INVITATION DESIGNS

Scripps Research scientists identify new class of antimalarial compounds

Findings point to the development of novel drugs that could eradicate the disease.

An international team led by scientists from the Genomics Institute of the Novartis Research Foundation (GNF) and The Scripps Research Institute has discovered a family of chemical compounds that could lead to a new generation of antimalarial drugs capable of not only alleviating symptoms but also preventing the deadly disease.

Caption: This is Elizabeth Winzeler, Ph.D. and Stephan Meister, Ph.D. Credit: Photo Scripps Research

In a study published yesterday November 17, 2011, in Science Express, the advance online publication of the journal Science, Elizabeth Winzeler, PhD, a Scripps Research associate professor and member of the GNF, and colleagues demonstrated that the class of compounds was more effective against malaria than some commercially available drugs.

Most antimalarial drugs are only effective during the blood stage, and those that do work in the liver have notable side effects. However, the new class of compounds identified by the team is highly effective against the parasite in both the blood and the liver.

“Because the parasite blood stages are more amenable to high-throughput screening, much research has focused on that area,” said Stephan Meister, PhD, a research associate in the Winzeler lab and first author of the new paper. “We’re excited to have found a class of compounds that appears to target a novel gene and is highly active against the liver stage parasites in mice. This compound class provided us with a lead for the development of novel anti-malaria drugs.”

A Complicated Lifecycle

Despite long-standing efforts to control malaria globally, the disease remains endemic in many parts of the world. According to the World Health Organization, malaria affected about 225 million people in 2009, and killed nearly 800,000. The disease, which tends to strike the poorest and most vulnerable populations in Asia, Africa, and the Americas, is caused by Plasmodium parasites transmitted through the bites of infected mosquitoes.

The Plasmodium parasite has a complicated lifecycle in two hosts—mosquitoes and humans (or other vertebrate). When a malaria-infected mosquito feeds on a person, the parasite enters the human body. Within 30 minutes, the parasite has infected liver cells, where it develops for about eight days without causing noticeable symptoms. In some cases it can even go into hiding in the liver and persist for several months to years.

When this period is over, however, the parasite (now in a different form) leaves the liver and enters red blood cells, where it grows and multiplies. When the infected red blood cells eventually burst, the parasite and Plasmodium toxins are released into the bloodstream, and the person feels sick. Symptoms include fever, chills, headache, and other flulike symptoms; in severe cases, patients can experience convulsions, coma, and liver and kidney failure, which can be fatal.

If a mosquito bites the infected person at this point, the parasite will enter the mosquito, where it will continue the cycle by maturing into a form that can infect the next human host.

Mining the Data

To find compounds to act against the parasite in more than one stage of its lifecycle, the team screened thousands of candidates that were already known to act against malaria parasites in the blood. Only 15 percent looked as if they might also work in the liver—a strong indication, Winzeler said, that “a lot of compounds that are active against blood stages probably aren’t going to do anything about eliminating malaria.”

The group then identified the strongest candidates for drug development by mining the data for groups of related compounds that all showed activity in the liver. In the end, they settled on a cluster related to the chemical imidazolopiperazine. “When we analyzed all of the data, we saw that multiple members of this imidazolopiperazine family were active in blood and liver stages,” Winzeler said.

The imidazolopiperazine family of compounds was especially attractive because it was chemically unrelated to existing antimalarial drugs, and therefore less likely to run into problems with existing resistance. “I wouldn’t want to base a multimillion dollar clinical trial on compounds for which there may be pre-existing resistance,” said Winzeler. “Ultimately, we want to have something that will still be effective in 10 years.”

The group used an automated system of their own design to see how these new compounds fared against malaria parasites incubated in liver cells in the lab. An imaging apparatus took multiple images of each collection of cells over time, and a computer script analyzed those images to see how well the various compounds inhibited the growth of the parasites.

In the end, the team was able to develop compounds that could be taken orally and would stay in the blood long enough to be a viable candidate for drug development. When it was given to mice, the compound provided complete protection against the parasite in the liver, and worked better in the blood than some commercially available drugs.

Spurring Drug Discovery

To better understand how the compound works, the team exposed successive generations of infected mosquitoes to low levels of the compound to produce resistant strains of parasites. They then sequenced the parasites’ whole genomes and looked for genetic changes. “Every [resistant] strain we looked at had a mutation in the same gene,” she said.

By offering a target for other new antimalarial drugs that can act in both the liver and the blood, that gene will provide other researchers fresh ammunition in the fight to eradicate the disease. So, too, will the decision by the team to make all of their data available online.

“We have been making all of our data available to the community to spur drug development,” Winzeler said. “The data on all of the compounds that were tested will eventually be released, and this will allow people at universities and research institutes around the world to mine this data, and to use it to guide their own drug discovery efforts.”

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In addition to Winzeler and Meister, the authors of the Science paper, titled “Exploring Plasmodium Hepatic Stages to Find Next Generation Antimalarial Drugs,” include Selina E Bopp, A. Taylor Bright, and Neekesh V. Dharia, of Scripps Research; David M Plouffe, Kelli L Kuhen, Ghislain MC Bonamy, and S.Whitney Barnes, of the Genomics Institute of the Novartis Research Foundation; and researchers at Columbia University Medical Center, UC San Diego, the Novartis Institute for Tropical Diseases and the Swiss Tropical and Public Health Institute.

Support for the study came from the Wellcome Trust, the Medicines for Malaria Venture, the Genomics Institute of the Novartis Research Foundation, the Swiss Tropical and Public Health Institute, and the Novartis Institute for Tropical Diseases.

Chew gum, lose weight

“Syracuse University scientist uses vitamin B12 to orally deliver appetite-suppressing hormone” 

Most people understand that serious weight loss requires changing attitudes toward what they eat and how often they exercise. But what if the process could be aided by simply chewing a stick of gum after meals? That’s the question a team of scientists, led by Syracuse University chemist Robert Doyle, is trying to answer. In a groundbreaking new study, Doyle’s team demonstrated, for the first time, that a critical hormone that helps people feel “full” after eating can be delivered into the bloodstream orally.

Doyle’s study was published online Nov. 4, 2011, in the American Chemical Society’s Journal of Medicinal Chemistry and is forthcoming in print. Doyle is an associate professor in the Department of Chemistry in SU’s College of Arts and Sciences. He collaborated on the study with researchers from Murdoch University in Australia.

The hormone, called human PYY, is part of a chemical system that regulates appetite and energy. When people eat or exercise, PYY is released into the bloodstream. The amount of PYY that is released increases with the number of calories that are consumed. Past studies have shown that people who are obese have lower concentrations of PYY in their bloodstream both when fasting and after eating than their non-obese counterparts. Additionally, intravenous infusion of PYY into a volunteer group of obese and non-obese individuals increased the serum levels of the hormone and lowered the number of calories both groups consumed.

“PYY is an appetite-suppressing hormone,” Doyle says. “But, when taken orally, the hormone is destroyed in the stomach, and that which isn’t destroyed has difficulty crossing into the bloodstream through the intestines.”

What’s needed is a way to disguise the PYY so that it can travel through the digestive system relatively unharmed. Several years ago, Doyle developed a way to use vitamin B12 as a vehicle for the oral delivery of the hormone insulin. B12 is able to pass through the digestive system with relative ease and carry with it insulin, or other substances, into the bloodstream.

Similarly, his research team attached the PYY hormone to his patent-pending vitamin B12 system. “Phase one of this study was to show that we could deliver a clinically relevant amount of PYY into the bloodstream,” Doyle says. “We did that, and we are very excited by the results.”

The next step involves finding ways to insert the B12-PYY system into such things as chewing gum or an oral tablet to create a nutritional supplement to assist individuals in losing weight in much the same way as nicotine-laced gum is used to help people stop smoking. “If we are successful, PYY-laced gum would be a natural way to help people lose weight,” he says. “They could eat a balanced meal, then chew a stick of gum. The PYY supplement would begin to kick in about three to four hours later, decreasing their appetite as they approach their next meal.”

Research sheds new light on body parts’ sensitivity to environmental changes

Research by a team of Michigan State University scientists has shed new light on why some body parts are more sensitive to environmental change than others, work that could someday lead to better ways of treating a variety of diseases, including Type-2 diabetes.

MSU assistant zoology professor Alexander Shingleton used fruit flies as a model for his research into why some body parts are more sensitive to environmental changes than others. Fruit flies use the same genes to control this process as humans do. Photo by G.L. Kohuth.

The research, led by assistant zoology professor Alexander Shingleton, is detailed in the recent issue of the Proceedings of the Library of Science Genetics.

In particular, Shingleton is studying the genetics of fruit flies and zeroing in on why some of the insects’ body parts will grow to full size even when suffering from malnutrition, while others will not. He uses fruit flies because they use the same genes to control this process as humans.

“The developmental mechanisms by which these changes in body proportion are regulated are really unknown,” Shingleton said.

Shingleton said that in humans, a person’s brain will grow to near full size despite malnutrition or other environmental, or nongenetic, problems.

If scientists can figure out why some organs or body parts are either overly sensitive or insensitive to environmental factors, then it’s possible that therapies could be developed to deal with any number of maladies.

“If we know how we can control sensitivity to environmental issues such as malnutrition, we can, in principle, manipulate genes that are regulating that sensitivity,” Shingleton said. “Genes can be activated so they can actually restore sensitivity.”

Type-2 diabetes is a good example of the body’s insensitivity to nongenetic issues. The most common form of diabetes, Type-2, occurs when the body becomes insensitive to insulin, which is released in response to blood sugar levels. The body needs insulin to be able to use glucose for energy.

“In diabetes, that response is suppressed,” Shingleton said. “We get desensitization. We know people become insulin resistant, but we’re not quite sure why.”

What Shingleton and colleagues discovered is that even when malnourished, the genitals of a male fruit fly continue to grow to normal size.

“The same developmental mechanism that a fly uses to make its genitals insensitive to changes in nutrition may be the same that we as humans use to modulate the responsiveness of individual body parts to changes in nutrition,” he said. “Our job is to try to understand why some body parts are responsive to changes in nutrition and others aren’t.”

Using the fruit fly for this type of research “gives us enormous information about how we as humans work and how we respond to our environment,” Shingleton said. “This provides information on biomedical issues that arise from things like malnutrition or insulin resistance.”

Shingleton’s research is funded by the National Science Foundation and MSU’s Bio/computational and Evolution in Action Consortium, or BEACON.

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Future prostate cancer treatments might be guided by math

Prostate cancer forms in the prostate gland, and can sometimes be felt on digital rectal examination. Photo: ADAM             Scientists have designed a first draft of a mathematical model that someday could guide treatment decisions for advanced prostate cancer, in part by helping doctors predict how individual patients will respond to therapy based on the biology of their tumors.

These decisions would apply to treatment of cancer that has already spread beyond the prostate gland or that has recurred after initial treatments, such as surgery or radiation. Patients with this more advanced prostate cancer receive a therapy called androgen ablation, which inhibits production of testosterone – the culprit that allows a tumor to keep growing.

Though the model’s outcomes remain theoretical at this point, the researchers have developed enough of a system to show that their incorporation of some personalized data – details about a patient’s tumor cell characteristics in particular – would give doctors more than they currently have to work with in making decisions about this stage of treatment.

“The model in its current form is proof of the concept that we can capture all of these different outcomes that are observed clinically. But we still need to refine the model with as much individual data as we can obtain,” said Harsh Jain, a postdoctoral fellow in Ohio State University’s Mathematical Biosciences Institute and lead author of the study.

“We envision that this model would be useful for clinicians who could keep feeding the equations with data about how a patient is responding to therapy, which would offer clues about how his cancer cells are mutating. Once you have an idea about that for the short or medium term, the model could predict the optimal therapy for that patient,” Jain said.

The model is described this week in the online early edition of the Proceedings of the National Academy of Sciences. Jain conducted the work with co-authors Steven Clinton, professor, and Arvinder Bhinder, assistant professor-clinical, in Ohio State’s division of medical oncology, and Avner Friedman, a Distinguished University Professor at Ohio State.

Prostate cancer is diagnosed in about 240,000 American men and leads to about 34,000 deaths each year, according to the National Cancer Institute.

The treatment of this cancer in its more advanced stages brings about chemical castration by targeting one of several mechanisms involved in the production of testosterone. In most patients, cancer cells develop castration resistance over time – on average, between 1½ and two years after the start of treatment. However, the overall range of resistance development spans from a few months to more than 10 years.

Jain said that some scientists have proposed that this treatment leads directly to castrate-resistant disease because once testosterone is removed from the body, mutant cancer cells that can survive in a no- or low-testosterone environment are able to take over the tumor.

Currently, continuous treatment to eliminate testosterone is the standard of care. But because clinicians know castration resistance is inevitable, a new approach is under study. A national clinical trial is assessing the benefits and risks of intermittent androgen ablation – keeping patients on the drugs until symptoms improve, and then giving men time off from the medication until the disease begins to progress again.

The math model developed by Ohio State scientists suggests that based on average clinical data currently available, such intermittent therapy could actually accelerate the development of castration resistance.

“In the same way that intermittent use of antibiotics gives a chance for bacteria that are resistant to the drug to take over, you might actually end up with intermittent anti-androgen therapy even more positively selecting for mutating cancer cells,” Jain said.

However, the averages don’t always apply, which is why the scientists are pursuing a system of differential equations to account for individual differences. For example, the “normal” levels of prostate-specific antigen, or PSA, in men’s blood cover a fairly broad range, Jain noted. Yet the PSA test remains the most common screening method for prostate cancer, and is used to gauge the effectiveness of treatments in advanced stages, as well.

“The PSA ranges are massive. It’s a very heterogeneous thing,” Jain said. “When we are talking about cancer, our point is that those variables should be personalized. Everyone’s cancer grows differently.

“There are a lot of questions. If you take an intermittent therapy route, how do you decide the scheduling of treatment? Is it based solely on PSA levels? Shouldn’t there be some incorporation of personal patient characteristics into these treatment decisions? Can you identify a subgroup of patients who are predicted to respond well to this, or are there conditions when one treatment vs. another could actually make things worse?”

Math offers some answers. The model’s foundation is based on existing animal and human data on prostate cancer characteristics. Beyond that, the researchers have selected parameters to plug into the equations that more specifically detail what could be going on in an individual tumor: cancer cell growth rates, cancer cell death rates, the level of activation of PSA in tumor cells, and how quickly one person’s PSA can travel from the prostate to the bloodstream.

The scientists even took into account the competitive power of individual types of cancer cells – for example, some mutated cancer cells aren’t as strong as their normal cancer cell counterparts. In those cases, the math model predicts, the best treatment option would be intermittent therapy because the stronger normal cancer cells would keep mutant cells in check during time off from the medication. With the cancer consistently dominated by cells that rely on the presence of testosterone, the treatment would continue to target those stronger cells that respond to androgen ablation therapy, Jain explained.

“That’s an important question with any therapy – is it making things better or worse in terms of allowing mutated cells to take over?” he said.

Jain and colleagues are now working to boost the model’s power by adding parameters that account for the blood vessel architecture in prostate tumors, a major indicator of how persistent the cancer will be. They also plan to add hundreds of individual patients’ case study data to make its predictions even more authentic.

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This research was supported by Ohio State’s Mathematical Biosciences Institute, the National Science Foundation, and a Molecular Carcinogenesis and Chemoprevention Program Grant from Ohio State’s Comprehensive Cancer Center.

New medical, research tool possible by probing cell mechanics

Researchers are making progress in developing a system that measures the mechanical properties of living cells, a technology that could be used to diagnose human disease and better understand biological processes.

This artist's conception depicts the use of an atomic force microscope to study the mechanical properties of cells, an innovation that might result in a new way to diagnose disease and study biological processes. Here, three types of cells are studied using the instrument: a rat fibroblast is the long slender cell in the center, an E coli bacterium is at the top right and a human red blood cell is at the lower left. The colored portions show the benefit of the new technique, representing the mechanical properties of the cells, whereas the gray portions represent what was possible using a conventional approach. (Purdue University image/Alexander Cartagena)

The team used an instrument called an atomic force microscope to study three distinctly different types of cells to demonstrate the method’s potentially broad applications, said Arvind Raman, a Purdue University professor of mechanical engineering.

For example, the technique could be used to study how cells adhere to tissues, which is critical for many disease and biological processes; how cells move and change shape; how cancer cells evolve during metastasis; and how cells react to mechanical stimuli needed to stimulate production of vital proteins. The technique could be used to study the mechanical properties of cells under the influence of antibiotics and drugs that suppress cancer to learn more about the mechanisms involved.

Findings have been posted online in the journal Nature Nanotechnology and will appear in the December print issue. The work involves researchers from Purdue and the University of Oxford.

“There’s been a growing realization of the role of mechanics in cell biology and indeed a lot of effort in building models to explain how cells feel, respond and communicate mechanically both in health and disease,” said Sonia Contera, a paper co-author and director of the Oxford Martin Programme on Nanotechnology and an academic fellow at Oxford physics. “With this paper, we provide a tool to start addressing some of these questions quantitatively: This is a big step.”

An atomic force microscope uses a tiny vibrating probe to yield information about materials and surfaces on the scale of nanometers, or billionths of a meter. Because the instrument enables scientists to “see” objects far smaller than possible using light microscopes, it could be ideal for “mapping” the mechanical properties of the tiniest cellular structures.

“The maps identify the mechanical properties of different parts of a cell, whether they are soft or rigid or squishy,” said Raman, who is working with doctoral student Alexander Cartagena and other researchers. “The key point is that now we can do it at high resolution and higher speed than conventional techniques.”

The high-speed capability makes it possible to watch living cells and observe biological processes in real time. Such a technique offers the hope of developing a “mechanobiology-based” assay to complement standard biochemical assays.

“The atomic force microscope is the only tool that allows you to map the mechanical properties – take a photograph, if you will – of the mechanical properties of a live cell,” Raman said.

However, existing techniques for mapping these properties using the atomic force microscope are either too slow or don’t have high enough resolution.

“This innovation overcomes those limitations, mostly through improvements in signal processing,” Raman said. “You don’t need new equipment, so it’s an economical way to bump up pixels per minute and get quantitative information. Most importantly, we applied the technique to three very different kinds of cells: bacteria, human red blood cells and rat fibroblasts. This demonstrates its potential broad utility in medicine and research.”

The technique is nearly five times faster than standard atomic force microscope techniques.

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The Nature Nanotechnology paper was written by Raman; Cartagena; Sonia Trigueros, a Senior Research Fellow in the Oxford Martin Programme on Nanotechnology; Oxford doctoral student Amadeus Stevenson; Purdue instructor Monica Susilo; Eric Nauman, an associate professor of mechanical engineering; and Contera.

Scientists develop brand new class of small molecules through innovative chemistry

Inspired by natural products, scientists on the Florida campus of the Scripps Research Institute have created a new class of small molecules with the potential to serve as a rich foundation for drug discovery.

Combining the power of synthetic chemistry with some advanced screening technologies, the new approach could eventually expand by millions the number of provocative synthetic compounds available to explore as potential drug candidates. This approach overcomes substantial molecular limitations associated with state-of-the-art approaches in small molecule synthesis and screening, which often serve as the foundation of current drug discovery efforts.

The study, led by Scripps Research Associate Professor Glenn Micalizio, was published Nov. 20, 2011, in an advanced online edition of the journal Nature Chemistry.

To frame the significance of this advance, Micalizio explains that high-throughput screening is an important component of modern drug discovery. In high-throughput screening, diverse collections of molecules are evaluated en masse for potential function in a biological area of interest. In this process, success is critically dependent on the composition of the molecular collections under evaluation. Modern screening centers maintain a relatively static collection of molecules, the majority of which are commercially available materials that have structures unrelated to natural products — molecules that are appreciated as validated leads for drug development.

“This divergence in structure between natural products and commercially available synthetics lies at the heart of our inquiry,” said Micalizio. “Why should we limit discovery of therapeutic leads to compound collections that are influenced by concerns relating to commercial availability and compatibility with an artificial set of constraints associated with the structure of modern screening centers?”

To expand the compounds available for investigation, the scientists embraced an approach to structural diversity that mimics nature’s engine for the discovery of molecules with biological function. This process, termed “oligomerization,” is a modular means of assembling structures (akin to the way that letters are used in a sequence to provide words with meaning) where a small collection of monomeric units can deliver a vast collection of oligomeric products of varying length, structure, and function (like the diversity of words presented in a dictionary).

Coupling this technique with a synthetic design aimed at generating molecules that boast molecular features inspired by the structures of bioactive natural products (specifically, polyketide-derived natural products, which include erythromycin, FK-506, and epothilone), the scientists established a new chemical platform for the discovery of potential therapeutics.

Micalizio points out: “The importance of oligomerization to drive discovery is well appreciated in chemistry and biology, yet a means to realize this process as an entry to small molecule natural product-inspired structures has remained elusive. The crux of the problem is related to challenges associated with the control of shape for each member of a complex oligomer collection — the central molecular feature that defines biological function.”

“It is the stability associated with the shape of these new compounds that lies at the heart of the practical advance,” he continued. “The unique features of this science now make possible the ability to synthesize large collections of diverse natural product-inspired structures that have predictable and stable three-dimensional shapes.”

Micalizio said that the science described represents a first step toward revolutionizing discovery at the interface of chemistry, biology, and medicine by embracing nature’s strategy for molecular discovery. Coupling this type of advance with modern screening technology that can handle the evaluation of large compound collections at low cost (such as work by Scripps Florida Professor Thomas Kodadek, a co-author of the new study), can dramatically enhance the future of pharmaceutically relevant science.

The potential of this vision was highlighted in the new study, in which a 160,000-member compound collection was employed to discover the first non-covalent small molecule ligand to the DNA binding domain of p53 — an important transcription factor that regulates a variety of genes involved in cell cycle control and cell death.

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The first author of the study, “A Biomimetic Polyketide-Inspired Approach to Small-Molecule Ligand Discovery,” is Claudio Aquino of Scripps Research. In addition to Micalizio and Kodadek, other authors include Mohosin Sarkar, Michael J. Chalmers, and Kimberly Mendes.

The study was supported by the Fidelity Biosciences Research Initiative, The State of Florida (The Florida Funding Corporation), and the National Institutes of Health.