SCIENCE & TECHNOLOGY
LASSOING YOUR TARGET UNUSUAL PEPTIDES with pharmacological
potential are amenable to engineering SARAH EVERTS, C&EN BERLIN
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 nooseshaped 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 atIle
Gly
Thr
Gly
Pro
Val Phe Val Pro HN
Ile His
Gly
Ala
Ser NH Gly
Tyr O
H HN N
O
O O
O OH
Gly Microcin J25
tached 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 chem-
ist 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 TYING THE KNOT This NMR structure shows cleavage and denaturation that lassolike they “behave more like small organ- capistruin’s the results of their systemfold, which only ic molecules than proteins,” wrote atic tweaking of the lasso nature can assemble. David Craik, a chemist at the Unipeptide’s backbone. The site of the versity of Queensland, in Brisbane, condensation reaction The team took four that forms the ring is Australia, in a recent commentary genes required to make a shown in red. (Chem. Biol. 2009, 16, 1211). lasso peptide called capYet unlike organic molecules istruin from the pathogen and other super-stable hooped Burkholderia thailandenpeptides such as the cyclotides, bench sis—one gene to provide the peptide sechemists can’t synthesize lasso peptides. quence, two for the enzymes that catalyze Bacteria make the lassos’ peptide backbone the lasso fold, and the fourth for a transby using the ribosome. Tying the knot in port protein that pumps the peptides outthe lasso fold requires two special bacteside the cell—and genetically engineered rial enzymes. One enzyme is thought to be them into Escherichia coli. responsible for cleaving a propeptide from With the model system in place, the the N-terminus to prepare it for cyclizateam systematically mutated residues in tion. The second enzyme is responsible for the first gene, which codes for the peptide the condensation reaction. backbone. The team found that only six of Strangely, “it appears that the lasso tail 19 amino acids are required to attain capistis already in the ring before cyclization ruin’s lasso fold, primarily near the seam of occurs,” Marahiel says, and it is possibly the macrolactam ring. In particular, a threopushed there by the physical features of the nine in the cleaved propeptide, the acidic enzymes. Bench chemists can make the lasresidue (in this case aspartic acid) to which sos’ peptide backbone with a peptide synthe N-terminus binds, the N-terminal glythesizer and even form the necessary ring, cine, and a tripeptide region nearby are all but they have not yet managed to slip the required for the lasso structure. Everything tail into that ring to form the lasso fold. else is incidental, Marahiel notes. His team Although the biomachinery used to make also found that capistruin’s C-terminal tail lassos from peptides is absolutely necessary can be lengthened by one residue or cut to achieve the three-dimensional structure, short by as many as three and still acquire the enzymes that catalyze the lasso fold are the lasso shape (Chem. Biol. 2009, 16, 1290). not particularly picky about their substrate. “This was a good sign,” because it opens Last December, Marahiel’s group reported the possibility of grafting pharmacologi-
“The noose that bacteria use to strangle their competitors might turn out to be a beneficial rope trick in pharmaceutical design.” WWW.CEN-ONLINE.ORG
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MARCH 1, 2010
Isomer Controlled Bromide Derivatives Pure starting material
Cost effective end product
MOHAMED MARAHIEL
Avoid purification steps by eliminating undesired isomers
cally 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 coopting and reprogramming such peptidemaking 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.
Some examples: 1-bromo-2-ethylbutane
Isomer A
Br
H3C H3C
Br
H3 C
CH3
H3C
H3 C
Br
H3C
>99.5%
Isomer B
H3C CH3
H3 C
H3C Br
Br >99.8%
Unique Isomer and Impurity Control
J
Large Scale Production
J
Custom Synthesis Capabilities
J
World Wide Product Distribution
