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Biological Chemistry

Lassoing Your Target

Unusual peptides with pharmacological potential are amenable to engineering

by Sarah Everts
March 1, 2010 | A version of this story appeared in Volume 88, Issue 9

TYING THE KNOT
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Credit: Mohamed Marahiel
This NMR structure shows capistruin’s lassolike fold, which only nature can assemble. The site of the condensation reaction that forms the ring is shown in red.
Credit: Mohamed Marahiel
This NMR structure shows capistruin’s lassolike fold, which only nature can assemble. The site of the condensation reaction that forms the ring is shown in red.

Of all the quirky molecules made by bacteria, lasso peptides stand out for their Wild West structure. Now, new research is revealing that the super-stable noose-shaped molecules—which resist proteases that can chop up proteins, as well as high temperatures—may also be easily amenable to protein engineering. This opens the possibility that the various antibiotic and otherwise bioactive lasso peptides that pathogenic bacteria produce might be tweaked to make drugs.

The lasso fold was first described in 2003, when three groups simultaneously published reports about the unusual tertiary structure of a natural product called microcin J25. These circular molecules feature a macrolactam ring produced when the peptide’s N-terminus, typically a glycine or a cysteine amino acid, is covalently attached to an acidic side chain eight or nine amino acids away in a condensation reaction. The remaining 10 or so residues near the peptide’s C-terminus tail are threaded through the loop like a bull-corralling lasso, while bulky side chains, often aromatics or arginines, block the tail from escaping the loop, explains Mohamed Marahiel, a chemist at Philipps University, in Marburg, Germany.

Like many other molecules exported by bacteria as a chemical defense against other microbes, these peptides have antibacterial properties. But they are also active against several HIV and cancer targets. What makes them particularly promising as drug leads is that their peculiar final fold is so resilient to proteolytic cleavage and denaturation that they “behave more like small organic molecules than proteins,” wrote David Craik, a chemist at the University of Queensland, in Brisbane, Australia, in a recent commentary (Chem. Biol. 2009, 16, 1211).

Yet unlike organic molecules and other super-stable hooped peptides such as the cyclotides, bench chemists can’t synthesize lasso peptides. Bacteria make the lassos’ peptide backbone by using the ribosome. Tying the knot in the lasso fold requires two special bacterial enzymes. One enzyme is thought to be responsible for cleaving a propeptide from the N-terminus to prepare it for cyclization. The second enzyme is responsible for the condensation reaction.

Strangely, “it appears that the lasso tail is already in the ring before cyclization occurs,” Marahiel says, and it is possibly pushed there by the physical features of the enzymes. Bench chemists can make the lassos’ peptide backbone with a peptide synthesizer and even form the necessary ring, but they have not yet managed to slip the tail into that ring to form the lasso fold.

Although the biomachinery used to make lassos from peptides is absolutely necessary to achieve the three-dimensional structure, the enzymes that catalyze the lasso fold are not particularly picky about their substrate. Last December, Marahiel’s group reported the results of their systematic tweaking of the lasso peptide’s backbone.

The team took four genes required to make a lasso peptide called capistruin from the pathogen Burkholderia thailandensis—one gene to provide the peptide sequence, two for the enzymes that catalyze the lasso fold, and the fourth for a transport protein that pumps the peptides outside the cell—and genetically engineered them into Escherichia coli.

With the model system in place, the team systematically mutated residues in the first gene, which codes for the peptide backbone. The team found that only six of 19 amino acids are required to attain capistruin’s lasso fold, primarily near the seam of the macrolactam ring. In particular, a threonine in the cleaved propeptide, the acidic residue (in this case aspartic acid) to which the N-terminus binds, the N-terminal glycine, and a tripeptide region nearby are all required for the lasso structure. Everything else is incidental, Marahiel notes. His team also found that capistruin’s C-terminal tail can be lengthened by one residue or cut short by as many as three and still acquire the lasso shape (Chem. Biol. 2009, 16, 1290). This work complemented research performed in 2008 by Richard H. Ebright and colleagues at the Waksman Institute of Microbiology at Rutgers University, who also reported that peptide residues near the condensation site of the lasso peptide microcin J25 are important for production, maturation, and export (J. Biol. Chem., 2008, 283, 25589).

“This was a good sign,” because it opens the possibility of grafting pharmacologically promising peptide sequences into the lasso fold, comments Craik, who was not involved in the research. “Peptides make great drug leads, but they are pathetic as drugs because they get chopped up by proteases,” Craik adds. This strategy could be used to avoid proteolysis, instead of opting for unnatural or d-amino acids to increase stability, as drug designers currently do.

Another benefit of lasso-based drugs is that they could be produced by bacterial ribosomes through fermentation. Researchers have long looked for ways to produce or engineer unusual, often cyclical peptides produced by microbes. In particular, one focus has been to reengineer the massive microbial machinery involved in polyketide synthesis and nonribosomal peptide synthesis to produce promising new drugs. But co-opting and reprogramming such peptide-making biomachinery has not been as easy as researchers had hoped, says Marahiel, who also works with nonribosomal peptide synthesis. Using ribosomally produced peptides such as lasso peptides as a scaffold for drug leads “may be a simpler strategy,” for making some therapeutic peptides, he adds.

To date, seven lasso peptides have been identified, some reinforced by disulfide bonds, some not. Because the lasso peptides were initially very hard to characterize—they are so sturdy that the peptides often don’t break apart in mass spectrometers—many more lasso architectures are probably out there to mine as scaffolds for therapeutic peptides, Craik notes.

But even as researchers look to the pharmacological applications of lasso peptides, a lot of basic biochemistry still needs to be ironed out, says Sylvie Rebuffat, a chemist at the Museum of Natural History in Paris who also works on these peptides. In particular, acquiring 3-D structures of the catalytic enzymes required to fold the lasso peptides would help scientists understand how the unique fold is achieved—and also help those wishing to engineer novel lasso peptides. She also points out that being able to produce these peptides synthetically, without the help of E. coli, would be another major achievement.

The long-term hope of all these studies is that “the noose that bacteria use to strangle their competitors might turn out to be a beneficial rope trick in pharmaceutical design,” Craik notes.

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