Resistance Eradicators: Researchers are poised to deploy a new weapon in the war against retroviral replication. By Roger Meissen
Resistance is the price paid for success in treating HIV/AIDS. Even as scientists develop successful combinations of drugs to combat the disease, the virus invents ways to confound them. Welcome to the world of the human immunodeficiency virus, the source of a global scourge that has killed millions. A constant stream of new approaches, compounds and drugs are needed to keep that total from moving ever higher.

Virologists at MU’s Bond Life Sciences Center are playing a key role in exploring the potential of resistance-subduing discoveries. Among the more notable is Stefan Sarafianos, an associate professor of molecular microbiology and immunology whose research team recently showed that EFdA, a compound that stops HIV from spreading, is effective in combatting tenofovir-resistant HIV. Tenofovir one of the world’s most heavily prescribed HIV drugs.

“HIV in patients treated with tenofovir eventually develops a K65R RT mutation that causes a failure of this first line of defense,” says Sarafianos. “Not only does EFdA work on resistant HIV, but it works 10 times better than if they are not tenofovir resistant.”

Sarafianos and a team of researchers found that EFdA, otherwise known as 4’-ethynyl-2-fluoro-2’-deoxyadenosine, is activated by cells more readily and isn’t broken down by the liver and kidneys as quickly as existing drugs. “These two reasons make it more potent than other drugs, and so our task is to look at the structural features that make it such a fantastic drug,” he says.

Not surprisingly, EFdA potential attracted the attention of some of the nation’s most prominent virologists, many of whom share Sarafianos’enthusiasm for its prospects.

For the past eight years, for example, Sarafianos’lab has worked closely with University of Pittsburgh biochemist Michael Parniak and the National Institutes of Health’s Hiroaki Mitsuya to explore the molecule’s potential. Mitsuya had a hand in discovering the first three drugs to treat HIV. Parniak has spent years evaluating HIV treatments using cultured white blood cells.

An estimated 36 million victims have succumbed to AIDS in the past 33 years, according to the World Health Organization. Since the beginning of the epidemic, around 75 million people have become infected with HIV.

The modern history of the disease began just over three decades ago. On June 5, 1981, the Centers for Disease Control’s Morbidity and Mortality Weekly Report described the unusual illnesses of five young people in Los Angeles. Each was a previously healthy man who reported having had sex with men. All had been diagnosed with Pneumocystis carinii, a rare form of pneumonia that preys upon weakened immune systems. By the time the report was released, two of the five were dead.

Within days calls flooded the CDC, and other opportunistic diseases were soon added to the roster of alarming reports from physicians. The list of infected, meanwhile, quickly widened to include women, infants and straight men, putting to rest the suspicion that only gay males could contract HIV. Intravenous drug use and blood transfusions joined unprotected sex as risk factors linked to infection.

By 1984, scientists in the U.S. and France identified versions of human immunodeficiency virus as the likely cause of the AIDS epidemic, noting that the virus attacked CD4+ T lymphocytes, a type of white blood cell vital to the immune system.

Although Congress initially allocted a paltry $15 million in 1982 to research the disease, total funding eventually climbed to hundreds of billions. In 2014, Congress appropriated $29.7 billion in HIV/AIDS-related funding, with 10 percent going toward research.

The secret to the success of HIV lies in the messiness of its replication. It can spawn billions of copies of itself within a day. This breakneck speed comes at the expense of genetic accuracy, with new copies of the virus often putting nucleoside building blocks in the “wrong” places.

“It ends up making all kinds of permutations of the virus to the point that it can change every single nucleoside of the virus every day,” Sarafianos says. “The vast majority of these changes will result in garbage viruses, but a few of these will help it survive certain pressures. In the case where you use a drug, it will help the virus survive the drug pressure.”

Rapid-fire replication, in other words, is HIV’s salvation, a process that allows it to create mutants with the machinery to slide by the defenses of each new drug combination.

Molecule illustration

virus fighter in action
After being incorporated into viral DNA strands (shown in green and red) by means of reverse transcriptase, EFdA, in yellow, is positioned to disrupt the process by which the HIV enzyme (blue surface) accomplishes replication of the virus.

Scientists and doctors found this out first hand in 1993 as resistance to zidovudin, the first effective HIV fighter, began appearing only six years after it was introduced. Today significant HIV resistance to zidovudine has greatly reduced its usefulness.

These days treatment guidelines prohibit the use of a single drug for fear of similar outcomes. Cocktails of three or more medicines are the norm in HIV treatment. But keeping ahead of HIV’s evolutionary dexterity means that the development of new drugs is critical. Because of its resistance-busting potential, many scientists believe EFdA could be our best hope yet.

EFdA’s path to “best hope yet” has been an unorthodox one. A Japanese soy sauce company patented the molecule, which looks like compounds used to make soy sauce taste better, in 2005. They recognized that this flavor enhancer fell into a family of compounds called “nucleoside analogues” that are similar to existing drugs used to disrupt replication of HIV and other viruses. Samples sent for further testing confirmed EFdA’s potential usefulness against HIV, and soon researchers across the globe were racing to pinpoint what made the compound special.

“It’s the most potent compound that’s ever been described for HIV,” Parniak says in his lab at the University of Pittsburgh. “The thing that’s interesting about EFdA is that it has activity against every resistant virus we’ve evaluated.”

EFdA is one of a class of compounds called nucleoside reverse transcriptase inhibitors (NRTIs). Among these are eight existing HIV drugs, including tenofovir and zidovudine. Like all NRTIs, EFdA hijacks the HIV replication process by tricking an enzyme called reverse transcriptase.

Reverse transcriptase helps build new DNA, assembling nucleoside building blocks into a chain. Normally reverse transcriptase starts its job soon after HIV infects. First it creates a new strand of DNA from the virus’existing RNA. Then it begins breaking down the RNA and coding for a matching DNA strand. The resulting new helix is inserted into the DNA of healthy cells, particularly CD4+ T lymphocytes. There it produces the proteins needed by the retrovirus.

Because EFdA looks like the nucleoside building blocks, reverse transcriptase is tricked into using the imposter. When this happens the virus’code cannot be added to the DNA of white blood cells it attacks. “NRTIs are called chain terminators because they stop the copying of the DNA chain, and once incorporated it’s like a dead end,” Sarafianos says.

Sarafianos is working to better understand how EFdA functions at the molecular level. He has used virology, crystallography and nuclear magnetic resonance to piece together the exact structure, bonding angles and configuration of the compound.

“The process started by putting different things at various sites on the drug, making EFdA a little different and then checking to see how the depth, the structural features, the parts of the molecule, impart these fantastic properties to it,” Sarafianos says. “This was done the hard way, working with medicinal chemists who make these EFdA analogues for us to evaluate biochemically, virologically and structurally.”

By looking at subtle differences in EFdA’s sugar-like ring, his lab identified a structure that looks the most like actual nucleosides, doesn’t break down easily and is activated readily by CD4+ T lymphocytes.

“The structure of this compound is very important because it’s a lock and key kind of mechanism that can be recognized by the target,” Sarafianos says. “We’re looking at small changes and the ideal scenario is a compound bound very efficiently by the target and activating enzyme, but not efficiently by the degrading enzymes.”

In research funded in part by the National Institutes of Health and published in the journals Retrovirology, Antimicrobial Agents and Chemotherapy and The International Journal of Pharmaceutics, Sarafianos, Parniak and Mitsuya have continued to demonstrate the magic of EFdA. They have shown, for example, that the drug worked incredibly well to treat the HIV equivalent in monkeys.

“These animals were so lethargic, so ill, that they were scheduled to be euthanized when EFdA was administered,” says Parniak. “Within a month they were bouncing around in their cages, looking very happy and their virus load dropped to undetectable levels. That shows you the activity of the molecule: it’s so active that resistance doesn’t come in as much of a factor with it.”

Among the more exciting elements of Parniak’s primate findings was the low level of EFdA needed to achieve optimal potency. “It starts out being so potent that even if you end up with a twenty-fold resistance of HIV, the therapeutic dose is still enough to eliminate replication,” Parniak says.

As therapies extend lifespans some 50 years beyond HIV diagnosis, prevention is the new frontier for HIV research and the focus of future collaboration. Thanks to formulation expert Lisa Rohan at the University of Pittsburgh, EFdA is now being tested in a vaginal film with a consistency similar to Listerine breath strips.

While AIDS in the U.S. and Europe occurs mostly in men, women are more often infected in some other parts of the world, notably Africa. Since a film has a better shelf life than creams or gels, it could be especially useful there. “We have nearly 30 drugs approved for treating HIV-infected individuals, but only one approved for prevention,” Sarafianos says. “Women in Africa would benefit from a formulation like this as a means to protect themselves.”

“The only way we are going to make a difference with HIV is prevention,” adds Parniak. “This approach could make a huge difference — when combined with existing therapies for people already infected — in minimizing the continued spread of the disease.”

Pharmaceutical giant Merck, meanwhile, licensed EFdA in 2012. It is currently in preclinical trials. Despite this success, Sarafianos and Parniak aren’t slowing down. “We want to understand how long EFdA stays in the bloodstream and cells,” Parniak says. “If we can understand structurally why this drug is so potent it will allow us to maybe develop additional molecules that are equally potent. A combination of those molecules could be a blockbuster.”

Reader Comments

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Alex Ber

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