New inhibitors of a cancer-causing protein may lead to targeted therapeutics

The activity of protein kinases, a large class of signaling molecules, must be closely regulated or signaling chaos arises within cells. Signaling chaos sets off a process that is implicated in the development of cancers, including solid tumors. Because protein kinases have a central role in cell signaling, researchers have devoted decades of investigation to developing protein kinase inhibitors.

One kinase inhibitor, a drug called Imatinib (or Gleevec), illustrates the profound potential of kinase inhibitors in cancer treatment. The hyperactivity of the Abl kinase is the most common cause of chronic myelogenic leukemia (CML). Imatinab is routinely used to treat CML. Since Imatinib’s Food and Drug Administration-approval in 2001, deaths related to CML have dropped significantly.

Src kinase inhibitors have been used in clinical trials as experimental treatments for many types of solid tumors. However, in contrast to the success of Imatinib for treating CML, a drug based on inhibiting the Src kinase has not been effective in treating solid tumors.

“Think of kinases as a traffic light system that is regulated to keep traffic flowing properly,” says Dr. Seeliger. “But if all lights on a road system are green, chaos occurs. The Src kinase is a dangerous ‘traffic light’ when it stays green. The challenge is to inhibit Src kinase, in other words make it a ‘red light,’ without stopping other Src family of kinases from staying ‘green’ to keep the traffic moving safely.”

In “Highly specific, bisubstrate-competitive Src inhibitors from DNA-templated macrocycles,” the researchers successfully completed two steps that are necessary for the development of new Src kinase-targeted drugs.

First, under the direction of David Liu, Ph.D., Professor, Department of Chemistry, Harvard University, the team developed chemical inhibitors of Src kinase. Dr. Seeliger’s laboratory at Stony Brook then determined the three-dimensional structure of these inhibitors bound to the Src kinase. This second step enabled the team to explain why the inhibitors work to stop the Src kinase but not the other kinases in cultured mammalian cells.

“Using this method, the precise molecular basis of the inhibitory mechanism and Src kinase are revealed,” says Dr. Seeliger. “These results provide new insights into the development of Src-specific inhibitors with potential therapeutic relevance.”

Provided by Stony Brook University (news : web)

Closing in on an ulcer- and cancer-causing bacterium

Writing in a Journal of Biological Chemistry "Paper of the Week," the scientists say the information they have obtained about the pathogen's clever employment of acid neutralizers may inform those who are designing new drugs to blunt H. pylori's effects across the globe.

H. pylori are the only bacteria known to thrive in the human stomach. It remains unclear how the pathogens are transmitted, although researchers suspect they could be spread through contaminated food or water. The damage the bacteria do to the mucous coating of the gut allows stomach acid to eat away at the sensitive organ lining, causing ulcers.

Although more than half of the world's population has the infection, for reasons still not quite understood most never develop ulcers. In fact, existing antibiotics can cure 80 to 90 percent of ulcers caused by the pathogen. However, H. pylori over the years have become increasingly resistant to antibiotics. Some experts have attributed that resistance to the fact that doctors are quick to prescribe antibiotics to kill it even when patients show no symptoms.

"There is a pressing need to develop new drugs and alternative strategies to fight against H. pylori infection before the prevalence of antibiotic resistance gets out of hand," says Ivan Fong, the lead author on the JBC paper and a graduate student at the Chinese University of Hong Kong whose research is focused on the biochemical makeup of protein complexes that assist in H. pylori's survival.

Ivan Fong, a graduate student at the Chinese University of Hong Kong, studies the biochemical makeup of protein complexes that assist in H. pylori's survival. Kam-Bo Wong is a professor at the institution and oversaw Fong's recent project. Credit: Chinese University of Hong Kong

It's the pathogen's ability to persist within the acid bath in the human stomach that has made it such a successful, albeit harmful, vector, says Fong. "The key is its use of an enzyme called urease to neutralize gastric acid," he explains.

H. pylori produce urease to spur the breakdown of urea, a naturally occurring chemical in the body, so that urea can release ammonia and make the gut an environment in which the pathogens can thrive. But, unlike most other enzymes, urease doesn't start doing its job immediately after being produced by the bacterium; instead, two nickel ions have to be delivered to it, and then the enzyme can mature, so to speak, and thus allow H. pylori to begin their damaging work.

"As the survival of H. pylori depends on active urease, this is a life-or-death issue for the pathogen to ensure nickel ions are delivered to the urease," says Kam-Bo Wong, a professor who oversaw the project at the institution.

It's not entirely clear how H. pylori make sure that urease can mature and then neutralize the surrounding acid. But Wong's team focused on four proteins that they suspect are helpers: UreE, UreF, UreG and UreH.

Using X-ray crystallography, "which essentially performs the function of a molecular microscope to visualize proteins with atomic resolution," Fong explains, the team took snapshots of UreF and UreH. What they saw was that UreH morphs the shape of UreF to enable UreF to recruit a third player, UreG, to form the UreF-UreH-UreG complex. In other words, the three proteins hook up to collectively deliver nickel ions to the right place on urease. Once the nickel ions are in place, they serve like a flint to ignite the breakdown of urea into ammonia, which then neutralizes the stomach acids.

"So, now we have a better understanding of how the machine can assemble itself, as if a skillful mechanic were there for the job, and deliver the nickel ions," says Fong.

Importantly, the team also discovered that disrupting the formation of the crafty UreF-UreH-UreG complex does, in fact, inhibit the synthesis of active urease. They hope that the information they've obtained about the molecular structures of UreF and UreH will help in the design of drugs that will essentially muck up the works of the molecular machine.

"As active urease is the key to survival of H. pylori, designing drugs that target this complex may well be a viable strategy to eradicate the pathogen," says Wong.

More information: The abstract for the paper, titled "Assembly of the preactivation complex for urease maturation in Helicobacter pylori: Crystal Structure of the UreF/UreH complex," is available at http://www.jbc.org … 830.abstract

Provided by American Society for Biochemistry and Molecular Biology

Cancer-causing virus exploits key cell-survival proteins

A cancer-causing retrovirus exploits key proteins in its host cells to extend the life of those cells, thereby prolonging its own survival and ability to spread, according to a new study by researchers at The Ohio State University Comprehensive Cancer Center – Arthur G. James Cancer Hospital and Richard J. Solove Research Institute (OSUCCC – James) and Ohio State's College of Veterinary Medicine.

The human T-lymphotropic virus type-1 (HTLV-1), which causes adult T-cell leukemia and lymphoma, produces a protein called p30 that is essential for the retrovirus to establish an infection. This study found that this viral protein targets two important cell proteins: ATM, a key player in a cell's response to DNA damage, and REG-gamma, which marks proteins within the cell for destruction.

"Our findings suggest that the p30 viral protein prolongs the survival of host cells through this interaction with ATM and REG-gamma, and the longer a virus-infected cell survives, the better chance the virus has to spread, " says principal investigator Michael Lairmore, DVM, PhD, professor of veterinary biosciences and associate director for shared resources at the OSUCCC – James.

The findings were published recently in the Journal of Biological Chemistry.

An estimated 20 million people worldwide are infected by HTLV-1, and about five percent of them will develop adult T-cell leukemia or lymphoma, or one of a variety of inflammatory disorders.

Lairmore and his colleagues used cell lines and a variety of biochemical assays to identify cellular binding partners of p30. They discovered the following:
p30 specifically binds to cellular ATM (ataxia-telangiectasia mutated), a key regulator of DNA damage responses and cell cycle control, and to REG-gamma, a nuclear proteasome activator.
Under stressful conditions, p30 levels are associated with lower ATM levels and increased cell survival.
The expression of p30 changes in concert with expression of REG-gamma, suggesting that overexpression of REG-gamma enhances levels of p30.
p30 forms a complex with ATM and REG-gamma.