Winning the War on Bacteria

The world is in the midst of an antibiotic crisis. As wily bacteria develop resistance to one drug after another, pharma companies are forced to play a game of antibacterial whack-a-mole, attempting to develop new drugs to kill them faster than they can mutate. "Every year, some 1.5 million people die from antibiotic resistant infections," says Neil Osheroff, John G. Coniglio Chair in Biochemistry at Vanderbilt University. "By 2050, it is estimated that that number will grow to 10 million."

At the same time, development of new classes of antibiotics has virtually dried up. It is challenging to develop drugs that are designed to be taken sparingly and need tight regulation in order to prevent resistance from taking hold. As a result, the vast number of antibiotics produced today are just tweaks on existing drugs; the last truly novel class of antibiotics was approved by the FDA in 2003, one of only a handful developed since the 1960s.

That changed in March, when Osheroff's lab played a key role in the approval of the first new class of antibacterials to treat uncomplicated urinary tract infections (uUTIs) in adolescent and adult females. The drug, gepotidacin, developed by GlaxoSmithKline under the brand name Blujepa, will be available starting later this year, potentially bringing relief to thousands of women. "Some 30 to 40 percent of women will experience urinary tract infections in their lifetime, leading to roughly 8 million emergency department visits and 100,000 hospitalizations each year in the United States," Osheroff says. And yet, studies have reported that up to 92 percent of the bacteria that cause those infections are resistant to at least one class of the most commonly prescribed antibacterials. All the "mechanism of action" data on gepotidacin that accompanied GlaxoSmithKline's application to the U.S. Food and Drug Administration were generated by the Osheroff lab.

In addition to its use for uUTIs, gepotidacin has also completed clinical trials for treating gonorrhea with positive outcomes. Gonorrhea, a sexually transmitted infection with more than 80 million annual cases worldwide, has developed high levels of resistance to many classes of antibiotics. Even more exciting, gepotidacin appears to target two separate enzymes in the bacteria that cause uUTIs and gonorrhea, which makes it less likely that these infections will develop resistance to it.

Along with their work on gepotidacin, Osheroff's lab has contributed important mechanistic data on zoliflodacin, a second new class of antibacterials that has completed clinical trials for treating gonorrhea with positive outcomes.

The extraordinary accomplishments that contributed to these breakthroughs cap Osheroff's 42-year career researching enzyme mechanisms at Vanderbilt. "By all standards, my lab has been very successful. I've been continually funded by federal sources since 1984, graduated 34 Ph.D.'s and authored close to 300 papers," Osheroff says. "But I've always lamented that we've never had a direct line from our research to human health. Our work on gepotidacin now provides this direct line, which is an amazing feeling."

"I am really heartened by the FDA approval of Blujepa, because this approval was made possible by fundamental research concerning its mechanism of action, carried out in Dr. Osheroff's lab. This is a wonderful example of how basic science research is leveraged into life-changing therapies," School of Medicine Basic Sciences Dean John Kuriyan said.

Unravelling DNA to Attack Bacteria

Neil Osheroff

As a postdoctoral fellow at Stanford decades ago, Osheroff became fascinated with a category of enzymes called topoisomerases, which work to untangle and unknot DNA within the cell. These enzymes would soon become the focus of Osheroff's career. Each one of our cells has approximately two meters of DNA, wrapped in tight double-helix spirals. Multiplied by the 10 trillion cells in our body makes some 12 billion miles of DNA, "enough in one person to stretch to the sun and back 65 times," Osheroff says.

With all those intricate spirals crammed into our nuclei, however, it's inevitable that some get twisted. "It's like with holiday lights or earbud cords," he says. "You put your earbuds in your pocket and pull them out, and they are tangled." DNA can experience both overwinding, where the strands are twisted too tight, as well as underwinding, where they are too loose; both can cause serious effects in cells. To deal with these issues, the body employs topoisomerases, which work diligently to untangle DNA and set it right again. These enzymes act by cutting the double helix for a split second, passing another double strand of DNA through, and then resealing the gap. "Topoisomerases are incredibly powerful-it's like if you could just sprinkle something on your earbuds and magically they're untangled," Osheroff says.

Despite their importance to a number of critical cellular processes, topoisomerases are also very dangerous, leaving DNA vulnerable for the milliseconds that its strands are cut and exposed. When a drug inserts itself during that crucial moment when the DNA is cut, it acts "like a molecular doorstop," Osheroff says. "You open the door and put your foot in, and it can't shut anymore." The action of the drug inhibits the catalytic activity of topoisomerases. More importantly, it converts these essential enzymes into "molecular scissors" that basically fragment the genome."

Shortly after he came to Vanderbilt University School of Medicine in 1983, Osheroff says that he got lucky when several anti-cancer drugs were found to act through topoisomerases. Although his laboratory worked with pharmaceutical companies with the idea of developing a new anti-cancer drug, nothing ever came of the idea.

Meanwhile, Bayer developed ciprofloxacin, better known as Cipro, which was part of a new class of antibacterials called fluoroquinolones. These drugs target two bacterial topoisomerases, known as gyrase and topoisomerase IV. Approved in 1987, fluoroquinolones include some of the most powerful and broad-spectrum antibacterials in the world. However, the fluoroquinolones come with a problem, as they tend to favor one of the two enzymes (usually gyrase) as their primary antibacterial target. Consequently, if there is a mutation in gyrase that undercuts its ability to bind the fluoroquinolone, then the "doorstop" won't work, and the pathogen becomes resistant. As a result, a number of infections that used to be treated with fluoroquinolones are now resistant and no longer respond to members of this drug class.

Osheroff's lab continued to study topoisomerases to better understand the way they worked and the way that they interacted with drugs. Starting about 15 years ago, doctoral student Katie Aldred, PhD'14, who is now an associate professor at the University of Evansville, became interested in examining how fluoroquinolones interacted with gyrase and topoisomerase IV. She identified specific amino acid residues in the enzymes that were involved in binding fluoroquinolones and determined how the binding interactions took place. "Katie's Ph.D. was a true tour-de-force that for the first time allowed us to understand how fluoroquinolones acted against their bacterial targets," Osheroff says.

Based on that expertise, GlaxoSmithKline approached Osheroff in 2016 to discuss his working with gepotidacin, which had shown efficacy in animal studies and was now in phase 2 clinical studies in humans for UTIs and gonorrhea. A crucial part of drug approvals by the FDA is to show "mechanism of action," that is, evidence on a molecular level of what a drug targets and how exactly it works. With his long expertise, Osheroff directed experiments by graduate students Elizabeth Gibson, PhD'19, now an associate director at Bristol Myers Squibb; Alexandria Oviatt, PhD'22, now a postdoctoral fellow at Michigan State; and Jessica Bauer (nee Collins), PhD'24, now an associate clinical trial manager at Medpace, that addressed the mechanism of gepotidacin.

Together they were able to demonstrate that gepotidacin interacted with different residues on gyrase and topoisomerase IV than were utilized by fluoroquinolones. Along with scientists from GlaxoSmithKline, they also discovered that unlike fluoroquinolones, gepotidacin equally favored the two enzymes. That means it would be much more difficult to generate resistant strains, as chances that both enzymes would simultaneously develop a mutation are remote, Osheroff says. "If you get a mutation that knocks out one of the targets, you can still kill the bacteria through the other," he explains.

With data in hand, gepotidacin was submitted to the FDA for priority review last October and was approved for treatment of uUTI in March-the first new class of antibacterials targeting the infection since 1996. Gepotidacin also has gone through phase 3 clinical trials for gonorrhea with promising results. "The expectations are that gonorrhea will be the next indication," Osheroff says.

Zoliflodacin has also shown promise in phase 3 trials for gonorrhea, meaning that it may soon join gepotidacin as the first two novel classes of antibacterial drugs approved in decades-greatly expanding the arsenal that clinicians have to treat infections that plague millions of people worldwide.

"Fluoroquinolones have been in the market for 40 years and are still working well against a number of infections. However, we need to have more arrows in the quiver," Osheroff says. He's proud of his lab for the part they've played in winning the war against antibiotic-resistant bacteria-noting that it's particularly fitting that three female graduate students played such a strong role in helping approve a new treatment for uUTIs, which are 30 times more likely to affect women than men. In conversations with the global medical lead for gepotidacin at GlaxoSmithKline, Osheroff says, the data the lab supplied were crucial in getting the drug over the hump of regulatory approval, and prescriptions are set to roll out later this year.

Looking back on his four-decade career at Vanderbilt, Osheroff notes that the institution has grown in its reputation and ability to effect real change in the medical field since he arrived in the 1980s. "I came to a very good school and am now at an outstanding school," he says. "The quality of the university and its national reputation has soared." The support he has received over the years has been crucial in helping him achieve what every medical researcher dreams about-having a lasting impact on alleviating human suffering.

"My lab has worked in an area with great clinical relevance for decades, but telling people how drugs work doesn't mean that it will change the way they use them, or help overcome resistance," he says. "Physicians have the privilege and responsibility of treating patients one at a time. Although researchers have the opportunity to affect thousands or even millions of lives, how often does that happen? I am proud to say my lab has been able to do that."