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All Natural Chemicals

The use of medicinals derived from nature is likely as old as humanity itself. Pollen evidence from caves in Kurdistan, Iraq suggests that Neanderthals may have taken advantage of the medicinal properties of plants. A Mesopotamian medical text from 2600 BC, written in cuneiform on clay tablets, lists a thousand medicinal plants. Even modern Western medicine owes a large fraction of pharmaceuticals, from aspirin to ziconotide, to natural products and their derivatives. The Molecular Targets Laboratory (MTL) works extensively with CCR investigators to allow them full advantage of nature’s bounty in their fight against cancers and infectious disease. Now, CCR wants to broaden its reach.

The Natural Products Repository on the NCI campus in Frederick, Md., houses about 200,000 extracts from terrestrial and marine organisms from around the world, such as the barrel sponge Xestospongia testudinaria.

The Natural Products Repository on the NCI campus in Frederick, Md., houses about 200,000 extracts from terrestrial and marine organisms from around the world, such as the barrel sponge Xestospongia testudinaria. (Photo: Natural Products Branch, DTP, DCTD, NCI)

On the submerged roots of mangroves in the warm shallow Caribbean island waters, live colonies of a tiny sea squirt, Ecteinascidia turbinate. In spring and summer, the colonies sexually reproduce, sending tadpoles out into the water column, but otherwise these are sedentary creatures at the mercy of their environment. Living in symbiosis with the sea squirt is a prokaryote, Candidatus Endoecteinascidia frumentensis, which produces a complex eight-ringed small molecule, trabectidin, for reasons not yet revealed to marine biologists. But, to oncologists, trabectidin, whose activity was first reported in a broad NCI anticancer screen of natural products, is known as Yondelis and is used for the treatment of soft tissue sarcomas and ovarian cancer.

“When marine animals settle out of the water column onto a substrate, they’re pretty much stuck there, just like plant seeds, and must fight, compete, and win in that environment,” said Kirk Gustafson, Ph.D., Senior Scientist in CCR’s MTL. “Thus they develop a lot of chemical strategies to deter predators, to prevent other larvae from dropping out and trying to settle on and overgrow them, to keep unwanted bacteria and fungi from colonizing them. A compound with growth inhibitor properties in that setting just might interact with a biological target in a similar manner to, say, affect cancer cell growth.”

On that premise, beginning in the 1950s, NCI created a worldwide collection program, focusing on terrestrial plants and marine organisms, and developed a repository, which currently houses some 200,000 natural product extracts. This repository is the largest, most diverse, public repository of natural products in the world (See “The Natural Products Repository: A National Drug Development Resource,” CCR connections Vol. 2, No. 2). The diversity of chemical structures springs from the diversity of organisms sampled. Among the other drugs discovered through this resource are paclitaxel, a compound found in the bark of the Pacific Yew, and noted on the World Health Organization’s List of Essential Medicines for the treatment of solid tumor cancers.

“Natural products provide a pool of structural diversity that is just unparalleled with anything that humans can think up and make. Synthetic chemists are very talented, but they don’t have the imagination and capabilities that Nature does,” said Gustafson. “Depending on whom you talk to, anywhere from 30 to 60 percent of drugs are either natural products or derived from Nature.”

Extracting and working with such a diversity of compounds requires unique expertise. Within CCR, MTL has decades of experience dedicated to turning these extracts into drugs to treat cancer and infectious disease.

Mining Small Molecules

“We’re in the mining business. Our main goal is to move the research of CCR scientists forward,” said James McMahon, Ph.D., MTL’s Chief. “We have built up a huge database and knowledge of the extracts that enables us to run high-throughput assays. The other thing we’ve done over the years is to build up a library of pure compounds, with a lot of interesting chemistry.”

Kirk Gustafson, Ph.D., and his team use nuclear magnetic resonance (NMR) spectroscopy to analyze compounds isolated from natural products.

Kirk Gustafson, Ph.D., and his team use nuclear magnetic resonance (NMR) spectroscopy to analyze compounds isolated from natural products. (Photo: R. Baer)

Natural products do not start out life as beautiful as the organisms that produce them. Materials are collected in the field, identified, dried, and sent to the Natural Products Branch of the Developmental Therapeutics Program (DTP) in Frederick, Md., part of NCI’s Division of Cancer Treatment and Diagnosis (DCTD), where they are ground and extracted with aqueous and organic solvents. “When we get one of these extracts, we’ve taken everything that is soluble from an organism. These are complex mixtures; if you make an organic extract of a plant, it looks like a bottle of black tar,” said Gustafson. “It’s not a pretty starting material.”

Gustafson is involved in the chemistry component of assay development and screening within MTL, focusing primarily on small molecules. In a typical collaboration, a CCR investigator has a target of interest and is looking for molecules that will interact in specific ways with that target. For example, Thomas Sayers, Ph.D., Senior Investigator in CCR’s Laboratory of Experimental Immunology in the Cancer and Inflammation Program, is studying the mechanisms by which the immune system destroys cancer cells. His team has found that cancer cells protect themselves from apoptosis mediated by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) with a family of anti-apoptotic proteins, known as cFLIP.

Working with assays from the Sayers laboratory, Gustafson and his colleagues were able to isolate a steroidal lactone, withanolide E, from the Cape gooseberry (Physalis peruviana), that enhanced apoptosis in a number of human cancer cell lines through reduction in the cFLIP proteins. A paper describing the activity of withanolide E has been published in Cell Death and Disease.

“Everything we do is a collaboration,” said Gustafson. “My work is irrelevant without the screening results that guide me to a particular extract and through chemical separation of an extract. The biological activity is our eyes and in a perfect world, it neatly tracks to one or a few compounds we can isolate as pure white or clear or yellow compounds. From the standpoint of the natural products chemist, the fun begins in figuring out what the entity is that we have isolated.”

Barry O’Keefe, Ph.D., and Lauren Krumpe, M.S., in the lab

Barry O’Keefe, Ph.D., and Lauren Krumpe, M.S., in the lab (Photo: R. Baer)

Gustafson and his colleagues use spectroscopic techniques: nuclear magnetic resonance (NMR) and mass spectrometry (MS). For noncrystalline compounds, NMR is the most powerful structure elucidation technique available. MS is used to identify the atomic components and define the molecular formula. “In a really perfect world, we end up solving a new type of molecular structure and assigning it a name.” Recently, a collaboration between MTL and William Figg, Sr., Pharm.D., Deputy Chief of CCR’s Genitourinary Malignancies Branch, has led to an entirely new class of alkaloids, derived from the marine sea squirt, Eudistoma, which they have termed eudistidines. One of these compounds is able to interfere with a protein/protein interaction critical to the ability of cancer cells to survive in the oxygen-poor milieu of tumors. Moreover, Martin Schnermann, Ph.D., Investigator in CCR’s Chemical Biology Laboratory (CBL), was able to work out an efficient five-step synthesis for the new class, meaning that its availability is no longer limited by the natural product. This work recently appeared in the Journal of the American Chemical Society.

“Small molecules that block protein/protein interactions are quite rare and these targets are usually considered undruggable. When you screen synthetic compound libraries, you often get no hits. When you screen natural product libraries, it seems that the chance of success is higher,” said Gustafson. “The difference is that these small molecules are not randomly derived molecular structures, they are the result of chemical evolution. Organisms have evolved structures that interact with biopolymers—proteins, nucleic acids, and the like—in a manner that produces a biologic effect.”

Prospecting for Proteins

“Kirk isolates small molecules that interact with cancer and HIV,” said Barry O’Keefe, Ph.D., Deputy Chief of MTL and Chief of DCTD’s Natural Products Branch. “My group concentrates on proteins and peptides.”

O’Keefe’s laboratory has two main parts. One part develops cell-free systems to study the interactions of natural products with biological molecules. These can be used both to screen for compounds that affect the function of proteins or nucleic acids as well as to define the thermodynamic and kinetic interactions of extracted compounds with their targets. Recently, a collaboration with John Schneekloth, Ph.D., a CBL Investigator, led to the identification of small molecules that selectively interact with a particular RNA structure—the HIV transactivation response (TAR) RNA hairpin. One class of compounds was also able to inhibit HIV-induced death of T cells in vivo.

O’Keefe is also known for his work in isolating bioactive proteins from natural products. Griffithsin, a lectin isolated from the eponymous red algae Griffithsin sp. found off the coast of New Zealand, is a potent HIV antiviral (See “By Land or by Sea: High-Yield Production of a Marine Anti-HIV Protein in Plants,” CCR connections Vol. 3, No. 1). Two large-scale clinical trials have now been funded to evaluate its use as a topical microbicide against HIV. The Population Council’s Center for Biomedical Research is leading one trial, funded by the U.S. Agency for International Development (USAID), for vaginal administration; the University of Louisville and Intracept Biomedicine are conducting an National Institute of Allergy and Infectious Disease (NIAID)-sponsored trial for rectal administration.

Having isolated the protein, the path to synthesis is quite distinct from small molecules. “Once griffithsin was isolated and we had the amino acid sequence, we could engineer its production in Escherichia coli,” said O’Keefe. “Then we began evaluating its mechanisms of action and extrapolated its use to other viruses.” Thus, griffithsin has shown activity against SARS, hepatitis C, Japanese encephalitis, and herpes simplex viruses. O’Keefe’s team has recently shown that another lectin isolated from cyanobacteria, scytovirin, is active in vivo against the Ebola virus.

“When we find a protein, we play with it. We modify it and see if we can improve on its native characteristics. For example, some modifications might improve its stability, immunogenicity, bioavailability, and shelf life,” said O’Keefe.

Foreign proteins have faced significant hurdles in drug development because they are automatically assumed to be immunogenic. However, there are many biotherapeutics now that are not native. The drug Exenatide, used for glucose control, was first isolated from the saliva of the Gila monster, a large venomous lizard found in Mexico and the southwestern U.S. Ziconotide, is an intrathecal pain medication, first isolated from cone snails found on tropical coral reefs. And of course the wrinkle-smoothing Botox is from the bacterium Clostridium botulinum.

“The hurdles for the therapeutic use of foreign proteins are much less than they were even 10 years ago,” said O’Keefe.

Sharing the Bounty

“It’s estimated that only one percent of existing natural products have been discovered,” said Joel Schneider, Ph.D., Chief of CCR’s CBL. “So, if we’ve got drugs that are affecting millions of people’ lives by only looking at the one percent, imagine what we have yet to discover.”

Screening of the Natural Products Repository has largely been limited to cancer and infectious disease. CCR is working to make this library more accessible, especially to those other disease areas.

The first step will be to make the extracts more user-friendly. Many screening centers are hesitant to use natural products because they aren’t compatible with existing instrumentation. Based on studies developed by Gustafson, the process of prefractionating the crude extracts may be the answer. The goal is to create a library of pre-fractionated extracts. “The plan is a one-million fraction library, a subset of current extracts separated based on polarity in an automated fashion. This will concentrate low percentage compounds to be more readily seen in assays and sequester nuisance compounds that routinely confound assays,” said O’Keefe.

The second step will be to aid external screening centers in their ability to identify the active compound. “Even when you narrow down the fraction to basically one peak on a chromatogram that is active, you still don’t know what the molecule is. We can help with that,” said Schneider.

A Precious Resource

“Back when Nixon declared war on cancer, there was a huge national effort in mining the natural product biome for active compounds,” said Schneider. “We’ve seen that effort wane over the last 20 years because of the promise of newer therapies. But, to this day, small molecules derived from natural products still represent the majority of cancer treatments. With that realization, we owe it to the patients and to the science to revitalize natural product discovery at CCR.”

New efforts are under way to curate a more diverse library and recognize new organisms that might provide even better molecules. “We want to build in a planned way to fill niches that we don’t currently have,” said O’Keefe.

“...small molecules derived from natural products still represent the majority of cancer treatments.”

To that end, McMahon and his colleague, John Beutler, Ph.D., a senior natural products chemist who serves as the MTL “librarian”, have been making trips to Kazakhstan to collect new plants that have never been tested. “Kazakhstan is a huge country; there are probably a thousand indigenous plants with ethnobotanical references from the native peoples that have never been tested,” said McMahon. The collaboration includes a mirror repository in Kazakhstan. “We’d like to do this with any country that’s willing,” urges McMahon.

“The world’s diversity is going away,” said McMahon. “And to me, as a scientist, that is criminal. We can get about 85 percent of our repository resupplied. The other 15 percent are just gone. The bottom line is you can only find what you have in your library. Natural products are Nature’s library.”

To learn more about collaborative Natural Product Research in the MTL, please visit their website at https://ccr.cancer.gov/Molecular-Targets-Laboratory.

To learn more about Dr. McMahon’s research, please visit his CCR website at https://ccr.cancer.gov/james-b-mcmahon.

To learn more about Dr. Gustafson’s research, please visit his CCR website at https://ccr.cancer.gov/kirk-r-gustafson.

To learn more about Dr. O’Keefe’s research, please visit his CCR website at https://ccr.cancer.gov/barry-r-okeefe.

To learn more about Dr. Beutler’s research, please visit his CCR website at https://ccr.cancer.gov/john-a-beutler.