RUBIK'S TUBES A given cyclic D,L--peptide sequence gives rise to several nanotube assemblies with different surface presentations through variations in the relative rotations of the cyclic subunits.
	BY KEVIN GUCKIAN AND REZA GHADIRI
	"As fast or even faster than bacteria can change their coats, we can change the nanotube coat."

Bacterial resistance to antibiotics is a global public health threat. Unless new approaches to antibacterial therapy are developed, humanity could again be defenseless against bacterial infections.

The lead compounds that have been developed into the current drugs may be characterized as having a narrow "sequence space." Only a few structural changes in a lead compound can be tolerated before it becomes inactive. For this reason, many lead compounds never yield drug candidates.

The best scenario involves active molecules with a large sequence space. That is, their mode of action is maintained despite gross structural changes. This situation cannot be achieved with the molecular approach to drug design. But it is possible with supramolecular assemblies such as the nanotubes designed by M. Reza Ghadiri, a chemistry professor at Scripps Research Institute.

THESE NANOTUBES are formed by self-assembly of cyclic peptides with an even number of alternating D- and L-amino acids. The cyclic peptides are stable in solution in an open, flat conformation, with all the side chains pointing outward. They stack by intermolecular hydrogen bonding through the amide backbone, forming a tube that resembles protein -sheets. The properties of the tube surface and inner pore can be varied by modifying the amino acid sequence, and the size is determined by the number of residues. Different 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 Guggenheim 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 [distance 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 synthetic 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 reasoned that nanotubes inserting themselves in a carpetlike manner might have antibacterial activity by exerting such an effect on bacterial cells. And because of the large sequence space, one can find combinations of residues that will form peptide nanotubes targeting only bacterial cells.

Ghadiri recently has shown that the cyclic peptide nanotubes could be the basis of potent and selective antibacterial agents [Nature, 412, 452 (2001)]. The study culminates six years of work, with contributions from graduate students Hui-Sun Kim and Keith M. Wilcoxen; postdocs Mercedes Delgado, Alisher Khasanov, Karin Kraehenbuehl, Georgina Long, and Dana A. Weinberger; technician Ellen C. Choi; former postdoc and visiting scholar Juan R. Granja, who is now an associate professor of chemistry at the University of Santiago de Compostela, in Spain; and Sara Fernandez-Lopez, a visiting graduate student from Granja's lab.

The team prepared a series of six- or eight-residue cyclic D,L--peptides designed to target bacterial membranes, and tested their activity 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 catastrophic; they die in minutes.

Experiments with control peptides support the hypothesis that the activity is due to self-assembled nanotubes. Biophysical and spectroscopic studies show that the cyclic peptides permeate the bacterial membranes and assemble into nanotubes oriented at a 70š tilt, consistent with carpetlike insertion leading to extensive membrane damage.

	GERM SLAYERS Delgado (left), Khasanov, Ghadiri, Fernandez-Lopez, Choi, Wilcoxen (back), and Weinberger.
	PHOTO BY JORGE SANCHEZ

ENCOURAGED BY these results, the team next tested the cyclic peptides in mice. Mice infected with MRSA and then treated with cyclic peptides survived for at least seven days. Untreated mice died within 48 hours.

"It's not very often that you see such creative synthetic work, excellent physical characterization, and serious in vivo studies all in one paper," comments Annelise E. Barron, an assistant professor of chemistry at Northwestern University. The thoroughness by which the Scripps researchers studied these nanotubes makes for a compelling case that they might be the basis for a new class of antibacterial peptides.

Samuel H. Gellman, a chemistry professor at the University of Wisconsin, Madison, is especially impressed with the results of the mouse studies. "The cyclic peptide nanotubes can cure infected mice without being toxic to the mice," he says. "Very few studies of synthetic antimicrobial peptides have demonstrated efficacy in animal models upon systemic administration."

Antibacterial peptides are not new, Gellman notes. The discovery in the 1980s that multicellular organisms, including humans, use short, membrane-disrupting peptides to fend off local infections has spurred the exploration of peptides as systemic antimicrobial agents. Structures based on natural peptides as well as new, protease-resistant classes of structures are being explored.

Gellman, for example, is studying -peptides as potential antimicrobial agents. Last year, his group reported that this class of peptides--which is made with -amino acids--shows potent in vitro activity against vancomycin- and methicillin-resistant bacteria (C&EN, April 10, 2000, page 14). Similarly, Barron is interested in the therapeutic potential of molecules called peptoids--peptidelike chains of amino acids bearing side chains on the backbone nitrogen atom rather than on the -carbon as seen in peptides.

Both -peptides and peptoids are protease resistant. So are Ghadiri's cyclic peptides because of the unnatural D,L--backbone configuration. All are membrane active, but they act differently.

"Ghadiri's cyclic peptides assemble to a higher order structure, and it's that structure that's killing the bacteria, not the single molecule," Barron says. Neither she nor 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."

"If you think about proteins, folding and function go hand in hand," Ghadiri explains. "The function of a natural peptide depends on its side chains. The primary structure dictates how the peptide will fold. You can't just use any side chain to get at that fold and that function. The sequence space that gives you that structure 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 tolerated to form the nanotube that performs the function.

"This system has a huge sequence space," Ghadiri says. Different sequences can assemble into the same tubular structure. It becomes a simple matter of screening libraries to select sequences with the properties required for a drug candidate. Coming up with candidates against specific targets will be much more rapid because the mode of action is preserved.

ON THE OTHER HAND, even small variations have profound effects on selectivity. For example, one basic amino acid can be the only difference between a sequence that acts only on bacterial cells and another that acts on both bacterial and mammalian cells. That's because the agent is not a single molecule but a supramolecule. One residue change in the peptide is amplified at the supramolecular level.

Another key aspect of this approach is that the specifics of supramolecular assembly are decided by the environment of the cyclic peptides. There is no one set way that molecules of a cyclic peptide will stack. How they stack will depend on what they encounter in the membrane.

"The membrane of a bacterium is itself a self-assembled supramolecular structure that is under dynamic flux," Ghadiri explains. Unlike an enzyme or a receptor, the membrane is not a constant well-defined entity. For this reason, it is difficult to target 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 membrane. Just like rotating the layers of a Rubik's cube, changing the relative rotations 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 dynamic self-sorting and self-organization gives the peptide nanotube a readout of its environment. Depending on the peptide sequence and the membrane being targeted, the readout can give rise to large selectivity differences between, for example, a bacterial membrane and a host mammalian membrane. That's how selectivity is achieved, which is greater than can be expected from simple adsorption of a small molecule onto the membrane."

That the mode of action is gross disruption of membrane integrity has implications for bacterial resistance. Cells must orchestrate several biosynthetic pathways to assemble the membrane. Bacteria could develop resistance to membrane-active antibacterial agents by evolving different membrane compositions. That requires modifying multiple biosynthetic pathways, not just one or a few mutations, and would likely be a more elaborate and time-consuming strategy for bacteria than simply mutating an enzyme or modifying the composition of one cell-wall constituent.

"In due course, bugs will [find] a way around these nanotubes because nature is wonderfully equipped to resist anything we can come up with," Ghadiri says. "However, our ace in the hole is that as fast or even faster than bacteria can change their coats, we can change the nanotube coat. We can select new sequences presenting new surface characteristics."

THAT THE ACTION is rapid and catastrophic has therapeutic implications. Like bleach, the peptide nanotubes kill on contact. They are bactericidal. Most antibiotics, on the other hand, are bacteriostatic. They only slow the growth of bacteria--for example, by interfering with cell-wall synthesis. It takes time for them to work. "If you're treating late in the infection," Ghadiri points out, "it's very important to have fast-acting drugs."

Whether antibacterial drugs will result from these supramolecular nanotubes is hard to predict. Ghadiri hopes the pharmaceutical industry will look into them. On the practical issues, such as cost and ease of production, they are attractive, he believes.

The cyclic peptides are small organic molecules with molecular weights ranging from 700 to 1,400. The starting materials, d- and l-a-amino acids, are readily available in bulk quantities. And synthetic methods for large-scale production are well developed, Ghadiri says. "They are simpler to synthesize than most drugs."

Furthermore, because the nanotubes assemble only at the site of action, a relatively small molecule can be administered, even though the active agent is large. In terms of drug delivery, small molecules usually are advantageous, notes Ronald N. Zuckermann, director of bioorganic chemistry at Chiron Corp., Emeryville, Calif., a biotechnology company with expertise in infectious diseases and vaccines.

"The potential applications of cyclic peptide nanotubes seem to be vast," Ghadiri says. "One should be able to target many of the current and emerging infectious diseases."

With funding from the National Institute of General Medical Sciences, Ghadiri is bent on optimizing sequences against specific bacterial infections and establishing the range of infectious agents the peptides can target selectively. "Over the next few years, we should be able to establish many useful properties for this class of compounds. We hope they will lead to new drugs that will save human lives. That would be one of the most satisfying outcomes of our adventures in basic science."

PEPTIDE STACKS Self-assembly of flat, cyclic, eight-residue D,L--peptides forms -sheet-like, tubular, open-ended supramolecular structures.

ADAPTED BY PERMISSION FROM NATURE © 2001

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