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MULT1VALENCY: STRENGTH IN NUMBERS
the mechanisms of multivalent recognition events and we are using these insights to develop new kinds of inhibitors and activators." Chemical scaffolds used as a basis for the design of multivalent ligands "range from large polymers to oligomers to dendrimers to small clustered ligands," she says. "Most people in the field are trying to use these multivalent ligands to occupy multiple binding sites on protein domains." Multivalency "is a new approach to drug design," comments chemistry proStu Borman ers are increasingly becoming interest- fessor George M. Whitesides of Hared in bringing the power of multivalen- vard University. With Mathai Mammen C&EN Washington cy to fruition in the rational design of and Seok-Ki Choi (now senior scientist and medicinal chemist, respectively, at any biological processes are set new drugs. into motion when two mole"Critical biological processes such as Advanced Medicine, South San Francules bind to each other. These antibody-antigen interactions and cell- cisco), Whitesides wrote an influential binding events are often monovalent in cell recognition processes are mediated review on the topic [Angew. Chem. Int. nature—such as when a single binding by multivalent interactions," says Laura £4,37,2754(1998)]. site on a ligand sticks to a single binding L. Kiessling, a professor of chemistry Multivalency "is just reaching the site on a receptor. And monovalent in- and biochemistry at the University of transition between university research teractions are often remarkably effec- Wisconsin, Madison. 'With our abilities and commercialization," Whitesides tive. But if monovalent interactions are as chemists to make a variety of scaf- says. Indeed, a number of multivalent good, wouldn't bivalent, trivalent, or folds for multivalent display, we are un- drugs are currently being tested. Howhigher valency interactions be better? covering new fundamental insights into ever, he notes that there is an important distinction to be made beFor some biological pro~"™—— tween oligomeric and polycesses, nature seems to ^ ~ " — ~ meric multivalent agents. have answered this quesDerivatized liposome is multivalent tion in the affirmative. InDivalent and trivalent inhibitor of flu virus deed, some multiple bindcompounds tend to have ing interactions between a low molecular weights and Influenza virus multivalent ligand and a to be orally active, just like ' membrane multivalent receptor are conventional drugs, he says. not only just a little better But polymeric multivalent but, in fact, are orders of species tend to be polydismagnitude better. perse (structurally heterogeneous) mixtures that are Multivalency is the sidifficult to characterize. In multaneous attachment of addition, they generally two or more binding sites Hemagglutinin have unfamiliar mechaon one biological molecule QH nisms of action and often reor organism (such as a proquire injection or other nontein or virus) to multiple reoral forms of administration. ceptor sites on another Therefore, he believes they (such as a cell surface). Scimm will probably be slower to entists have long known reach clinical practice. "It is that multivalent interacnot obvious that the Food & tions play an essential role Drug Administration will in biological processes. But Liposome ever be relaxed about letthey have recently become ting high-molecular-weight, more aware of the imporNeuraminidase polydisperse polymers be tance of these interactions used as systemic drugs," and have begun to focus on Whitesides says. "There multivalency as a separate Wong and coworkers have created liposomes with 3-deoxy3-fluorosialic acid groups—shown once as a chemical strucare all sorts of potential issubdiscipline of chemical ture and otherwise represented by pink ovals. The liposues here." biology. somes bind hemagglutinin and neuraminidase on thefluviMost multivalency studMultivalency can be rus surface, inhibiting viral attachment to host cells. The ies have involved the ratioviewed as a means nature potency of the multivalent inhibitor is about 1,000 times nal design of carbohydrate has found to use weak greater than that of the monomelic sialic acid, an enhanceligands. "Multivalent carbinders to make strong atment caused by the multivalent cluster effect bohydrate-protein interactachments. And research-
Enormous affinity enhancements afforded by multivalent binding may have implications for drug design
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OCTOBER 9,2000 C&EN
multivalency. "It started with tions are at the front edge of some water-soluble polyglycobiology today," says Multivalent neoglycoconjugates mers," Roy says. "Sialic acid is chemistry professor Rene Roy come in many shapes and sizes one of the carbohydrate liof the University of Ottawa, in gands we attached to polyOntario. "Nature uses carbomers, back in 1987. Then we hydrates in a multivalent fashstarted to make water-soluble ion," he says, "and we're trycarbohydrate polymers, which ing to mimic Mother Nature in I call glycopolymers. making carbohydrate-protein interactions better." " 'Neoglycoconjugates' is the general term for artificial or Medicinal chemists "norsynthetic carbohydrate molemally design drugs that have cules," Roy says, "and here very high affinity for a recepwe're talking about multivalent tor—nanomolar or picomolar neoglycoconjugates." Multivabinding affinity," Roy says. lent compounds that have been "But in the carbohydrate field assembled by Roy's group inwe are generally stuck with clude random-coil glycopolylow affinities—millimolar at mers, hyperbranched polybest. So we were never big mers, glycodendrimers (sugarwinners in the design of drugs containing branched spherical based on saccharides or mimstructures), glycodendrons ics of saccharides. This is why (carbohydrate-derivatized halfwe have collectively jumped spherical treelike compounds), into this concept of multivaMultivalent neoglycoconjugate structures synthesized by Roy and coworkers include (clockwise, and comblike dendrigrafts lent interaction." from top right) a comblike dendrigraft (with (compounds in which comblike The affinity enhancements comblike branches on a common polymer backbranches are grafted onto a polyafforded by multivalent bindbone), clusters attached to a polymer, a random-coil mer backbone). ing are often referred to as the glycopolymer, a hyperbranched polymer, and a glycluster effect. In the cluster efWith all of these comcodendrimer. Each ball is a carbohydrate group. fect, "you get an increase in pounds, the cluster effect "is overall affinity above and beclearly there," Roy says. "We yond the multiplication factor of valen- thesize artificial glycopeptides that have observed extremely high binding cy—considerably better than if you bound nearly as well as natural glyco- with clusters and polymers." would just add up the binding affinity peptides," Lee says. "In essence, the per ligand," Roy explains. For example, spatial positioning of the terminal galac- Viral inhibitors a pentavalent inhibitor may bind its tar- tose in the branched structure of N-glySome of the most notable demonstraget with an affinity millions of times cans must fit the combining sites of the tions of the promise of multivalency are higher than that of the corresponding receptor molecules perfectly. Any devia- studies showing how it can be used to monovalent ligand. tion from such an arrangement results fight influenza virus. Several research in a lesser enhancement of binding. The groups have synthesized sialic acidEarly multivalency work key criterion is the fitness of the sugars based polymers that discourage flu viFrom a historical standpoint, Roy and combining sites in multivalent bind- ruses from sticking to cell surfaces—a prerequisite for viral infection of cells. says, biology professor Yuan C. Lee of ing situations." Johns Hopkins University was a pioneer Flu viruses attach to cell surfaces via Whitesides notes that three other rein the study of multivalent interactions searchers carried out pioneering studies multivalent interactions between triin carbohydrate chemistry. "He essen- on multivalency: Nicolai V. Bovin, head of mers of hemagglutinin (a carbohydratetially started it all with small clusters, the Laboratory of Carbohydrate Chemis- binding protein, or lectin, found on viral containing two to three sugar residues," try at Shemyakin & Ovchinnikov Insti- surfaces) and sialic acid (a terminal glyRoy says. tute of Bioorganic Chemistry, Moscow, coprotein found on cell surfaces). This In a 1983 study, Lee and coworkers whom Whitesides calls "a pioneer in the is the interaction that's inhibited by the found tremendous affinity enhance- development of synthetic polymers pre- sialic acid-based polymeric agents. ments in a study on galactose or N- senting multiple ligands as antiviral Early work on the use of sialic acidacetylgalactosamine binding with the agents"; professor emeritus of biological based polymers to inhibit flu virus recepmammalian hepatic carbohydrate re- chemistry Nathan Sharon at Weizmann tor-binding activity was carried out by ceptor [/. Biol. Chern., 2 5 8 , 199 Institute of Science, Rehovot, Israel, who, three groups: Bovin and coworkers (1983)]. 'The binding affinity of mono- Whitesides notes, "realized the impor- [FEBS Lett., 2 7 2 , 209 (1990)], Whitevalent, divalent, and trivalent ligands tance of polyvalency in carbohydrates in sides and coworkers \J. Am. Chem. Soc, increased from 1 to 1,000 and then to the 70s"; and professor Ruth Duncan of 113,686 (1991)], and Roy's group [Can. 1 million, in a rough estimation," Lee the Welsh School of Pharmacy at Cardiff J. Microbiol, 37,233 (1991)]. says. He first used the term "glycoside University, in Wales, a "moving force in The affinity enhancements observed cluster effect" to describe such binding polymeric pharmaceuticals," according in these studies are impressive. Accordenhancements related to multivalency. to Whitesides. ing to Whitesides and coworkers, the "Subsequently, we were able to synRoy's group also did early work on most effective inhibitor of this type de-
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§y::u
ters hemagglutination (cross-linking Chem., 1 0 , 271 (1999)], and we're tive treatment for Shiga-like toxin once of erythrocytes by a virus) at a concen- about to go into animal testing," Toma- it enters the bloodstream. The ligand designed initially by Buntration of only 35 picomolar, whereas lia says. dle's group complexes with only one site the corresponding monomeric interac(site 2) of Shiga-like toxin's three binding tion requires concentrations in the mil- Toxin inhibitors limolar range [Chern. Biol., 3 , 757 Two other groups, working indepen- sites, but the researchers have since in(1996)]. This 108-fold enhancement is dently, recently designed multivalent in- vestigated the possibility of creating a lithe greatest increase in activity "for hibitors that successfully inhibit two dis- gand capable of also binding to site 1, the any synthetic polymeric polyvalent ease-causing bacterial agents: Shiga- site with the next highest affinity. "We have some preliminary data on a secondsystem," Whitesides says. Such find- like toxin and enterotoxin. ings could have implications for the Chemistry professor David R. Bundle generation inhibitor that suggests that a development of highly potent antiviral of the University of Alberta and cowork- design that places an oligosaccharide in drugs. ers designed a multivalent carbohydrate site 1 as well as site 2 yields even more In one set of experiments, Whitesides that inhibits a pathogenic Shiga-like bac- active inhibitors," Bundle says. "We have recently completed a preand coworkers used sialic acid-contain- terial toxin closely related to cholera toxing liposomes to inhibit flu \J. Am. Chem. in [Nature, 403,669 (2000); C&EN, Feb. liminary investigation into the scale-up Soc, 114, 7303 (1992)]. More recently, 14, page 20]. The carbohydrate ligand of our synthesis of the Starfish molecule chemistry professor Chi-Huey Wong and has two trisaccharide units at the tips of to the multigram level, with a view to coworkers at Scripps Research Institute five tethers that radiate from a central conducting animal trials," Bundle tells created a sialic acid liposome and a ly- glucose core. The decavalent inhibitor C&EN. "At the same time, we have insoganglioside/poly-L-glutamic acid con- binds to multiple binding sites on the tox- vestigated modified designs for the moljugate as multivalent picomolar inhibitors in—preventing the toxin from attaching ecule and find that several changes preof flu virus hemagglutinin. The liposome itself to carbohydrates on cell surfaces, serve activity while simplifying the synactually inhibits not only hemagglutinin its usual mechanism of action. The li- thesis. We are in discussions to either but also the viral surface protein neur- gand's molecular shape led Bundle and form a start-up biotechnology company or license this invention, which we find aminidase, an enzyme required for viral coworkers to name it Starfish. replication. The liposome's activity is The in vitro potency of the multiva- has several other potentially attractive about 1,000-fold higher than that of the lent Starfish inhibitor was 10 million applications." corresponding monomeric sialic acid li- times higher than that of the correA group led by research assistant progand, and the activity of the lysoganglio- sponding monovalent ligand-toxin inter- fessor Erkang Fan and professor of bioside is about a millionfold higher than action—suggesting that it could point logical structure and Howard Hughes that of the monomeric ligand [Angew. the way toward development of a potent Medical Institute investigator Wim G. J. Chem. Int. Ed., 3 7 , 1524 (1998); C&EN, antitoxin. Currently, there is no effec- Hoi of the University of Washington deJune 15,1998, page 9]. signed a multivalent inhibitor for — § another bacterial toxin—an enA group led by Donald A. — o Tomalia, scientific director of the @ terotoxin related to cholera toxin Starfish inhibitor binds two Center for Biologic Nanotechnolo| \J. Am. Chem. Soc, 122, 2663 Shiga-like toxin molecules gy at the University of Michigan 1 (2000)]. When ingested, the enMedical School, also has succeed| terotoxin's five identical binding ed in inhibiting viral attachment § sites recognize and bind to ganby employing multivalency. "We 1 glioside carbohydrate groups in make nanodecoys—fake biologiI the gut lining. cal cells consisting of dendrimers £ Designing a multivalent lidecorated with sialic acid groups," gand capable of inhibiting entero| Tomalia says. 'They literally act toxin was not easy because the | as decoys to attract viruses away * toxin's binding sites are widely from healthy cells. If s an artificial separated. Fan, Hoi, and coworkimmunosystem." ers solved this problem by deThe idea, Tomalia says, "is to signing "octopus" ligands that use these nanodecoys either as have five arms—that is, pentavasprays in the mucous membrane lent ligands with long linkers that or as a coating on an absorbent extend five carbohydrate-binding filter that one might have in a resgalactose "fingers" out toward piratory device. Viruses would the toxin binding sites. The best The ciystal structure of a complex between Bundle stick to this like flypaper before pentavalent ligand had submicroand coworkers' Starfish ligand (ball-and-stick repthey even got into the respiratory molar affinity for the toxin—a levresentation, center) and two Shiga-like toxin moletract." Tomalia and coworkers el of potency 105 times higher cules (top and bottom) shows how the toxin is neubelieve nanodecoys can potenthan that of the corresponding tralized by the multivalent carbohydrate ligand. In tially be used to protect against monovalent ligand. the protein, 0-strands are shown as broad arrows bioterrorism or other cases of 'We have not sought commerand oc-helices as coiled ribbons. Some atoms of the large-scale pathogen release. ligand are not visible in the X-ray structure, makcial development for our coming the inhibitor molecule look discontinuous. "We're encouraged by the in pounds so far," Fan tells C&EN, vitro results on this [Bioconjugate "primarily because we want to 50
OCTOBER 9,2000 C&EN
Pentavalent ligand inhibits bacterial enterotoxin Finger Linker
Receptor binding site Pentavalent ligand
Enterotoxin pentamer
Complex
Source: Erkang Fan, Wim Hoi, and coworkers, University of Washington
binding cell-adhesion receptors. "Our approach," Stoddart says, "is to establish a flexible synthetic strategy that will aid the modular construction of a diverse range of suitably branched oligosaccharide-based glycodendrimers, designed to carry specific carbohydrate ligands to bind targeted proteins in a multivalent fashion." Stoddart, graduate student David Fulton, and coworkers are also trying to develop simple and efficient strategies for attaching carbohydrate residues to different parts of cyclodextrin molecules. "We have employed the wellknown photoaddition of thiols to allyl ethers in an anti-Markovnikov fashion to yield thioethers as the key step in the divergent attachment of carbohydrates High affinity and specificity to cyclodextrin cores," Stoddart says Whitesides and coworkers demon- [Organic Letters, 2,1113 (2000)]. strated a multivalent route to high-affiniHe adds that he and his coworkers ty systems when they showed that a tri- "have it in mind to introduce our experimeric vancomycin derivative could bind ence with mechanically interlocked sysa trimeric compound based on the tems into the business of probing multidipeptide D-Ala-D-Ala, a sequence found valency. We would like to establish if in bacterial cell walls, to which vanco- glycodendrimers, coated with sialylmycin binds [Science, 280,708 (1998)]. oligosaccharides and attached to (3-cyThe binding affinity was very strong, clodextrin cores, will thread automatiwith a dissociation constant of about cally onto appropriately constituted 10"17 M for the complex. This makes polyester chains in a thermodynamicalthe complex one of the most stable li- ly driven self-assembly process." Stodgand-receptor pairs based on small or- dart believes the resulting multivalent liganic molecules that is known to chem- gands could find "the ideal spacings and istry—with 25 times higher affinity than orientations to interact with Siglecs on in biotin-avidin binding, one of the cell surfaces." strongest known biological interactions. Chemistry professor Daniel E. Kahne Organic chemistry professor J. Fra- and coworkers at Princeton University ser Stoddart, postdoc Bruce Turnbull, made their own unique contribution to and coworkers at the University of Cali- the field of multivalency by constructing fornia, Los Angeles, currently are work- multivalent compound libraries on resin ing in collaboration with molecular biol- beads. Almost all other studies in the field ogy professor James C. Paulson and co- have involved the synthesis of soluble workers at Scripps Research Institute to multivalent constructs. develop multivalent sialoside probes The Princeton group made a large specific for Siglecs, a recently identified number of resin-bound carbohydrates family of mammalian carbohydrate- in a combinatorial fashion and found make much better ligands before we put them into practical use." Because of the ligands' modular design, "we can independently optimize any module and incorporate it into a new ligand with enhanced affinity toward enterotoxin," Fan says. "Right now, we are focusing on designing better finger modules. We have in hand easily synthesizable fingers that are 100 to 1,000 times better than the one we described in our original JACS paper. We are very optimistic that we will soon be able to synthesize pentavalent ligands against enterotoxin with dissociation constants [down to the] picomolar range. At that time, we will initiate commercial development of our compounds."
that the bead-bound carbohydrates exhibited multivalent behavior, just as soluble multivalent ligands do. When the researchers screened a resin-bound library against a carbohydrate binding protein, they found that the protein bound with high selectivity to only one carbohydrate among more than 1,300 closely related compounds in the library [Science, 274,1520(1996)]. In collaboration with Whitesides and coworkers, Kahne has since shown that the selectivity of carbohydrate binding depends not only on the level of multivalency of the bead-bound carbohydrate ligands but also on the surface density of the ligands [Proc. Natl. Acad. Sci. USA, 9 6 , 11782 (1999)]. "Surface density may be another way in which biological responses are tuned," Kahne says. Wong and coworkers at Scripps have been looking at multivalency from yet another perspective. In an effort to better understand multivalent sugar-receptor interactions on cell surfaces, they developed a dimeric version of the tetrasaccharide cell-adhesion ligand sialyl Lewis X as an inhibitor of the carbohydrate-binding protein E-selectin, which is associated with inflammatory reactions. The researchers also have investigated the role of tyrosine sulfonation in the multivalent interaction that occurs between sialyl Lewis X and P-selectin, another carbohydrate-binding protein. Their study shows that adding a sulfate group to a key tyrosine residue significantly enhances the ligand-receptor interaction and acts as a switch to turn off biosynthesis of the sugar ligand. In addition, Wong and coworkers have found that multivalent inhibitors can be used to target not only proteins but also RNA. They recently developed a bifunctional dimeric aminoglycoside antibiotic that binds to bacterial RNA approximately 1,000 times more tightly than the natural antibiotic does and that inhibits an antibiotic-resistance mechanism—the bacterial enzyme-catalyzed modification of aminoglycoside drugs [/. Am. Chern. Soc, 122, 5230 (2000); C&EN, May 29, page 12]. This new antibiotic is active against several bacteria, including tobramycin-resistant strains.
Multivalent effectors Like most other groups that specialize in multivalency, Wisconsin's Kiessling and coworkers have devoted considerable effort to the development of multivalent inhibitors. But they give uniquely strong emphasis to developing OCTOBER 9,2000 C&EN
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science/technology multivalent effectors as well. Although there are lots of multivalent inhibitors around, there are only a few examples of synthetic multivalent ligands that active ly promote or activate biological pro cesses and cellular responses. In 1998, Kiessling's group showed it could use a multivalent ligand to influ ence the concentration ofL·selectin,a carbohydrate-binding protein that, like E-selectin, is associated with inflamma tion. "Normally, the protein's on the cell," Kiessling says, "but when we en gage the protein with this multivalent li gand, the concentration of protein on the cell surface is diminished. A straight inhibitor would act just by inhibiting L·selectin, whereas this actually takes the whole protein off the cell. It's better than an inhibitor. " Other examples of multivalent ligands that act as effectors are carbohydrate vac cines, such as the anticancer vaccines be ing developed by chemistry professor Samuel J. Danishefsky and coworkers at Memorial Sloan-Kettering Cancer Center and Columbia University. "When you make a vaccine," Kiessling says, "you're making ligands that act as effectors be cause you're trying to giveriseto a partic ular cellular signal—the signal to pro duce antibodies." Kiessling and coworkers use ringopening metathesis polymerization (ROMP) to synthesize defined ligands with multiple recognition elements as po tential inhibitors or effectors. "We've fo cused on using ROMP because it allows us to make a variety of structured multi valent ligands just by simple variations in monomer structure and polymerization conditions," she says. 'Then what we can do is systematically change the valency of a ligand and ask how that affects the in hibitory activity and the effector function of that ligand. We think thaf s really im portant because thaf s whaf s going to al low us to create ligands with very tailored properties that can inhibit or activate a particular signal in a cell." One of the questions she and her co workers have asked concerns the ef fects of using multivalent ligands to change the extent of clustering of pro teins on a cell surface. For example, if dealing with a signaling protein, can the signal be changed by changing the clus tering or pattern of distribution of that protein on the cell surface? The researchers have shown that they can use that approach to systemati cally vary the signal from a signaling protein involved in bacterial chemotax52
OCTOBER 9,2000 C&EN
Multivalent ligand clusters and removes protein Ligand
t^SHs?
L-selectin
Kiessling and coworkers have found that a multivalent ligand can be used to remove Dselectin proteinfromcell surfaces. is, the migration of bacteria toward nu trients. The technique "allows you to ac tually control signal strength by control ling the structure of your ligand. To our knowledge no one's ever done this be fore," Kiessling says. Kiessling and her coworkers are cur rently exploring potential applications for control of protein clustering. "Signal ing cascades are essential processes in the bacterial life cycle," she notes. "So you could imagine that if you influence those signal transduction cascades you could generate antibacterial agents. Be yond bacteria, you could imagine that in the case of human neutrophils it might be useful to have more potent chemoattractants that could recruit neutro phils to specific sites, but it might also be useful to inhibit recruitment. Those are obvious applications." Control of clustering also may be useful for controlling immune respons es. One solution to the shortage of hu man organ donors for transplantation is xenotransplantation—the use of animal organs—but transplanted animal or gans are normally destroyed by the hu man immune system. The major cause of rejection is binding of the human an tibody "anti-Gal" to "α-Gal" carbohy drate antigenic groups on surfaces of animal cells. Kiessling's group has been working with Biotransplant, Charlestown, Mass., to inhibit the production of anti-Gal. "Bcells are cells that make antibodies," she explains, "and every B-cell has on its surface a B-cell receptor that has the same ligand specificity as the antibody it secretes. The idea is if you can cluster the B-cell receptor—if you can make multivalent ligands that can do that— you can prevent antibody production. We have made some ligands that have
that property, and we actually see very good diminution of antibody response. You can knock down about 90% of anti body production using this strategy." Associate professor Peng George Wang and coworkers at Wayne State University, Detroit, used a related ap proach to design multivalent agents that inhibit the immune response to trans plantation \J. Am. Chern. Soc, 121, 8174 (1999)]. They designed and syn thesized carbohydrate polymers conju gated with varying densities of α-Gal epitopes in hopes that the multivalent cluster effect would enhance the bind ing of α-Gal to anti-Gal, which isn't nor mally a strong interaction. Their polymer ligands caused bind ing enhancements of up to 10,000-fold. And Wang and coworkers subsequently demonstrated that the α-Gal polymers can be used in vitro to prevent cytotoxic immunorejection of animal cells, tis sues, and organs by human blood se rum. "We envision that such polymeric α-Gal carbohydrates can be highly use ful in tissue or organ transplantations," Wang says. "We are also working on us ing the α-Gal conjugates in gene thera py and in development of better tumor and viral vaccines." Of course, not all multivalent ligands are carbohydrates. For example, polyamide "chemobodies" have been syn thesized by professor of medical bio chemistry Keith Rose and coworkers at the University of Geneva, in Switzer land. "Chemobody" refers to a chemi cally synthesized antibody-like mole cule. Chemobodies are synthetic mole cules that display multiple peptide subunits that can bind simultaneously to complementary structures. "We wish to exploit the enhanced avidity [multivalent affinity] offered by
binding through two or more subunits simultaneously—multivalent binding— and the flexibility of synthetic chemistry to create molecules of high avidity and selectivity," Rose says. 'When several copies of the same peptide are present in a single multimeric molecule, avidities and bioactivities can be greatly enhanced over the affinity or activity of the monomelic peptide." Using oxime chemistry, Rose and coworkers have successfully attached up to eight peptides to a core molecule. To be able to bind through two or more peptide groups simultaneously, the units must be spaced at appropriate distances and the linking moiety must be flexible. The researchers synthesized a new class of polyamide chains that contain a variable but controllable number of monomer units and used them as flexible linkers to control spacing of ligands in their chemobodies \J. Am. Chem. Soc, 121, 7034 (1999)]. Each chemobody resembles a molecular octopus, with one copy of a peptide on the tip of each of several "tentacles" (polyamide linkers). Rose hopes chemobodies will prove useful for potential diagnostic, therapeutic, and sensing applications. His polyamide chemistry is being further developed by researchers at Gryphon Sciences, South San Francisco.
chelate-type binding but rather from an aggregation or precipitation process that follows the initial intermolecular binding step. 'The mechanism of the enhancement in affinity isn't just an esoteric discussion," Toone says. Rather, it determines "the issue of whether or not multivalency will ever be useful in a biologically relevant setting." If increases in affinity are attributable to intramolecular binding, then affinity will be independent of ligand concentration and multivalent ligands will be active in the low concentration conditions found in vivo, he says. But if the affinity increases because of aggregation, apparent affinities will be strongly dependent on concentration. "Our results suggest that multivalency effects, or cluster glycoside effects, have aggregation events as their origin. If this finding is general, there will be no affinity or avidity at biologically relevant concentrations," thus limiting the use of multivalent compounds as systemic drugs. Harvard's Whitesides agrees that "aggregation and precipitation of cells, bacteria, and viruses by molecules presenting multiple ligands certainly plays a role in the activity of these systems in some in vitro assays." However, he emphasizes that the degree to which aggregation affects in vivo bio-
logical responses has not yet been established. 'There is still some controversy on the origin of this better binding, and I don't think anyone has a clear answer," the University of Ottawa's Roy says. 'We all know multivalent binding is better, but why is a controversial aspect. And thaf s the fun. That makes every one of us work harder to come up with new assays and new designs that help us collectively understand whaf s going on." And even Toone concedes that the verdict isn't totally in on the enhancement mechanism and that more research is needed.
Drug development
"Divalent and trivalent species are going to lead to important pharmaceuticals fairly soon," Whitesides predicts, "and multivalent polymers will lead to new biological activities and eventually also to new pharmaceuticals—although I am not sure for what indications or when." GelTex Pharmaceuticals, Waltham, Mass., is testing a multivalent polymer (GT160-246) that neutralizes the toxin produced by the bacterium Clostridium difficile, the most common cause of antibiotic-associated diarrhea. And Synsorb Biotech, Calgary, Alberta, has two multivalent support-bound trisaccharide M e c h a n i s m s of multivalency agents in clinical trials: Synsorb Pk, a treatment for infection with bacteMeanwhile, scientists are atria such as Escherichia coli tempting to model the underly" — 0157:H7, which produces veroing molecular mechanisms of Chemobody displays peptides toxin (the toxin in '"hamburger dismultivalency to account for the on polyamide 'tentacles' ease"); and Synsorb Cd, a treatoutsize affinity enhancements ofment for C. difficile-associated ten observed in multivalent binddiarrhea. ing interactions. However, the source of the enhancements reWhitesides notes that antimains uncertain. body-based drugs, some of which have already been approved, Some researchers believe that bind their targets bivalently, and multivalent binding is similar to a that a multivalent polymer has chelation process. In this model, been approved for treatment of an initial intermolecular associamultiple sclerosis. tion event between ligand and receptor is followed by intramolecIdeally, Roy says, "every one ular association events between of us is hoping to hit the jackpot individual ligand binding sites on the medicinal chemistry, and individual receptor binding whereby multivalent molecules sites. could prevent cancer cell metastasis, inhibit inflammation, or But associate professor of prevent bacterial adhesion. chemistry Eric J. Toone of Duke Multivalent chemobody designed by Rose and coWe're all looking for a ligand that University and coworkers recentworkers displays four copies of a peptide on polycan be a vaccine, prevent flu or ly proposed a different model amide "tentacles." Flu = acetaminofluorescein, Cy AIDS virus attachment, or treat [Tetrahedron: Asymmetry, 1 1 , 95 = cysteamine linker, ox = oxime linker, s = succinyl [gastrointestinal] infection. (2000)]. They believe that most group, p = NHCH2CH2CH2(0CH2CH2)3CH2NH. Single capital letters are standard single-letter These are the potential applicaof the enhancement in activity designations for amino acids. tions of multivalency that are in observed in multivalent binding the air."^ arises not from intramolecular OCTOBER 9,2000 C&EN
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