Spider peptides battle superbugs and cancer

As antibiotic resistance rises and fears over superbugs grow, scientists are looking for new treatment options. One area of focus is antimicrobial peptides (AMPs), which could someday be an alternative to currently prescribed antibiotics, many of which are becoming increasingly useless against some bacteria. Now, a team reports in ACS Chemical Biology that they have improved the antimicrobial — and anticancer — properties of an AMP from a spider.

According to the U.S. Centers for Disease Control and Prevention, 2 million people become infected with antibiotic-resistant bacteria in the U.S. each year. Because no known antibiotics work against these bacteria, patients simply have to hope that their natural defenses eventually overcome the infection. But some patients experience severe symptoms, landing them in a hospital, and in extreme cases, they could die. Researchers are trying to find alternatives to traditional antibiotics, and one such possibility is a group of peptides called AMPs. These peptides are found in all plants and animals as a type of immune response and have been shown to be potent antibiotics in the laboratory. Gomesin, an AMP from the Brazilian spider Acanthoscurria gomesiana can function as an antibiotic, but it also has anticancer activity. When gomesin was synthesized as a circle instead of as a linear structure, these characteristics were enhanced. Sónia Troeira Henriques and colleagues wanted to further boost the peptide’s traits.

The team made several variations of the cyclic gomesin peptide and found that some of these were 10 times better at killing most bacteria than the previously reported cyclic form. In other experiments, the new AMPs specifically killed melanoma and leukemia cells, but not breast, gastric, cervical or epithelial cancer cells. The researchers determined that the modified peptides killed bacteria and cancer cells in a similar way — by disrupting the cells’ membranes. The group also notes that the modified AMPs were non-toxic to healthy blood cells.

Researching Radiosensitizers, a New Class of Drugs That Would Make Tumors More Vulnerable to Radiation Therapy

Two out of three cancer patients are treated with radiation, but the therapy often fails to wipe out the tumor or slow its growth. Southern Research is working to develop a new class of drugs that will help the radiation deliver a more powerful punch to the disease.

Dr. Bo Xu, M.D., Ph.D., Distinguished Fellow and Chair of Southern Research’s Oncology Department, said a radiosensitizer would greatly benefit cancer patients by improving the success rate of radiation by reducing resistance to the treatment.

“Our project focuses on making those tumor cells more vulnerable to radiation by targeting a critical survival mechanism that allows them to recover from the effects of radiation,” Xu said.

It’s a challenging project, in the works for almost a decade. It got started when Southern Research scientists began looking at fundamental biology concepts to identify a pathway that could play a role in the ability of cancer cells to survive radiation.

They discovered that disrupting the tumor’s self-protection mechanism – in this case, an interaction between two specific proteins – makes the cancer more sensitive to radiation treatment, Xu said.

“The whole idea is to use this strategy to find a new drug that can be used by patients who receive radiation. This drug wouldn’t have toxicity because if it got into the cell it wouldn’t mess up the major functions of the protein network,” he said.

“It would only work when radiation is delivered, and that radiation would be more effective. It’s like a catalyst.”

Using funding from the Alabama Drug Discovery Alliance (ADDA), a partnership with the University of Alabama at Birmingham, Southern Research scientists recently scanned thousands of compounds to identify potential drug candidates. The focus now is to validate the results of those scans and to identify lead compounds for more testing.

“Our hope is that in three years, we can identify a novel class of radiosensitizers that can help the approximately two-thirds of cancer patients who will eventually receive radiotherapy,” Xu said.

While some forms of cancer, such as lymphoma, are sensitive to radiation therapy, many others are not. Solid tumors with a low supply of oxygen, called hypoxic tumors, are tough to treat with radiation. So are cancer cells with a high DNA-repair capability.

To develop a radiosensitizer, Xu is taking aim at a protein that binds to DNA and recognizes the damage being done by radiation. The protein then joins forces with an enzyme to initiate a molecular repair job.

“If that recruitment is successful, then the DNA damage will be repaired, and the cancer cell will survive,” Xu said. “What we’re trying to do is to block this protein from finding the other one, so that the repair process will be diminished or affected. That way, the tumor cells will die.”

To prevent the DNA repair job from getting started, Xu is investigating a small peptide mimic, a small sequence of amino acids that is similar to a human protein but just a fraction of its size. These strands get to the site to block the interaction of the two natural, full-size proteins.

“This interference makes the cancer cell more vulnerable to radiation treatment,” he said.

Radiosensitizers are in demand, but they have proved difficult to develop. While the concept has been around for half a century, very few radiosensitizers have actually become available, according to Xu.

“While there are compounds that work synergistically with radiation, there are few drugs that were developed as a pure radiosensitizer,” he said.

In addition to the ADDA, the National Institutes of Health and the Department of Defense prostate cancer program have provided Southern Research with funding for this research over the years.

Regenerative bandage heals diabetic wounds faster

At some point in their lives, 15 percent of people with diabetes will develop a painful and hard-to-treat foot ulcer. Twenty-four percent of those affected will require a lower-leg amputation because of it. And, in some instances, what seems like a harmless sore might even lead to death.

A Northwestern University team has developed a new treatment for this severe and potentially deadly complication of diabetes. Called a “regenerative bandage,” the novel material heals diabetic wounds four times faster than a standard bandage and has the added benefit of promoting healing without side effects.

“Foot ulcers cause many serious problems for diabetic patients,” said Guillermo Ameer, professor of biomedical engineering in Northwestern’s McCormick School of Engineering and surgery in the Feinberg School of Medicine. “Some sores don’t heal fast enough and are prone to infection. We thought that we could use some of our work in biomaterials for medical applications and controlled drug release to help heal those wounds.”

An expert in biomaterials and tissue engineering, Ameer’s research was published online last week in the Journal of Controlled Release. Yunxiao Zhu, a PhD student in Ameer’s laboratory, is the paper’s first author. Northwestern Engineering’s Hao F. Zhang, associate professor of biomedical engineering, and Feinberg’s Robert Galliano, associate professor of surgery, also contributed to the work.

Diabetes can cause nerve damage that leads to numbness in the feet. A diabetic person might experience something as simple as a blister or small scrape that goes unnoticed and untreated because they cannot feel it to know that its there. As high glucose also thickens capillary walls, blood circulation slows, making it more difficult for these wounds to heal. It’s a perfect storm for a small nick to become a life-threatening sore.

Some promising treatments for these chronic wounds exist, but they are costly and can come with significant side effects. One gel, for example, contains a growth factor that has been reported to increase cancer risk with overuse.

“It should not be acceptable for patients who are trying to heal an open sore to have to deal with an increased risk of cancer due to treating the wound,” Ameer said.

Ameer’s laboratory previously created a thermo-responsive material — with intrinsic antioxidant properties to counter inflammation — that is able to deliver therapeutic cells and proteins. His team used this material to slowly release into the wound a protein that hastens the body’s ability to repair itself by recruiting stem cells to the wound and creating new blood vessels to increase blood circulation.

“We incorporated a protein that our body naturally uses to attract repair cells to an injury site,” Ameer said. “When the protein is secreted, progenitor cells or stem cells come to the wound and make blood vessels, which is part of the repair process.”

The thermo-responsive material is applied to the wound bed as a liquid and solidifies into a gel when exposed to body temperature. When the same amount of the protein was directly applied all at once, no benefit was observed. This demonstrates the importance of slow release from the thermo-responsive material. Ameer believes that the inherent antioxidant properties within the material also reduce oxidative stress to help the wound heal.

“The ability of the material to reversibly go from liquid to solid with temperature changes protects the wound,” Ameer said. “Patients have to change the wound dressing often, which can rip off healing tissue and re-injure the site. Our material conforms to the shape and dimensions of the wound and can be rinsed off with cooled saline, if needed. This material characteristic can protect the regenerating tissue during dressing changes.”

In collaboration with Zhang, Ameer imaged diabetic wounds to discover that they were much healthier after application of the regenerative bandage. The blood flow to the wound was significantly higher than in those without Ameer’s bandage.

“The repair process is impaired in people with diabetes,” Ameer said. “By mimicking the repair process that happens in a healthy body, we have demonstrated a promising new way to treat diabetic wounds.”

An Engineered Protein Can Disrupt Tumor-Promoting ‘Messages’ in Human Cells

Over a century of research has shined light on the once-murky innards of our cells, from the genes that serve as our “blueprints” to the proteins and other molecules that are our cellular taskmasters.

Building on this basic knowledge, the search is underway for cellular mechanisms that could serve as gateways for new therapies. These could lead to precise treatments for disease — targeting a specific cellular function or gene with fewer unintended side effects. Ideally, these effects would also be temporary, returning cells to normal operation once the underlying condition has been treated.

A team of researchers from the University of Washington and the University of Trento in Italy announced findings that could pave the way for these therapies. In a paper published July 18 in Nature Chemical Biology, they unveiled an engineered protein that they designed to repress a specific cancer-promoting message within cells.

And that approach to protein design could be modified to target other cellular messages and functions, said senior author and UW chemistry professor Gabriele Varani.

“What we show here is a proving ground — a process to determine how to make the correct changes to proteins,” he said.

For their approach, Varani and his team modified a human protein called Rbfox2, which occurs naturally in cells and binds to microRNAs. These aptly named small RNA molecules adjust gene expression levels in cells like a dimmer switch. Varani’s group sought to engineer Rbfox2 to bind itself to a specific microRNA called miR-21, which is present in high levels in many tumors, increases the expression of cancer-promoting genes and decreases cancer suppressors. If a protein like Rbfox2 could bind to miR-21, the researchers hypothesized, it could repress miR-21’s tumor growth effects.

But for this approach to be successful, the protein must bind to miR-21 and no other microRNA. Luckily, all RNA molecules, including microRNAs, have an inherent property that imbues them with specificity. They consist of a chain of chemical “letters,” each with a unique order or sequence. To date, no other research team had ever successfully altered a protein to bind to microRNAs.

“That is because our knowledge of protein structure is much better than our knowledge of RNA structure,” said Varani. “We historically lacked key information about how RNA folds up and how proteins bind RNA at the atomic level.”

UW researchers relied on high-quality data on Rbfox2’s structure to understand, down to single atoms, how it binds to the unique sequence of “letters” in its natural RNA targets. Then they predicted how Rbfox2’s sequence would have to change to make it bind to miR-21 instead. Elegantly, altering just four carefully selected amino acids made Rbfox2 shift its attachment preference to miR-21, preventing the microRNA from passing along its tumor-promoting message.

The UW team spent several years proving this, since they had to test each change individually and in combination. They also had to make sure that the modified Rbfox2 protein would bind strongly to miR-21 but not other microRNAs. Since microRNAs have many functions in cells, it would be counterproductive to repress miR-21 while disrupting other normal microRNA-mediated functions.

The researchers also engineered a second protein that should clear miR-21 from cells entirely. They did this by grafting the regions of Rbfox2 that bound to miR-21 onto a separate protein called Dicer. Dicer normally chops RNAs into small chunks and generates functional microRNAs. But the hybrid Rbfox2-Dicer protein displayed a specific affinity to slice miR-21 into oblivion.

Varani and his team believe that Rbfox2 could be redesigned to bind to microRNA targets other than miR-21. There are thousands of microRNAs to choose from, and many have been implicated in diseases. The key to realizing this potential would be in streamlining and automating the painstaking methods the team used to model Rbfox2’s atomic-level interactions with RNA.

“This method relies on knowledge of high-quality structures,” said Varani. “That allowed us to see which alterations would change binding to the microRNA target.”

Not only would these be useful laboratory tools to study microRNA functions, but they could — in time — form the basis of new therapies to treat disease.

When it comes to developing stem cell treatments, seeing is half the battle

Stem cell therapies hold great promise in treating a variety of diseases, but in order to develop them researchers must first be able to monitor them inside the body. Enter the contrast agent.

“Often things go wrong right away when stem or therapeutic cells are injected into the body because some cells can migrate, while other dying cells are eaten by the body and expelled,” says Xiao-an Zhang, an assistant professor of chemistry at U of T Scarborough.

“It’s important to know if these cells are being injected in the right place, where they go once inside the body and whether they’re still alive. The only way to do that is with an effective contrast agent.”

Conventional contrast agents are directly injected into the body to make some tissues or diseases more clearly visible on MRI scans. The one developed by Zhang and his team, including Ph.D. student Inga Haedicke, U of T Assistant Professor Hai-Ling Margaret Cheng and two research groups (Dr. Timothy Scholl and Dr. Paula Foster) from Western University, can improve monitoring at the cellular level, which is a crucial element in measuring the effectiveness of stem and therapeutic cell treatments.

While still early in development, stem and therapeutic cells may one day offer effective treatments against diseases, particularly cancer. But one major hurdle in developing these treatments is an inability to effectively monitor them once inside the body.

“Current MRI methods are not great at looking at what’s going on at the cell or sub-cellular level. It’s just not powerful enough – the resolution and sensitivity needs to be higher and the contrast needs to be greater in order to distinguish one type of cell from another,” says Zhang.

“Traditional MRI can’t do quite what we need it to do right now, but there’s huge opportunity to improve the technology.”

Magnetic resonance imaging (MRI) uses a magnetic field and radio waves to produce a detailed image of tissues within the body. MRI is ideal for medical imaging because it’s milder than techniques like CT scans or X-rays since it’s not as invasive and does not rely on ionizing radiation.

There are quite a few technical challenges when developing an effective contrasting agent for cellular imaging. For one, it needs to be non-toxic or low in toxicity so it doesn’t damage cells. It also needs to be able to enter cells and accumulate in order to provide a clear enough contrast from the rest of objects in the image, notes Zhang.

The agent developed by Zhang and his team addresses these challenges by using manganese porphyrin. Manganese is a micronutrient that the body’s cells can handle and forms a very stable complex with the porphyrin, a type of pigment molecule that can help to achieve the necessary properties. Using a new porphyrin developed by Haedicke, the agent can be delivered to cells more efficiently in smaller concentrations and can accumulate more effectively than current alternatives.

“The real positive aspect of this design is that it’s cell permeable, and that the new mechanism allows it to stay inside the cell and accumulate,” says Haedicke. “This is extremely important for labelling which cells are being monitored.”

The research, which will be published in the journal Chemical Science, received funding from the Canada Foundation for Innovation (CFI) and the Natural Sciences and Engineering Research Council of Canada (NSERC).

The next steps for Zhang and his team is to focus mostly on using the agent on different types of stem cells and to test them in vivo.

No symptoms, but could there be cancer? Our chemosensor will detect it!

Many cancers could be successfully treated if the patient consulted the doctor sufficiently early. But how can a developing cancer be detected if it doesn’t give rise to any symptoms? In the near future, suitably early diagnosis could be provided by simple and cheap chemical sensors – thanks to special recognizing polymer films developed at the Institute of Physical Chemistry of the Polish Academy of Sciences in Warsaw.

These days, cancer is no longer a death sentence for the patient. However, the best chances of recovery are when the correct treatment is undertaken at an early stage of the disease. This is where the trouble starts, because many tumours develop over a long period without any symptoms. One solution to this problem could be diagnostic tests available to everyone that could be performed by people themselves and on a relatively regular basis. A step bringing us closer to this sort of personalized medical diagnosis and cancer prophylaxis is the chemical sensor devised and fabricated by Prof. Wlodzimierz Kutner’s group from the Institute of Physical Chemistry of the Polish Academy of Sciences (IPC PAS) in Warsaw using a grant from the National Science Centre, in collaboration with the team of Prof. Francis D’Souza of the University of North Texas in Denton TX, USA.

The most important element of the chemosensor devised at the IPC PAS is a thin film of the polymer that detects molecules of neopterin. Neopterin – in chemical terminology known as 2-amino-6-(1,2,3-trihydroxypropyl)-1H-pteridin-4-one) – is an aromatic compound present in human body fluids, such as serum, urine, and cerebrospinal fluid. Produced by the immune system, it is regarded as a universal marker in medical diagnosis. The concentration of this biomarker rises significantly particularly in the case of certain neoplastic diseases, e.g., malignant lymphoma, although elevated levels of neopterin are also seen in some viral and bacterial infections, as well as in diseases of parasitic aetiology. In turn, in transplant patients, increased levels of neopterin signal probable rejection.

“How can we detect the presence of neopterin? A reasonable approach is to use special recognizing materials for this purpose, prepared by molecular imprinting. This technique involves ‘stamping out’ molecules of the desired compound – their shape, but also at least some of the chemical characteristics – in a carefully designed polymer,” explains Dr. Piyush Sindhu Sharma (IPC PAS), the lead author of an article published in the Biosensors and Bioelectronics journal.

During the preparation of the polymer film, molecules of the substance being detected – in this case neopterin – are in a working solution in which their binding sites have to link with recognizing sites of so-called functional monomers. In turn, these monomers should be able to form connections with another monomer, a cross-linking agent which together, after polymerization, form a rigid support structure of the polymer. Next, the molecules of the compound used as a template are washed out from the structure. The result is a durable polymer with molecular cavities of a shape and chemical properties ensuring the capture of molecules of the desired compound from its surroundings.

The basic difficulty in molecular imprinting is the selection of the appropriate functional and cross-linking monomers as well as solvents, their proportions and reaction conditions. PhD student Agnieszka Wojnarowicz (IPC PAS) explains: “With the aid of quantum-chemical calculations, we first check whether there is bonding between our template molecule and selected functional monomers, and whether they will be stable in the solvent used. We also check whether the molecular cavities formed are sufficiently selective, i.e., whether they will primarily capture the molecules we are detecting, and not any that are similar to them. When the calculation results confirm our expectations, that is when we proceed to their experimental confirmation.”

At the IPC PAS a recognizing polymer film with molecular cavities from neopterin has been produced on the surface of an electrode. After immersion in artificial blood serum spiked with neopterin, the film on the electrode captured molecules of the latter, thus leading to a decrease in electrical potential in the connected measuring system. The tests showed that the molecular cavities of the polymer were almost entirely filled with molecules of neopterin despite the presence of molecules of similar structure and properties. This result means that the probability of false positive detection (detecting the presence of neopterin in body fluids not containing it) is negligibly small. The new chemical sensor therefore mainly reacts to what it should react to – and nothing else.

“At present, our chemosensor is a piece of laboratory equipment. However, the production of its key element, that is, the recognizing polymer film, does not pose major problems, and the electronics responsible for electrical measurements can easily be miniaturized. There is nothing standing in the way of building simple and reliable diagnostic equipment, based on our development, in just a few years’ time, which would be affordable not only for medical institutions and doctors’ surgeries, but also for the public in general,” predicts Prof. Kutner (IPC PAS).