Structure discovered of cancer drug candidate

Structure discovered of cancer drug candidate

Chemists at The Scripps Research Institute (TSRI) have determined the correct structure of a highly promising anticancer compound approved by the U.S. Food and Drug Administration (FDA) for clinical trials in cancer patients.

The new report, published this week by the international chemistry journal Angewandte Chemie, focuses on a compound called TIC10.

In the new study, the TSRI scientists show that TIC10's structure differs subtly from a version published by another group last year, and that the previous structure associated with TIC10 in fact describes a molecule that lacks TIC10's anticancer activity.

By contrast, the correct structure describes a molecule with potent anticancer effects in animals, representing a new family of biologically active structures that can now be explored for their possible therapeutic uses.

"This new structure should generate much interest in the cancer research community," said Kim D. Janda, the Ely R. Callaway Jr. Professor of Chemistry and member of the Skaggs Institute for Chemical Biology at TSRI.

A new report shows the structure of a promising anticancer compound, TIC10, differs subtly but importantly from a previously published version.
Credit: Figure courtesy of The Scripps Research Institute.

Antitumor Potential

TIC10 was first described in a paper in the journal Science Translational Medicine in early 2013. The authors identified the compound, within a library of thousands of molecules maintained by the National Cancer Institute (NCI), for its ability to boost cells' production of a powerful natural antitumor protein, TRAIL. (TIC10 means TRAIL-Inducing Compound #10.)

As a small molecule, TIC10 would be easier to deliver in a therapy than the TRAIL protein itself. The paper, which drew widespread media coverage, reported that TIC10 was orally active and dramatically shrank a variety of tumors in mice, including notoriously treatment-resistant glioblastomas.

Tumors can develop resistance to TRAIL, but Janda had been studying compounds that defeat this resistance. The news about TIC10 therefore got his attention. "I thought, 'They have this molecule for upregulating TRAIL, and we have these molecules that can overcome tumor cell TRAIL resistance - the combination could be important,'" he said.

The original publication on TIC10 included a figure showing its predicted structure. "I saw the figure and asked one of my postdocs, Jonathan Lockner, to make some," Janda said.
A new report shows the structure of a promising anticancer compound, TIC10, differs subtly but importantly from a previously published version.
Credit: Image courtesy of The Scripps Research Institute.

Although the other team had seemingly confirmed the predicted structure with a basic technique called mass spectrometry, no one had yet published a thorough characterization of the TIC10 molecule. "There were no nuclear magnetic resonance data or X-ray crystallography data, and there was definitely no procedure for the synthesis," Lockner said. "My background was chemistry, though, so I was able to find a way to synthesize it starting from simple compounds."

Surprising Inactivity

There was just one problem with Lockner's newly synthesized "TIC10." When tested, it failed to induce TRAIL expression in cells, even at high doses.

"Of course I was nervous," remembered Lockner. "As a chemist, you never want to make a mistake and give biologists the wrong material."

To try to verify they had the right material, Janda's team obtained a sample of TIC10 directly from the NCI. "When we got that sample and tested it, we saw that it had the expected TRAIL-upregulating effect," said Nicholas Jacob, a graduate student in the Janda Laboratory who, with Lockner, was a co-lead author of the new paper. "That prompted us to look more closely at the structures of these two compounds."

The two researchers spent months characterizing their own synthesized material and the NCI material, using an array of sophisticated structural analysis tools. With Assistant Professor Vladimir V. Kravchenko of the TSRI Department of Immunology and Microbial Science, Jacob also tested the two compounds' biological effects.

The team eventually concluded that the TIC10 compound from the NCI library does boost TRAIL production in cells and remains promising as the basis for anticancer therapies, but it does not have the structure that was originally published.

The Right Structure

The originally published structure has a core made of three carbon-nitrogen rings in a straight line and does not induce TRAIL activity. The correct, TRAIL-inducing structure differs subtly, with an end ring that sticks out at an angle. In chemists' parlance, the two compounds are constitutional isomers: a linear imidazolinopyrimidinone and an angular imidazolinopyrimidinone.

Ironically, Lockner found that the angular TRAIL-inducing structure was easier to synthesize than the one originally described.

Now, with the correct molecule in hand and a solid understanding of its structure and synthesis, Janda and his team are moving forward with their original plan to study TIC10 in combination with TRAIL-resistance-thwarting molecules as an anticancer therapy.

The therapeutic implications of TIC10 may even go beyond cancer. The angular core of the TRAIL-inducing molecule discovered by Janda's team turns out to be a novel type of a biologically active structure - or "pharmacophore" - from which chemists may now be able to build a new class of candidate drugs, possibly for a variety of ailments.

"One lesson from this has got to be: don't leave your chemists behind," said Janda.

Worse side effects from chemotherapy experienced when biological rhythms interrupted

Worse side effects from chemotherapy experienced when biological rhythms interrupted

Patients receiving chemical treatment for cancer often suffer fatigue and body weight loss, two of the most worrying effects of this therapy linked to the alteration of their circadian rhythms.

The circadian system, better known as our biological clock, is responsible for coordinating all the processes that take place in our organism.

If it does not function correctly, what is known as a circadian disruption or chronodisruption, has for years been linked to an increased incidence of cancer, obesity, diabetes, depression, cognitive problems or cardiovascular diseases.

"Also, circadian disruption in cancer patients aggravates the prognosis of the disease and the chance of survival for these patients diminishes," Elisabet Ortiz Tudela, a researcher at the University of Murcia, told SINC.

The expert is the author of a study published in the International Journal of Cancer, which reveals the importance of assessing how the circadian system works in order to prevent chronodisruption and to implement measures to strengthen the biological clock in people whose system is damaged.

However, measuring how the biological clock works is not easy in humans given that the "machinery" is located within the suprachiasmatic nucleus in the hypothalamus, deep within the brain.

"Therefore, it is impossible to directly assess how the clock works," adds Ortiz. "Today, biological rhythms are studied which are clock "interruptions" and which enable us to indirectly assess the status of the circadian system".

One of the most studied "interruptions", which can be measured with non-invasive techniques and during long periods of time, is the activity-rest rhythm.

Chronotherapy for cancer

Researchers characterised the evolution of the circadian system in cancer patients submitted to a standard chronotherapy protocol (synchronisation of medication with natural rhythms). The activity-rest rhythm was recorded in 49 patients with advanced cancer while being given a standard chronomodulated chemotherapy cycle, which resulted in circadian disruption.

All the parameters calculated according to the activity-rest rhythm worsened considerably with the chemotherapy. Also, the existence of circadian disruption during the treatment was linked to greater fatigue and body weight loss, two of the most worrying associated factors for doctors.

"After the treatment and at the end of the study, the mean values of all the parameters were recovered to near baseline values," stated the scientist from the institution in Murcia.

The results show four different patterns with regard to the evolution of the circadian system in response to the treatment: in 9.5% of the patients the activity-rest rhythm remained stable despite the chemotherapy, 14.3% of patients showed some improvement, 31% suffered alterations in response to the treatment and recovered completely at the end of the study and in 45% of these deterioration sustained, possibly through inadequate dosing or incorrect timing.

This effect shows the great differences between individuals, which could affect the effectiveness of the treatment.

"Minimising circadian disruption through the personalisation of chronotherapy delivery could help to improve clinical tolerability and potentially contribute towards the treatment being more effective," concludes Ortiz.

Understanding of how Taxol works could lead to better anticancer drugs

Understanding of how Taxol works could lead to better anticancer drugs

University of California, Berkeley, scientists have discovered the extremely subtle effect that the prescription drug Taxol has inside cells that makes it one of the most widely used anticancer agents in the world.

The details, involving the drug's interference with the normal function of microtubules, part of the cell's skeleton, could help in designing better anticancer drugs, or in improving Taxol and other drugs already known to disrupt the workings of microtubules.

The findings are reported in the journal Cell.

"Efforts towards understanding these chemotherapeutics better are very important, because there are some microtubule differences in cancer cells versus normal cells that maybe we can exploit," said principle author Eva Nogales, a biophysicist, UC Berkeley professor of molecular and cell biology and senior faculty scientist at Lawrence Berkeley National Laboratory (LBNL). "We are not there yet, but this is the kind of analysis we need to get there."

Taxol, originally extracted from the bark of the Pacific yew tree, is one of the mostly commonly used drugs against solid tumors, and is a front-line drug for treating ovarian and advanced breast cancer. The drug is known to bind to microtubules and essentially freeze them in place, which prevents them from separating the chromosomes when a cell divides. This kills dividing cells, in particular cancer cells, which are known for rapid proliferation.

Nogales, a Howard Hughes Medical Institute investigator, has worked on microtubules since she was a doctoral student in England in the early '90s, using techniques such as X-ray scattering and cryoelectron microscopy to study how Taxol and other anticancer agents affect microtubules. Later, during her postdoctoral work at LBNL with Ken Downing, she was the first to discover exactly where Taxol binds the basic building block, called tubulin, of the microtubule polymer.

Microtubules are the cell's skeleton

Work by many scientists around the world has shown the microtubule network inside cells, called the cytoskeleton, to be very different from rigid animal skeletons. Microtubules are polymer filaments that constantly grow and shrink, and in doing so push and pull things around the cell, including the chromosomes. Scientists call this dynamic instability. The microtubules also provide a highway for transporting the cell's organelles and other packages around the cell.

Tubulin, the basic structural unit of the microtubule, is a complex of two proteins - alpha and beta tubulin. Tubulin units stack one atop another to form strips that align with other strips and then zip up to form a hollow tube, the microtubule.

"Tubulin, the cytoskeletal protein that self-assembles into microtubules, is absolutely essential for the life of every eukaryotic cell, which is why it has become a major target of anticancer agents," Nogales said. "It's amazing how microtubules probe and try new things almost at random, but there is a level of control built into the cell that ultimately makes sense of this chaos, and the cell survives and prospers."

Microtubules grow from their free end at about 1 micron per minute by continually adding more tubulin (around 20 tubulin molecules per second). But if they stop growing, they rapidly peel apart like the skin of a banana, releasing tubulin for recycling into other microtubules. This peeling, or depolymerization, takes place at up to 15 microns per minute, or about 300 tubulin molecules falling off per second, Nogales said.

Microtubules are like compressed springs

Nogales has now discovered why microtubules peel apart so rapidly. When they assemble, the strips of tubulin are put under intense strain, but prevented from bending and pulling apart by the growing cap of tubulin on the end. Once growing stops and that cap disappears, the restrained tension rips the microtubule apart.

The tension is created when the tubulin complex, which has a small energy molecule called GTP (guanosine triphosphate) attached, becomes hydrolyzed and the GTP turns into GDP (guanosine diphosphate). This chemical reaction compacts the alpha and beta subunits, much like compacted vertebrae, keeping the tubulin stack under tension as long as the microtubule is growing at its end.

"It had been proposed that tubulin had to be constrained, but no one had proved it," Nogales said. "What we have seen is that as GTP hydrolysis happens, the tubulin structure gets stuck in a strained state, like a compressed spring. The end subunits are holding the whole thing together."

When growth stops, the tension is unleashed, and the strips peel apart rapidly.

"This work represents a major step forward on a problem with a long history," wrote Tim Mitchison in a commentary in the same issue of Cell. Mitchison, a Harvard University professor of systems biology, was the first to show the importance of GTP hydrolysis in destabilizing microtubules. The model proposed by Nogales and her team, he added, "provides our first glimpse into (the) destabilization mechanism."

Nogales also found that Taxol inserts itself into the tubulin protein and prevents compaction of the alpha and beta subunits, so that no tension builds up. As a result, even if the microtubule stops growing, it remains intact, basically frozen in place, unable to peel apart, or depolymerize, and carry out its normal function.

"Taxol reverses the effects of GTP hydrolysis," she said.

Nogales and her team discovered these structural changes by pushing the limits of cryoelectron microscopy, a technique in which samples are frozen and probed with a high-powered electron beam. They have now achieved a resolution sufficient to see details smaller than 5 angstroms (one-tenth of a nanometer) across, which is about the size of five hydrogen atoms. While most information to date about the structure of tubulin inside the microtubule has come from the study of artificial, flat sheets of aligned strips of tubulin, Nogales was able to probe three-dimensional microtubules frozen into their natural state, with and without Taxol bound to tubulin. This comparison clearly showed the effect Taxol has on microtubule structure.

For dysplastic Barrett's esophagus, radiofreqeuncy ablation and complete endoscopic resection equally effective

For dysplastic Barrett's esophagus, radiofreqeuncy ablation and complete endoscopic resection equally effective

According to a new systematic review article, radiofrequency ablation and complete endoscopic resection are equally effective in the short-term treatment of dysplastic Barrett's esophagus, but adverse event rates are higher with complete endoscopic resection. The article comparing the two treatments appears in the May issue of GIE: Gastrointestinal Endoscopy, the monthly peer-reviewed scientific journal of the American Society for Gastrointestinal Endoscopy (ASGE).

Barrett's esophagus is a condition in which the lining of the esophagus changes and becomes more like the lining of the small intestine. It is believed that Barrett's esophagus (BE) occurs because of chronic inflammation resulting from long-standing Gastroesophageal Reflux Disease (GERD). Barrett's esophagus is more common in Caucasian males older than the age of 50 who have had GERD for greater than five years. Most patients with Barrett's esophagus will not develop cancer. However, in some patients further precancerous change in the tissue, called dysplasia, will develop. Those patients that develop dysplasia, especially high grade dysplasia, are significantly more likely to develop esophageal cancer.

Esophagectomy (surgery to remove part or all of the esophagus) has previously been the recommended treatment for BE with high grade dysplasia (HGD) or intramucosal cancer (cancer limited to the most superficial layer of the esophagus), but this surgery is associated with significant morbidity and mortality. As a result, endoscopic therapies for treatment of HGD or superficial cancers have been developed which minimize treatment-related morbidity. Ideally, endoscopic treatments need to target the entire segment of Barrett's mucosa (lining of the esophagus) in order to maximally reduce the risk of developing esophageal cancer.

To date, two distinct endoscopic approaches have been widely used for this purpose. The first is complete endoscopic mucosal resection (EMR) where the BE mucosa is resected or removed endoscopically. This has the advantage of providing a large histologic specimen, which can then be evaluated for unrecognized, more advanced pathology, and may be curative. The other approach is ablation of the BE mucosa by using a variety of techniques such as photodynamic therapy, argon plasma coagulation (APC), and more recently, radiofrequency ablation (RFA). RFA uses a focal heat process to destroy the Barrett's tissue. In recent years, RFA has become the ablative treatment of choice in the management of dysplastic BE, with early studies suggesting excellent efficacy and low rates of adverse events.

"Only one trial to date has directly compared complete EMR and RFA in treating dysplastic BE. The aim of this systematic review was to compare the efficacy and safety of these two techniques. This is important because RFA is substantially more expensive than complete EMR and may require multiple procedures over six months or more, making it less acceptable to patients. Therefore, in order to justify the use of RFA in the future it must be convincingly proven to be superior to complete EMR, in terms of both efficacy and risk of adverse events," said study lead author Georgina Chadwick, MRCP, The Royal College of Surgeons of England. "We found that RFA and complete EMR are equally effective in the short-term treatment of dysplastic BE, but adverse event rates are higher with complete EMR."

Methods

This article was a systematic review of literature to compare the efficacy and safety of complete EMR and radiofrequency ablation in the treatment of dysplastic BE. Patients had a diagnosis of BE with HGD or intramucosal cancer treated with either complete EMR or RFA. Main outcome measurements included complete eradication of dysplasia and intestinal metaplasia at the end of treatment and after more than 12 months' follow-up, as well as short and long-term adverse event rates associated with either treatment.

Results

A total of 22 studies met the inclusion criteria. Only one trial directly compared the two techniques; most studies were observational case series. Dysplasia was effectively eradicated at the end of treatment in 95 percent of patients after complete EMR and 92 percent after RFA. After a median follow-up of 23 months for complete EMR and 21 months for RFA, eradication of dysplasia was maintained in 95 percent of patients treated with complete EMR and 94 percent of patients treated with RFA. Short-term adverse events were seen in 12 percent of patients treated with complete EMR, but in only 2.5 percent of those treated with RFA. Esophageal strictures were long-term adverse events in 38 percent of patients treated with complete EMR, compared with 4 percent of those treated with RFA. Progression to cancer appeared to be rare after either treatment, although follow-up was short.

The authors concluded that both complete EMR and RFA have proven efficacy in eradication of BE with HGD or intramucosal cancer, but both short and long-term adverse events are significantly greater after complete EMR. The results of this review suggest that RFA, with prior resection of any nodules, is the endoscopic treatment of choice for dysplastic BE. But further research needs to be done to prove the long-term durability of both treatments in order to confirm their superiority over surgery in the management of dysplastic BE. Though low, the risk of recurrence of dysplasia and intestinal metaplasia after treatment reiterates the need for continuing endoscopic surveillance. Further research needs to determine the optimal surveillance regimen after successful eradication.