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Bicoastal discoveries from Scripps
September 2015
by Mel J. Yeates  |  Email the author
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JUPITER, Fla.—Scientists from The Scripps Research Institute (TSRI) Florida campus have shown an “unprecedented mechanism” for how a natural antibiotic with antitumor properties incorporates sulfur into its molecular structure. This new discovery could open the way to incorporating sulfur into other natural products, potentially advancing new therapies for indications beyond cancer.
 
The study, which was led by TSRI professor Ben Shen, was recently released online ahead of print by the journal Proceedings of the National Academy of Sciences. “We found a novel mechanism to incorporate sulfur into natural products,” according to Shen. “Until our study, we didn’t really know how sulfur atoms are incorporated into a natural product—now we have discovered a new family of enzymes and have a workable mechanism to account for sulfur incorporation into a larger class of polyketides that include many drugs such as erythromycin (antibacterial) and lovastatin (cholesterol-lowering).”
 
The study is focused on leinamycin (LNM), a sulfur-containing antitumor antibiotic produced by a species of the soil-dwelling bacterium Streptomyces. The Shen laboratory has been studying the potential of this natural compound for development of anticancer drugs.
 
“In an earlier study, we have also showed that an engineered analogue of LNM (LNM E1) could act as a prodrug activated by cellular reactive oxygen species to cause DNA damage and cancer cell death,” says Shen. “The LNM scaffold and its unprecedented sulfur-mediated mode of action will serve as the basis for further drug discovery efforts.”
 
“The sulfur-incorporation mechanism discovered in our study revealed the novel function of a polyketide synthase, greatly expanding our understanding of its chemistry,” said Ming Ma, one of the co-first authors of the study and a member of the Shen lab. “Since polyketide synthases are a large family of enzymes that have been proven amenable for polyketide structural diversity and drug discovery, it is particularly exciting that this new discovery now provides the possibilities of adding sulfur atoms to compounds similar to leinamycin or other polyketide natural products.”
 
Polyketide synthases (PKSs) link directly to a complex series of chemical reactions that ultimately add sulfur to leinamycin. Shen tells DDNews that the discovery is based on long-term research into key steps in LNM biosynthesis. Their discovery of the C-S bond cleaving polyketide synthase domain in LNM expands the current toolbox for developing novel polyketide natural products.
 
Leinamycin is a unique natural product not only for its chemical structure but also for its antitumor mechanism. The unique features of LNM are mainly concentrated in its sulfur-containing dithiolane moiety, which kills by interfering with the cancer cell’s DNA. PKSs are the key enzymes responsible for the construction of the polyketide backbones of the polyketide family of natural products from short carboxylic acid precursors. Advances in polyketide biosynthesis and engineering have demonstrated that PKSs are quite amenable to metabolic pathway engineering to generate polyketide structural diversity.
 
“Our study not only reveals how the first sulfur in the dithiolane moiety of LNM is incorporated, but also the novel function of a PKS domain (LnmJ-SH domain), expanding our understanding of the chemistry and enzymology of PKSs,” says Shen. “The functional characterization of LnmJ-SH domain opens the door for new LNM-like natural product discovery by means of homologic gene cluster screening or biosynthetic pathway engineering, particularly since LnmJ-SH represents a large family of PKS domains whose functions have never been characterized before. LNM is a promising anticancer drug lead, and we have also demonstrated in an earlier study the therapeutic potential of LNM E1 by showing it to be effective against two prostate cancer cell lines, which are known to exist under high oxidative stress and with increased levels of reactive oxygen species.”
 
In a broad sense, this new discovery could be further developed into enabling technologies to increase natural product structural diversity and accelerate new natural product discovery. Because few sulfur-containing natural products are known, this particular enzyme and its gene could now be useful tools to probe ecological niches for the discovery of other sulfur-containing natural products.
 
In addition to Shen and Ma, other authors of the study, “C-S Bond Cleavage by a Polyketide Synthase Domain,” are Jeremy R. Lohman (co-first author) of TSRI and Tao Liu of the University of Wisconsin, current and former members of the Shen lab.
 
Meanwhile, TSRI’s original campus in La Jolla, Calif., has been a busy place as well, with a new study exploring a bacterial enzyme that might be used as a drug candidate to help people quit smoking. The research shows that this enzyme can be recreated in lab settings and possesses a number of promising characteristics for drug development.
 
“Our research is in the early phase of drug development process, but the study tells us the enzyme has the right properties to eventually become a successful therapeutic,” said Kim Janda, the Ely R. Callaway Jr. Professor of Chemistry and member of the Skaggs Institute for Chemical Biology at TSRI.
 
The new research, published in the Journal of the American Chemical Society, offers a possible alternative to current smoking cessation aids, which are shown to fail in at least 80 to 90 percent of smokers. The idea behind an enzyme therapy would be to seek out and destroy nicotine before it reaches the brain, depriving a person of the “reward” of nicotine that can trigger a smoking relapse.
 
For more than 30 years, Janda and his colleagues have struggled to create such an enzyme in the lab, but they recently ran across a potential enzyme found in nature: NicA2, from the bacteria known as Pseudomonas putida. This bacterium consumes nicotine as its sole source of carbon and nitrogen. Janda tells DDNews that the NicA2 was first discovered from a soil sample in a field under continuous tobacco cropping in Shandong, China. “The bacterium is like a little Pac-Man,” according to Janda. “It goes along and eats nicotine.”
 
In the new study, the TSRI researchers characterized the bacterial enzyme responsible for nicotine degradation and tested its potential usefulness as a therapeutic. The enzyme’s catalytic efficiency may be what will prevent nicotine from reaching the brain, as it will degrade nicotine to non-psychoactive substances, says Janda.
 
The researchers first combined blood serum from mice with a dose of nicotine equivalent to one cigarette. When they added the enzyme, the nicotine’s half-life dropped from two to three hours to just nine to 15 minutes. Janda said a higher dose of the enzyme—with a few chemical modifications—could reduce the half-life of nicotine even further and keep it from ever reaching the brain. Next, the researchers subjected the enzyme to a barrage of tests to determine its practicality as a drug candidate. “It was a long shot,” according to Janda. “If it didn’t have the right metrics, it would be a bust.”
 
The results were encouraging. The enzyme stayed stable in the lab for more than three weeks at 98 degrees Fahrenheit, which Janda called “pretty remarkable.” Importantly, the researchers detected no toxic metabolites produced when the enzyme degraded nicotine. “The enzyme is also relatively stable in serum, which is important for a therapeutic candidate,” said Song Xue, a TSRI graduate student and first author of the new study. “Hopefully we can improve its serum stability with our future studies, so that a single injection may last up to a month.”
 
The next step is to alter the enzyme’s bacterial makeup, which will help mitigate potential immune liabilities and maximize its therapeutic potential.
 
“Two points will need to be addressed moving forward. First, the enzyme must be made 'immunosilent,'” says Janda. “Because it is of bacterial origin, it undoubtedly will elicit an immune response. The enzyme will need to have any T cell epitopes removed to prevent an immune response. The enzyme will also need to be reengineered so as to increase its half-life in vivo to weeks.”
 
In addition to Janda and Xue, Joel E. Schlosburg of TSRI was an author of the study, “A new strategy for smoking cessation: Characterization of a bacterial enzyme for the degradation of nicotine.”

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