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The UCSD researchers have devised a way to cut the time it takes to determine the structure of peptides derived from natural com pounds from six months or a year to as little as one day. This advance may assist drug discovery researchers - who need to know as much as possible as quickly as possible about the natural products with antibiotic, antiviral and other pharmacologically interesting properties that they are probing.
According to the researchers, it is currently difficult, time consuming and costly to determine the molecular structure of a class of natural com pounds called nonribosomal peptides (NRPs) that are intensely studied for their drug potential.
To address this issue, they developed a quick, automated and inexpensive way to determine the structure of NRPs through an innovative collaboration between mass spectrometry experts at the UCSD Skaggs School of Pharmacy and Pharmaceutical Sciences and bioinformatics experts and computer scientists from UCSD's Jacobs School of Engineering.
If you imagine the structure of an NRP as a cyclic string of beads, then the new algorithms both decipher the mass of each bead based on the mass spectrometry and determine the order of the beads within the ring - crucial pieces of information for uncovering both the structure of the molecule and its pharmacological activities (Fig.1).
In addition to screening for new drugs and studying natural com pounds , the authors say this work may aid biosynthetic engineering efforts to reprogram E. coli strains in order to turn them into NRP assembly lines, now that researchers have a rapid method for characterising the resulting NRPs.
NRPs such as penicillin, and other natural products, have an unparalleled track record in pharmacology: nine out of the top 20 best-selling drugs were either inspired by or derived from natural products, the authors say.
NRPs evolved over million s of years and often serve chemical defence and communication purposes for the organisms that manufacture them, explained first author Nuno Bandiera, a UCSD postdoctoral researcher and successful PhD candidate from the computer science department at UCSD's Jacobs School of Engineering.
It is notoriously difficult to determine the structure of NRPs because the usual peptide sequencing tools do not work. The cyclic structures of NRPs, the prevalence of non-standard amino acids that thwart database lookups, and the lack of structural information directly inscribed in the genomic DNA due to the nonribosomal nature of the peptides are all major contributors to the roadblock. Researchers have had to rely on slow, manual, expensive and not always reliable approaches to deciphering the structure of NRPs.
"This work removes a particularly troublesome bottleneck in the drug discovery pipeline for this class of therapeutics," said Pieter Dorrestein, assistant professor in the Skaggs School of Pharmacy and Pharmaceutical Sciences and the Departments of Pharmacology, Chemistry and Biochemistry. "We have shown a way to quickly, structurally characterise nonribosomal peptides. Our next step is to replicate our findings with newly discovered, potentially therapeutic peptides."
The UCSD researchers have shown that it is possible to break NRP rings apart and then break the resulting peptide strings into smaller and smaller subunits of the original ring using multiple passes with a mass spectrometer. This approach - called multistage mass spectrometry - allowed the UCSD Skaggs School researchers to collect data on the weights of ring fragments as these fragments got progressively shorter and more numerous with each pass of the mass spectrometer.
The UCSD Jacobs School computer scientists designed algorithms that literally pick up the pieces from here. The algorithms glue the overlapping pieces together until they have reassembled a series of possible original ring structures, explains Julio Ng, a graduate student in UCSD's Interdisciplinary Bioinformatics PhD programme and paper co-author.
The algorithms make use of data on the weights of the various NRP ring fragments collected at each stage using mass spectrometry. This work is an extension of an award-winning automated approach Bandeira and colleagues used to reconstruct snake venom peptides.
"Our Recomb 2008 paper represents the first demonstration of de novo sequencing of nonribosomal peptides. Without knowing the structure of the original compound, we can determine it," explained computer science professor Pavel Pevzner, director of UCSD's Centre for Algorithmic and Systems Biology.
Cheap screening of synthetic molecules
Meanwhile, researchers at the Dallas-based UT Southwestern Medical Centre have developed a simple and inexpensive method to screen small synthetic molecules and pull out a handful that might treat cancer and other diseases less expensively than current methods.
In one screen of more than 300000 such molecules, called peptoids, the new technique quickly singled out five promising candidates that mimicked an antibody already on the market for treating cancer. One of the com pounds blocked the growth of human tumours in a mouse model.
Antibodies are molecules produced by the body to help ward off infection. Natural and manmade antibodies work by latching onto very specific targets such as receptors on the surface of cells.
"Many new drugs being made today are antibodies, but they are extremely expensive to make. Financially, the US health care system is going to have a difficult time accommodating the next 500 drugs being antibodies," said Thomas Kodadek, chief of translational research at UT Southwestern and senior author of the study, which appears online and in an upcoming issue of the Journal of the American Chemical Society.
"Our results show that a peptoid can attack a harmful receptor in the body with the same precision as an antibody, but would cost much less to develop," he added.
Peptoids are designed in the laboratory to resemble chains of natural molecules called peptides. Some peptides are used as medications, such as insulin or antibodies used to treat some cancers, but because the stomach digests them, most cannot be taken by mouth and must be injected instead.
By contrast, peptoids are resistant to the stomach enzymes that degrade natural peptides, so it is possible that they could be swallowed as a pill. Peptoids are much less expensive and easier to manufacture than antibodies, Kodadek said. They are also much smaller than antibodies, so they might be better at penetrating tumours or other disease sites.
"Our technique is simple and fast, works with existing chemicals and needs no high-tech instrumentation, except for a microscope to detect the fluorescent colours we use to sort the com pounds ," said D Gomika Udugamasooriya, postdoctoral researcher in internal medicine and lead author of the study.
The new technique also has major advantages over traditional screening techniques that are commonly used to discover biologically active com pounds from large collections. These screens, which require extensive automation, generally cost US$40000 or more; the new method can be conducted for less than US$1000.
The researchers screened about 300000 peptoids to see which ones would interact with VEGFR2, a type of molecule on the surface of human cells. VEGFR2 is essential in creating new blood vessels through interaction with the hormone VEGF, which is normally a helpful process but is harmful to the body when the new blood vessels are nourishing a growing tumour.
A commercially produced antibody is used to treat some cancers by blocking the VEGF-VEGFR2 interaction and thus starving the tumour, but it costs a patient about US$20000 a year, said Kodadek.
This screen, which took a couple of days, isolated five peptoids out of approximately 300000 screened, showing that the process was an effective way to quickly narrow down a search.
The researchers further tested one of the five peptoids that bound most tightly to VEGFR2 and found that it blocked VEGFR2's action in cultured cells. When they gave it in low doses to mice with implanted human bone- and soft-tissue cancer, the peptoid slowed the growth of the tumours and reduced the density of blood vessels leading to them.