K. et al. (2000) AB(5) toxins: structures and inhibitor design. Curr. Opin. Struct. Biol. 10, 680–686 Pukin, A.V. et al. (2007) Strong inhibition of cholera toxin by multivalent GM1 derivatives. ChemBioChem 8, 1500–1503 Branderhorst, H.M. et al. (2007) Strong inhibition of cholera toxin binding by galactose dendrimers. Chem. Commun. 5043–5045 Oyelaran, O. and Gildersleeve, J.V. (2009) Glycan arrays: recent advances and future challenges. Curr. Opin. Chem. Biol. 13, 406–413 Wu, C.Y. et al. (2009) New development of glycan arrays. Org. Biomol. Chem. 7, 2247–2254 Park, S. et al. (2008) Chemical tools for functional studies of glycans. Chem. Soc. Rev. 37, 1579–1591 Horlacher, T. and Seeberger, P.H. (2008) Carbohydrate arrays as tools for research and diagnostics. Chem. Soc. Rev. 37, 1414–1422 Branderhorst, H.M. et al. (2008) Multivalent carbohydrate recognition on a glycodendrimer-functionalized flow-through chip. ChemBioChem 9, 1836–1844
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Vol. 6, No. 1–4 2009
Editors-in-Chief Kelvin Lam – Pfizer, Inc., USA Henk Timmerman – Vrije Universiteit, The Netherlands DRUG DISCOVERY
TODAY
TECHNOLOGIES
Non Protein Therapeutics
Toward multivalent carbohydrate drugs Roland J. Pieters Department of Medicinal Chemistry and Chemical Biology, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, P.O. Box 80082, 3508 TB Utrecht, The Netherlands
Proteins that bind and/or convert carbohydrate structures represent a vast potential for therapeutic development. The use of multivalency is nature’s solution to
Section Editor: Peter Timmerman – Pepscan Therapeutics B.V., PO Box 2098, 8203 AB Lelystad, The Netherlands
overcome the limited affinities of carbohydrate ligands and has also proven to be a successful strategy for inhibitor development. Suitable protein targets are those with closely spaced binding sites such as the cholera toxin B-subunit. However, also protein targets of unknown tertiary and quaternary structure, such as the adhesion protein of the pig pathogen Streptococcus suis, have experimentally proven to be highly suitable. To enhance the search process for new multivalent ligands for new targets, a glycodendrimer microarray technology is developed which rapidly identifies multivalency effects using little precious protein and ligand material.
Introduction Considering the involvement of carbohydrates in many biological processes, interference with its recognition processes is a natural route for the development of new therapeutics. There are numerous protein targets that are involved in disease whose natural ligands are carbohydrate structures [1]. It is a fact that most of these targets represent a vast but unharnessed potential for drug development [2]. This is due to several challenges that are linked to the natural carbohydrate ligands or inhibitors of these types of proteins. E-mail address: R.J. Pieters ([email protected]) 1740-6749/$ ß 2010 Elsevier Ltd. All rights reserved.
DOI: 10.1016/j.ddtec.2009.12.001
First of all, carbohydrates are very polar and exhibit poor oral availability. Furthermore, when in circulation they are subject to rapid renal clearing. Besides these pharmacokinetic issues, the affinities of individual carbohydrate ligands for their binding sites are typically too low for use as therapeutics. Several solutions for the mentioned issues have been found in selected cases or are challenges to be tackled in research. To prolong the circulation time, the binding of a carbohydrate to plasma proteins can be beneficial. This is taking place for compound 1 (ArixtraTM [3]), which functions in the blood as an anticoagulant and is protected from elimination since it is bound to the AT-III protein. Oral availability can also be improved by modifying the carbohydrate so it becomes a glycomimetic, which still can make all the intermolecular contacts of the original carbohydrate ligand, but all unneeded polar hydroxyls have been removed, and additional hydrophobic interactions have been added. A prominent example for this is the prodrug compound 2 (TamifluTM) whose free carboxylic acid is an inhibitor of viral neuraminidase [4]. In comparison to its functional relative 3 (RelenzaTM [5]), the structure of 2 was modified and was made more drug-like, resulting in oral availability. Compound 4, the Gaucher disease drug ZavescaTM [6], is also orally active and this lipophilic iminosugar is even able to pass the blood– brain barrier [7]. For certain applications, no oral availability is required, such as application in the intestines, in the lungs or on the skin. To tackle the other major drawback of carbohydrates as therapeutics, their typical low affinity, a strategy e27
Drug Discovery Today: Technologies | Non Protein Therapeutics
Vol. 6, No. 1–4 2009
Figure 1. Examples of carbohydrate (derived) drugs on the market.
that nature itself frequently uses in carbohydrate–protein interactions, that is multivalency, can be used. Multivalent ligands can bind much more strongly to their targets than their monovalent counterparts [8–12]. Especially when chelation is possible (Fig. 2), affinities can be orders of magnitude increased due to multivalency. In those cases the linking units between the subligands have the appropriate length to effectively bridge between adjacent carbohydrate binding sites of a multisite target protein. Despite all the challenges faced by scientists working on carbohydrate-derived therapeutics, several drugs have made it to the market or are in clinical trials. In Fig. 1, several examples are shown. Strikingly, the size of these compounds varies from a relatively lipophilic monosaccharide to a highly polar and charged pentasaccharide. As mentioned, different solutions for the pharmacokinetic challenges were found in each case. Besides these purely carbohydrate (derived) examples, numerous examples exist of (potential) therapeutics that contain one or more carbohydrate moieties, in addition
to a noncarbohydrate part, that can play an important role in the functioning of the compound. Examples include the glycopeptide antibiotics such as for example vancomycin [13] and also the DNA binding abilities of topoisomerase I inhibitor rebeccamycin are affected by the sugar [14].
Glycodendrimers One major problem that is likely to become far worse is the increase in the occurrence of antibiotic-resistant pathogens [15]. This problem represents a multifaceted challenge for medicinal chemistry researchers to develop new therapeutics or even new methods to deal with the bacterial threat. One such new method that holds promise is to interfere with the adhesion process of the bacteria [16–18]. For bacteria the adhesion is often required to establish an infection or to proceed to invasion. Bacteria have evolved adhesion proteins or adhesins for this purpose that are specific for carbohydrate structures present on the specific host tissues to be infected. To inhibit the adhesins would mean to inhibit the bacterial
Figure 2. Schematic representation of a monovalent receptor–ligand interaction (a) contrasted to a divalent interaction (b), based on a chelation-type mechanism that allows simultaneous binding of the two subligands.
e28
www.drugdiscoverytoday.com
Vol. 6, No. 1–4 2009
Drug Discovery Today: Technologies | Non Protein Therapeutics
Figure 3. Structures of multivalent inhibitors that exhibited greatly valency-dependent inhibitory potencies. Compounds 5–7a are inhibitors of the adhesion of bacterial pathogen Streptococcus suis (6a being 167-fold more potent than a monovalent ligand). Compounds 5–7b containing the GM1oligosaccharide (GM1os) inhibit the cholera toxin B-subunit (CTB5), with an up to 47,500-fold potency increase. Compounds 5–7c are simplified or partly glycomimetic CTB5 inhibitors with potencies for 6c and 7c that rival that of the endogenous GM1os-based ligand.
infection, without actually killing the pathogen, and consequently with a reduced likelihood of resistance build-up. The compounds 5–7a as well as related structures [19] are inhibitors of the pathogen Streptococcus suis, which usually infects pigs and represents a major cost burden for the pig industry (Fig. 3). However, the pathogen can also cause meningitis in humans. It binds to the galabiose sequence (Gala1,4Gal). In an assay reporting on the inhibition of bacterial binding it was shown that the tetravalent 6a was a ca. 167-fold more potent inhibitor than a monovalent galabiose derivative [20]. Cholera bacteria that cause the cholera disease are producers of a toxin, the cholera toxin (CT). This toxin consists of a single disease-causing A-subunit that is surrounded by five lectin-like B-subunits. The B-subunits are responsible for attaching the toxin to the intestinal surface by binding to the exposed GM1-oligosaccharide (GM1os) moieties [21]. The five B-subunits represent a well-defined multivalent protein ˚ target with binding sites for the GM1os units spaced ca. 30 A apart. The advent of ‘click’ chemistry in combination with chemoenzymatic synthesis of the complex carbohydrate allowed us to assemble multivalent versions of the GM1os into glycodendrimers 5–7b [22]. In the evaluation of these compounds the divalent 5b was a dramatically more potent inhibitor with a close to 10,000-fold potency increase relative to a monovalent GM1os derivative. With an additional increase observed for 6b, the octavalent 7b was 380,000-fold more potent (IC50 = 50 pM), which after division by the eight ligands, that is correction for valency, still remains a strong 47,500-fold. The galactose dendrimers 5–7c represent a simplified or a partly glycomimetic version of the multivalent GM1 compounds 5–7b. The pentasaccharide of GM1os was reduced to
a simple monosaccharide while the rest of the hexose rings were replaced by PEG modules. The inhibitory potency did suffer because of this transformation because the relatively large binding site of the CT remains partly unoccupied. Nevertheless, the multivalency effects were still very strong and the compounds 6–7c were competitive with the natural GM1os ligand regarding their inhibitory potency [23]. Carbohydrate binding sites are not usually as large as the CT site and it can be expected that complicated carbohydrate structures can be reduced to a mere mono- or disaccharide without the loss of potency. Presenting these mono- or disaccharides on a dendrimer in a multivalent fashion can subsequently lead to vastly increased potency for certain target proteins.
Glycodendrimer chips Carbohydrates have been displayed on microarray chips in many cases. They are being screened by large series of carbohydrate binding proteins, antibodies and also pathogenic organisms [24–27]. These studies yield a wealth of information for the design of new therapeutics without using a lot of material. In short, these studies are very efficient. Such an increase in efficiency is also needed for the study of multivalent carbohydrate–protein interactions. One step forward is the display of glycodendrimers on a microarray surface. As can be seen in Fig. 4, glycodendrimers can be displayed on a microarray surface in such a way that the number of the carbohydrates on each spot is the same and only the valency differs from spot to spot [28]. Evaluation of these spots with fluorescent lectins showed that in the case where a multivalency effect was expected because of the presence of multiple closely spaced binding sites, this could indeed be observed in a single experiment, using only minute quantities of www.drugdiscoverytoday.com
e29
Drug Discovery Today: Technologies | Non Protein Therapeutics
Vol. 6, No. 1–4 2009
Figure 4. On the left, a cartoon depiction of the glycodendrimer array surface is shown, with ever increasing valency yet with a carbohydrate content that is the same in all cases. On the right, the actual structures of the mannose dendrimers are shown.
synthetic and biological materials. The mannose structures were incubated with fluorescent mannose binding proteins, the lectins ConA and GNA. In the case of ConA strong signals were seen but no major differentiation with respect to valency. By contrast, the very weakly binding GNA showed a much stronger binding to the higher valent mannosides on the chip and seemed to benefit much more from the multivalent presentation than ConA. This result correlated well with the possibility for chelation, which is unlikely to occur for ConA. The distance between binding sites is too large, ˚ . We whereas in GNA binding sites are spaced as close as 14 A believe that further developments along these lines will lead to the identification of many instances of a strong multivalent binding, and thus will yield novel lead compounds.
Conclusions Proteins that bind and/or convert carbohydrate structures represent a vast potential of target structures for the development of therapeutics. Whether such therapeutics will emerge depends on our ability to overcome the challenges ahead. Aspects such as bioavailability or clearance can be overcome either by creating glycomimetics or by using tricks involving transporter or plasma proteins while the issue of low affinity can be tackled by multivalency. Multivalent presentation has a great potential in the modulation of protein carbohydrate interactions, because nature itself has evolved carbohydrates to act in a multivalent fashion to reach biologically relevant potency levels. Glycodendrimer technology has proven to be an excellent platform to further explore these interactions. In selected cases very high potency increases have been observed with these dendrimers. The modular synthesis allows convenient variation of e30
www.drugdiscoverytoday.com
valency and also of spacer length, a crucial factor in potency optimization. To become of interest to the drug development community, still more efficiency is needed in both the synthesis and the evaluation of multivalent systems. To this end glycodendrimer microarray technology may play an important role. It allows the rapid evaluation of several multivalency effects without using large quantities of synthetic and biological materials. While ultimately all challenges for the development of glyco(mimetic) therapeutics need to be solved, multivalency studies with glycodendrimers promise to greatly facilitate the needed affinity increase.
References 1 Werz, D.B. and Seeberger, P.H. (2005) Carbohydrates as the next frontier in pharmaceutical research. Chem. Eur. J. 11, 3194–3206 2 Ernst, B. and Magnani, J.L. (2009) From carbohydrate leads to glycomimetic drugs. Nat. Rev. Drug Dis. 8, 661–677 3 Petitou, M. and van Boeckel, C.A.A. (2004) A synthetic antithrombin III binding pentasaccharide is now a drug! What comes next? Angew. Chem. Int. Ed. 43, 3118–3133 4 Kim, C.U. et al. (1997) Influenza neuraminidase inhibitors possessing a novel hydrophobic interaction in the enzyme active site: design, synthesis, and structural analysis of carbocyclic sialic acid analogues with potent anti-influenza activity. J. Am. Chem. Soc. 119, 681–690 5 von Itzstein, M. et al. (1993) Rational design of potent sialidase-based inhibitors of influenza-virus replication. Nature 363, 418–423 6 Platt, F.M. et al. (1994) N-Butyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis. J. Biol. Chem. 269, 8362–8365 7 Aerts, J. et al. (2006) Substrate reduction therapy of glycosphingolipid storage disorders. J. Inherit. Metab. Dis. 29, 449–456 8 Mammen, M. et al. (1998) Polyvalent interactions in biological systems: implications for design and use of multivalent ligands and inhibitors. Angew. Chem. Int. Ed. 37, 2755–2794 9 Lundquist, J.J. and Toone, E.J. (2002) The cluster glycoside effect. Chem. Rev. 102, 555–578 10 Pieters, R.J. (2009) Maximising multivalency effects in protein– carbohydrate interactions. Org. Biomol. Chem. 7, 2013–2025
Vol. 6, No. 1–4 2009
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Lee, R.T. et al. (1989) Binding characteristics of N-acetylglucosaminespecific lectin of the isolated chicken hepatocytes: similarities to mammalian hepatic galactose/N-acetylgalactosamine-specific lectin. Biochemistry 28, 8351–8358 Kamitakahara, H. et al. (1998) A lysoganglioside poly-L-glutamic acid conjugate as a picomolar inhibitor of influenza hemagglutinin. Angew. Chem. Int. Ed. 37, 1524–1528 Gerhard, U. et al. (1993) The role of the sugar and chlorine substituents in the dimerization of vancomycin antibiotics. J. Am. Chem. Soc. 115, 232–237 Bailly, C. et al. (1999) Calories from carbohydrates: energetic contribution of the carbohydrate moiety of rebeccamycin to DNA binding and the effect of its orientation on topoisomerase I inhibition. Chem. Biol. 6, 277–286 Alekshun, M.N. and Levy, S.B. (2007) Molecular mechanisms of antibacterial multidrug resistance. Cell 128, 1037–1050 Imberty, A. et al. (2008) Glycomimetics and glycodendrimers as high affinity microbial anti-adhesins. Chem. Eur. J. 14, 7490–7499 Sharon, N. and Ofek, I. (2000) Safe as mother’s milk: carbohydrates as future anti-adhesion drugs for bacterial diseases. Glycoconj. J. 17, 659– 664 Pieters, R.J. (2007) Intervention with bacterial adhesion by multivalent carbohydrates. Med. Res. Rev. 27, 796–816
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