COVER STORY
RU Β IK'S TUBES A given cyclic D,i_-a-peptide sequence gives rise to several nanotube assemblies with different surface presentations through variations in the relative rotations of the cyclic subunits.
BACTERIA: BEWARE! Self-assembled cyclic peptide nanotubes potentially offer a powerful new approach to treating bacterial infections The current approach is largely based on single molecules interacting with bac terial cells in very specific ways. Because N ARGENTINA, INFANTS ARE BECOMING INFECTED WITH DYSENthese interactions typically involve molec tery-causing bacteria that are resistant to third-generation ular recognition, a drug molecule's struc and function are mtertwined. The spe antibiotics. In Taiwan, over the pastfiveyears, there has been ture cific interaction, which depends on the an increase in pneumonia caused by bacteria that are fre molecule's structure, ensures that the drug acts only against its target. Function can be quently resistant to many antibiotics. In the U.S., infections lost with just a slight change in either of with methicillin-resistant Staphylococcus aureus (MRSA), whichthe interacting moieties. Such changes occur easily in bacteria. previously had been associated with stays in hospitals or chronic The lead compounds that have been developed into the current drugs may be care facilities, maybe becoming endemic. characterized as having a narrow "sequence Bacterial resistance to antibiotics is a global public health threat. space." Only a few structural changes in a compound can be tolerated before it Unless new approaches to antibacterial therapy are developed, lead becomes inactive. For this reason, many lead humanity could again be defenseless against bacterial infections. compounds never yield drug candidates. A.MAUREEN ROUHI, C&EN WASHINGTON
I
HTTP://PUBS.ACS.ORG/CEN
C&EN
/ AUGUST
6,
2001
-41
COVER STORY The best scenario involves active mol ecules with a large sequence space. That is, their mode of action is maintained despite gross structural changes. This sit uation cannot be achieved with the molec ular approach to drug design. But it is pos sible with supramolecular assemblies such as the nanotubes designed by M. Reza Ghadiri, a chemistry professor at Scripps Research Institute. THESE NANOTUBES are formed by selfassembly of cyclic peptides with an even number of alternating D- and L-amino acids. The cyclic peptides are stable in solu tion in an open, flat conformation, with all the side chains pointing outward. They stack by intermolecular hydrogen bond ing through the amide backbone, forming a tube that resembles protein β-sheets. The properties of the tube surface and inner pore can be varied by modifying the amino acid sequence, and the size is deter mined by the number of residues. Differ ent combinations of residues will form the same tubular structure. Ghadiri originally conceived of these nanotubes as a way to reach confined spaces in which to do chemical reactions. The idea of stacking circular disks to form a tube was inspired by a visit to the Gug genheim Museum in New York City. "The inside of that museum is basically a helix," he tells C&EN. A tightly wound helix with a sufficiently large radius would be a tube. However, he had rejected that approach as impractical. "But as I was standing there, I realized that a helix with zero pitch [dis tance a helix rises along its axis per turn} is a circle. As soon as that simple idea came to me, it quickly became clear how I would make my nanotubes. And we have since done many things with them." The nanotubes are membrane active. They readily insert themselves into syn thetic lipid bilayers, and the manner of insertion can be modified by design. If the side chains are largely hydrophobic, the nanotubes insert themselves individually If some of the side chains are hydrophilic, several nanotubes insert themselves as a bundle or form carpetlike aggregates that open huge pores in the bilayer. Cells die instantly if their membranes become permeable and leaky Ghadiri rea soned that nanotubes inserting themselves in a carpetlike manner might have anti bacterial activity by exerting such an effect
for a new class of antibacterial peptides. on bacterial cells. And because of the large Samuel H. Gellman, a chemistry pro sequence space, one can find combinations fessor at the University of Wisconsin, Mad of residues that will form peptide nano ison, is especially impressed with the results tubes targeting only bacterial cells. of the mouse studies. "The cyclic peptide Ghadiri recently has shown that the nanotubes can cure infected mice without cyclic peptide nanotubes could be the basis being toxic to the mice," he says. "Very few ofpotent and selective antibacterial agents [Nature, 412, 452 (2001)}. The study cul studies of synthetic antimicrobial peptides have demonstrated efficacy in animal mod minates six years of work, with contribu els upon systemic administration." tions from graduate students Hui-Sun Kim and Keith M. Wilcoxen; postdocs Mer Antibacterial peptides are not new, Gell cedes Delgado, Alisher Khasanov, Karin man notes.The discovery in the 1980s that Kraehenbuehl, Georgina Long, and Dana multicellular organisms, including humans, A. Weinberger; technician Ellen C. Choi; use short, membrane-disrupting peptides former postdoc and visiting scholarJuan R. to fend off local infections has spurred the Granja, who is now an associate professor exploration ofpeptides as systemic antimi of chemistry at the University of Santiago crobial agents. Structures based on natural de Compostela, in Spain; and Sara Fer peptides as well as new, protease-resistant nandez-Lopez, a visiting graduate student classes of structures are being explored. from Granja's lab. Gellman, for example, is studying βpeptides as potential antimicrobial agents. The team prepared a series of six- or eight-residue cyclic D,L-a-peptides designed to target bacterial membranes, and tested their activ ity against Escherichia coli, MRSA, and mammalian cells. They found that some cyclic peptides are preferentially active against bacterial cells compared with mammalian cells. The effect on bacteria is rapid and cata strophic; they die in minutes. Experiments with control pep tides support the hypothesis that the activity is due to self-assem- GERM SLAYERS Delgado (left), Khasanov, bled nanotubes. Biophysical and Ghadiri , Fernandez-Lopez, Choi, Wilcoxen spectroscopic studies show that (back), and Weinberger. the cyclic peptides permeate the bacterial membranes and assemble into Last year, his group reported that this class nanotubes oriented at a 70° tilt, consistent ofpeptides —which is made with β-amino with carpetlike insertion leading to exten acids —shows potent in vitro activity sive membrane damage. against vancomycin- and methicillin-resistant bacteria (C&EN, April 10, 2000, ENCOURAGED BY these results, the team page 14). Similarly, Barron is interested in the therapeutic potential of molecules next tested the cyclic peptides in mice. called peptoids—peptidelike chains of Mice infected with MRSA and then amino acids bearing side chains on the treated with cyclic peptides survived for backbone nitrogen atom rather than on at least seven days. Untreated mice died the α-carbon as seen in peptides. within 48 hours. Both β-peptides and peptoids are pro "It's not very often that you see such cre tease resistant. So are Ghadiri's cyclic pep ative synthetic work, excellent physical tides because of the unnatural D,L-backcharacterization, and serious in vivo stud bone configuration. All are membrane ies all in one paper," comments Annelise active, but they act differently E. Barron, an assistant professor of chem "Ghadiri's cyclic peptides assemble to istry at Northwestern University The thor a higher order structure, and it's that struc oughness by which the Scripps researchers ture that's killing the bacteria, not the sin studied these nanotubes makes for a com gle molecule," Barron says. Neither she nor pelling case that they might be the basis
As fast or even faster than bacteria can change their coats, we can change the nanotube coat." 42
C&EN
/ AUGUST
6,
2001
HTTP://PUB5.ACS.ORG/CEN
anyone else looking at peptide and peptidelike agents "thinks they're creating highly ordered superstructures," she says. "Ghadiri's peptides self-assemble and kill. That's an entirely new concept." "Ifyou think about proteins, folding and function go hand in hand," Ghadiri ex plains. "The function of a natural peptide depends on its side chains. The primary structure dictates how the peptide will fold. % u can't just use any side chain to get at that fold and that function. T h e sequence space that gives you that struc ture and function is limited." With these supramolecular nanotubes, assembly is not dictated primarily by the identity of the amino acid residues. Large variations in the side chain can be toler ated to form the nanotube that performs the function. "This system has a huge sequence
what they encounter in the membrane. "The membrane of a bacterium is itself a self-assembled supramolecular structure that is under dynamic flux," Ghadiri ex plains. Unlike an enzyme or a receptor, the membrane is not a constant well-defined entity For this reason, it is difficult to tar get the membrane selectively with single molecules. But because peptide nanotubes form on-site and are held together by noncovalent forces, they can arrange and rearrange, so as to interact maximally with the mem brane. Just like rotating the layers of a Rubik's cube, changing the relative rota tions of the peptide subunits in a nanotube leads to different-looking nanotubes with different surface presentations. "In a sense, the molecule is deciding what is its best topological isomer in the given environment," Ghadiri says. "This HO
H?
0
J Η Η ο
g H
0 Ο''Η
/*>
*r*
HN L NH
Self-assembly
Ho
R,.„|D
0
, J}
fci^kf
L NH
^\L
H
9 Η
yU0
ΗΝ 0
