How Entasis Pivoted to Engineer a Novel Antibiotic

For years, Entasis Therapeutics had been designing a molecule to target Pseudomonas aeruginosa, an opportunistic bacterium that rapidly evolves to evade antibiotics and kills an estimated 334,000 people annually around the world. As the arsenal of antibacterial drugs to treat P. aeruginosa has diminished, the CDC and WHO have both identified it as a top health threat.

Midway into their project, Entasis had engineered a molecule that could inhibit a common antibiotic target in P. aeruginosa, but it failed to enter the bacterium to kill it. After months of tweaking the molecule to try to improve uptake, the team hit a wall. Dr. Ruben Tommasi gathered his biology and chemistry teams and encouraged them to abandon this approach. “It was a provocative and difficult decision to move away from something that was so biochemically active,” says Dr. Tommasi, Entasis’ Chief Scientific Officer. “But we knew what we had to accomplish structurally. And lo and behold, three to four months later, we were seeing molecules like ETX0462 coming out of our chemistry program that had biochemical activity and the desired cellular uptake.”

Last fall, Entasis published its significant findings about ETX0462 in Nature, an internationally renowned, peer-reviewed journal. ETX0462 is a novel antibiotic that broadly targets P. aeruginosa and other pathogens that the CDC has flagged as national security risks, including Bacillus anthracis (Anthrax), Yersinia pestis (Plague), Francisella tularensis (Tularemia), and two species of Burkholderia that cause glanders and melioidosis.

In developing ETX0462, the goal was to create a novel antibiotic that shared the safety profile of beta-lactams, and that was not susceptible to beta-lactamase resistance mechanisms which are becoming increasingly common. Since the 1940s, when penicillin was introduced, beta-lactams have been favored by physicians. These small molecules with characteristic beta-lactam rings can be prescribed at high doses without the risk of toxic side effects. That’s key, because unlike drugs in other therapeutic areas, small antibacterial molecules must often be prescribed at higher doses to kill bacteria and avoid promoting resistance.

Beta-lactams work by binding penicillin binding proteins (PBPs) which are essential for bacteria to synthesize new cell walls. When PBPs are bound, cell wall production halts, stopping the bacteria in their tracks. Over time, some bacteria have evolved to produce beta-lactamase enzymes which break apart beta-lactams. Many companies have engineered beta-lactamase inhibitors (BLIs) to work in combination with non-effective beta-lactams to restore their function.

Engineering ETX0462

As Dr. Tommasi’s team witnessed how quickly beta-lactams were getting turned over and inactivated, they felt they could design something better than another BLI.

They took inspiration from a promising and potent molecule, NXL-105, that exhibited strong activity against P. aeruginosa in vitro, but not in vivo. “Somehow the bacteria could circumvent the selective inhibition in vivo, and the infection could continue to grow when that pressure was gone because the animal cleared the drug,” says Dr. Tommasi. His team figured out that NXL-105 was targeting PBP2, which is a less desirable molecular target in the cell wall synthesis pathway.

Dr. Tommasi’s team imagined that if they could develop a molecule with broader PBP activity, they could achieve in vitro and in vivo activity. They decided to transform NXL-105 and avibactam, molecules within the diazabicyclooctane (DBO) class which contain strong carbonate bonds that could prevent them from getting turned over by beta-lactamases. Noting that avibactam could freely enter P. aeruginosa–and act as a BLI in combination with ceftazidime–the team imagined that part of avibactam’s structure was required for bacterial permeation. As they engineered a hybrid molecule to redirect activity from BLIs toward PBP1 and PBP3, they had to add a second methyl group. But as soon as they did, the molecule lost a hydrogen bond, and it could no longer enter P. aeruginosa.

Dr. Tommasi described this as a turning point. Over his career, he had witnessed too many projects fail because chemists could not understand the structural reason why their compound couldn’t get in. “As scientists, we’re supposed to fix it.”

The Entasis team forged ahead, studying the P. aeruginosa porins, channels which permit molecules to pass through the outer membrane of the bacterial cell. Unlike E. coli, which has several large porins that enable a wide array of molecules to diffuse through its membrane, P. aeruginosa porins are highly specific. Some are designed to take up an individual amino acid, making generalized diffusion a challenge.

After performing a battery of porin assays, they figured out how to transform the DBO scaffold, originally represented by NXL-105, which contained a pyrazole ring at the top of the molecule. “By redesigning the dimethyl amide on the northwest piece, it mimics that hydrogen bond donor you need for uptake and gives you the potency,” says Dr. Tommasi. “I’ve been working in infection since 2006. It was the first time I’ve witnessed a team say, ‘my compound isn’t getting in well, and this is what I need to do about it.’”

The resulting molecule, ETX0462, has direct activity against PBP1 and PBP3 and is not subject to hydrolysis by beta-lactamase enzymes. The single-agent drug does not require the help of BLI molecules to kill bacteria, and it avoids efflux pumps, the transport proteins that can eject beta-lactams, a major cause of resistance.

Dr. Tommasi says that while ETX0462 is in early stages of preclinical development, it is a promising proof of concept on a couple of levels. “One, you can find PBP inhibitors that do work in in vivo models of infection and are not beta-lactams. That really hadn’t been shown before. And you can design molecules to get into bugs better, if you build the right assays.”

A Strong Team and Pipeline

Dr. Tommasi credits the success to his team who personify the company’s name, Entasis, a Greek architectural term that describes how columns are designed to endure strength under stress.

When Entasis spun out of AstraZeneca in 2015, they downsized from a 200-person anti-infective division on a Friday to a 16-person start-up the following Monday. Today the tight team comprises an array of experts–biology, chemistry, biochemistry, microbiology, and bioanalytics–all in the same lab. Unlike at many large pharmaceutical companies, Dr. Tommasi says an Entasis chemist can walk around the hood and talk to a microbiologist about their plans and designs. “Everyone on the team has grown because of that. We are all Entasis scientists working on the same problem together.”

ETX0462 is only one part of Entasis’ success story. It joins a strong roster of three antimicrobials–ETX0282, zoliflodacin and durlobactam–moving through different stages of clinical development. Two of the programs are oral agents, which could enable patients to be treated at home.

ETX0282 was among the first projects to enter the CARB-X portfolio in 2017. Currently in Phase 1 clinical trials, ETX0282 is an oral BLI to be used in combination with cefpodoxime. It targets complicated urinary tract infections (UTIs) caused by drug-resistant Enterobacteriaceae, affecting many women, in particular, who must be hospitalized to access medications that work.

The novel oral antibiotic, zoliflodacin, is a gyrase inhibitor that targets uncomplicated gonorrhea which affects an estimated 82 million people annually around the world. Since only one injectable class of antibiotics remains effective against Neisseria gonorrhoeae, the CDC has designated it as a top public health threat. While zoliflodacin is in Phase 3 clinical trials, Entasis has partnered with the non-profit GARDP to make it available for use in low- and middle-income countries, where resistant gonorrhea infections are rampant. “So hopefully we’ll finish that study soon, and have good news,” says Dr. Tommasi.

After successfully completing human trials for sulbactam-durlobactam (SUL-DUR), Entasis is now filing the regulatory package to be considered by the FDA for approval. Durlobactam targets multidrug-resistant Acinetobacter baumannii infections that have high mortality rates up to 50%. “Acinetobacter infections are tough to handle with resistance rates that vary from 30% up to 90% depending on the geography,” says Dr. Tommasi. “It’s an underappreciated medical need, and resistance will continue to evolve, so we need new therapies.”

A two-time CARB-X award recipient, Entasis received $5.8 million for preclinical development and its first-in-human program of ETX0282, and an additional $9.8 million to develop ETX0462. “The collaboration with CARB-X has allowed us to evolve our portfolio at a time when it’s difficult to leverage VC money in the discovery space,” says Dr. Tommasi. Beyond the funding, Dr. Tommasi appreciates the camaraderie of CARB-X’s in-house R&D team. “While making our proposal, they helped us think through problems that might arise, and plan further ahead than we typically would feel comfortable doing.”

As Entasis progressed ETX0462 from the bench to a development candidate, CARB-X R&D Chief Dr. Erin Duffy identifies two critical elements that can be thought of as a horse and jockey. “The horse is the scientific plan that has elevated ETX0462 for targeting multidrug-resistant P. aeruginosa, a goal which has eluded scores of antibiotic drug discoveries,” says Dr. Duffy. “The jockey is the experienced, close-knit team that–through years of working together–removed the traditional scientific silos and instead set their sights on a singular goal. The delivery of new antibiotics that will be meaningful to patients of the future requires excellence in both, and we are proud to have supported Entasis on this journey and the journey of our recent graduate, ETX0282.”

Ruben Tommasi
Chief Scientific Officer

Dr. Ruben Tommasi obtained a doctorate degree in organic chemistry from SUNY Albany in 1992 with professor Frank M. Hauser. He completed two postdoctoral assignments at the University of Colorado Boulder with professor Gary Molander and at Upjohn, where his work on the dihydropyrone class of HIV protease inhibitors led to the discovery of Tipranavir. In 1994, Dr. Tommasi joined Ciba-Geigy (now Novartis) as a medicinal chemist, working in several therapeutic areas including arthritis, bone metabolism and infection. During his 17-year tenure at Novartis, Dr. Tommasi led his teams to advance two candidates to Phase 2 clinical studies (HCV protease inhibitor–BZF961; EF-Tu inhibitor for C. difficile–LFF571). In 2011, he joined AstraZeneca to lead the Infection Chemistry team. Currently, Dr. Tommasi is the Chief Scientific Officer of Entasis Therapeutics, the spinout of AstraZeneca’s infection franchise. These efforts have resulted in three Entasis clinical stage development candidates (two Phase 3 and one Phase 1). Among Dr. Tommasi’s main interests is to improve understanding of factors that affect drug permeation into Gram-negative organisms. Dr. Tommasi is the co-author of 44 papers and co-inventor of 40 patents.

For more information, www.entasistx.com