In 1928, Alexander Fleming discovered penicillin, which would become the first commercialized antibiotic. Ever since, germs have looked for ways to survive and resist new drugs. Such antimicrobial resistance, which threatens the effective prevention and treatment of an ever-increasing range of infections, has been accelerating over the past several decades as the misuse and overuse of antimicrobials has spread around the globe.

Resistance arises through one of three mechanisms: natural resistance in certain types of microbes, genetic mutation, or horizontal gene transfer. All classes of microbes, including bacteria, viruses, fungi, and protozoans, can develop resistance. Antimicrobials increase selective pressure in microbial populations, causing vulnerable microbes to die and conferring a growth advantage to resistant microbes. As a result, the medicines become ineffective and infections persist in the body, increasing the risk of spread to others.

To make matters worse, these same selective pressures act independently of a given microbe’s resistance status for other antimicrobials, so a pathogenic microbe already resistant to one medicine can just as easily acquire resistance to others through these same processes. The survivors of this process can emerge resistant to a wide spectrum of antimicrobials. When they do, they are said to be multidrug resistant.

Antimicrobial resistance has the potential to affect people at any stage of life. Infections caused by antimicrobial-resistant germs are difficult and sometimes impossible to treat, leading to an estimated 700,000 to several million deaths per year globally. Antimicrobial-resistant infections also typically require extended hospital stays, additional follow-up doctor visits, and costly and toxic alternative drugs. It also significantly impacts the veterinary and agriculture industries, making it one of the world’s most urgent public health problems.

New discovery platforms, novel screens, and innovative approaches are essential for the development of new classes of antimicrobials and for ceasing the dangerous trend of multidrug microbial resistance.

The possibilities and challenges of this important field were discussed at the Antibacterial Discovery and Development track of the Discovery on Target conference held recently in Boston. Several of the scientists who spoke at the conference have since shared with GEN their thoughts on how antibacterial development may be advanced.

Persistence resistance

Bacteria don’t have to resist antibiotics to defy them. There is a much simpler, more common, and largely unappreciated way called bacterial persistence. While the vast majority of antibiotics work by killing bacteria that are actively growing and multiplying, tiny fractions of bacterial populations can go into a metabolically quiescent state in response to antibiotics and other stressors. “So, as most of the population gets wiped out from one or another of these threats, there will be some persisters that survive and become the seeds of future infections,” said Diane Joseph-McCarthy, PhD, senior vice president, discovery and early development at Boston-based EnBiotix.

“It’s widely accepted within infectious disease circles that bacterial persisters are a real problem from two perspectives: first, as the seeds of chronic, recurrent infections; second, as a repository from which bona fide resistant bugs can emerge,” Joseph-McCarthy added. “The need to eradicate bacterial persisters is, therefore, imperative.”

EnBiotix has developed a product candidate based on the observation that certain classes of antibiotics, such as aminoglycosides, depend on proton-motive force to be transported across the cell wall to reach their targets of action. This type of transport does not occur in metabolically quiescent cells.

EnBiotix researchers hypothesized that if they could find an agent to prime cell metabolism, they could potentially jumpstart the necessary transport machinery. Then co-administering that agent with an aminoglycoside could lead to the eradication of bacterial persisters. EnBiotix first demonstrated this concept with gentamicin and mannitol and have since translated that work into EBX-001, an inhaled drug in preclinical trials for cystic fibrosis lung infection that is a combination of tobramycin (the aminoglycoside) and fumarate (the cell metabolism primer).

Forging a new path

“Many companies today are looking at the existing classes of antibiotics and making small, incremental changes,” said Zachary Zimmerman, PhD, CEO and co-founder of San Diego-based Forge Therapeutics. “Once a bacterium has become resistant to generations one, two, and three, it’s going to become resistant to generation four. It’s just a matter of time. What we really need are novel classes of antibiotics.”

In its search for antibiotics that won’t face immediate resistance problems, Forge’s proprietary discovery platform combines traditional medicinal chemistry with bioinorganic chemistry targeting metalloenzymes. When metalloenzymes are inhibited in Gram-negative bacteria, the bacteria die. “The reason why we don’t have drugs against these targets is because of chemistry limitations,” Zimmerman explained. “These are chemistry problems that Forge’s platform can fix.”

Forge’s lead target is LpxC, a protein responsible for forming Gram-negative bacteria’s outer membrane, without which the microorganisms would become compromised and die. Though LpxC has been in the crosshairs of the pharma industry for more than two decades, traditional chemistry (such as hydroxamate-based inhibitors) has resulted in candidates with poor pharmacokinetics, poor drug-like properties, and even toxicity.

Forge’s first-ever nonhydroxamate inhibitor of LpxC, FG-LpxC-UTI, is focused on urinary tract infections (UTIs) caused by Escherichia coli and Klebsiella pneumonia. “This is a very large, unmet need,” Zimmerman noted. “New drugs against UTIs have been developed, but they’re only intravenously administered.” Forge’s drug candidate will be both intravenous, for hospital stays, and oral, for outpatient treatment. Forge is currently nominating a development candidate and moving into investigational new drug (IND)-enabling studies. It plans to file an IND and start Phase I studies next year. It is also looking beyond UTIs. Like EnBiotix, Forge is aiming at lung infections in cystic fibrosis patients. The company has modified its drug candidate target a different Gram-negative bacterium, Pseudomonas, which is often the cause of these infections.

Double shot

Inspired by cancer immunotherapies, Cidara Therapeutics developed Cloudbreak, an antibody-drug conjugate platform that combines surface-acting antimicrobial agents with immune engagers in a single molecule. The immune system is targeted by stably fusing multiple copies of the antimicrobial agent to the Fc domain of the human IgG1 antibody. The antimicrobial agents are engineered to target conserved regions of the pathogen where mutations often incur major fitness costs. “By adding the synergistic immune-mediated killing mechanism to the direct action of the antimicrobial, we see enhanced activity that should also help minimize the probability of developing resistance, because you have these orthogonal killing mechanisms,” said Les Tari, PhD, Cidara’s senior vice president of research.

Cidara is also using Cloudbreak to develop an antiviral drug to fight seasonal and pandemic influenza, which kills over 600,000 people each year worldwide. Because the antiviral agent is conjugated to an Fc domain, the platform provides a dramatic improvement in half-life compared to conventional small-molecule approaches. Cidara has protected mice from lethal influenza infections for a month with a single low dose of antiviral Fc conjugates.

“Also, because of the way we’re targeting the virus, we’re going to cover strains that are missed by the vaccine and will have broad spectrum activity that covers influenza A and B,” Tari asserted. He added that Cidara has selected a clinical development antiviral candidate based on preclinical data demonstrating potent antiviral activity against both influenza A and B viruses, and that the company is progressing IND-enabling studies this year.

Triangulating causal links

“The standard approach to fight bacterial infection for the last 75 or so years has been to mine natural products,” said Neil Surana, MD, PhD, an assistant professor of pediatrics at the Duke University School of Medicine. “In contrast, we’ve been trying to identify bacterial products that induce endogenous host defenses to either prevent and/or treat infectious diseases in a pathogen-agnostic manner.”

One of the more successful implementations of this approach is the use of fecal microbiota transplantations to improve colonization resistance in Clostridium difficile infections. According to Surana, the difficulty in extending this approach to novel bacterial products has been a lack of causal linkages between microbes and host phenotypes.

To tease out such causal relationships, Surana and colleagues have developed a discovery platform that he calls microbe-phenotype triangulation, or MPT. They use MPT to compare the gut microbiomes of several groups of mice harboring different populations of intestinal bacteria. In one study, the researchers found that mice harboring human microbes were protected against intestinal inflammation, while mice with typical mouse bacteria developed severe symptoms. To “triangulate” the suspect’s identity, Surana and his team looked for microbes that were either scarce or abundant across mice with varying colitis severity.

Surana and his group have also recently used MPT to identify two different conventional bacteria that induce host expression of an antimicrobial peptide called Reg3 gamma, which is expressed in the small intestine, as well as multiple other mucosal surfaces. The researchers are currently inducing Reg3 gamma expression in an attempt to prevent infection by antimicrobial-resistant organisms such as entercocci, C. difficile, or methicillin-resistant Staphylococcus aureus.

A new platform

“We’ve been working with a single reliable platform—screening Streptomyces for antibiotics—for over half a century, with mostly just ad hoc discoveries,” said Kim Lewis, PhD, a molecular microbiologist at Northeastern University. “What we need is a new platform for antibiotic discovery.”

Lewis noted that there are two general possibilities for a new platform: natural products and synthetic compounds. Lewis, along with Northeastern colleague Slava Epstein, PhD, formed the Cambridge-based company Novobiotics to explore the former. The two have developed a thumb drive–sized device called the iChip to overcome a stubborn problem. Of the untold billions of bacterial species in nature, only 1% will grow in the lab. iChip grows and cultures bacteria within a natural environment. A soil sample from the environment is sandwiched between two semipermeable membranes, and then, a short time later, the sample is returned to the environment. “Everything diffuses through this chamber, and of course, bacteria grow because they’re tricked,” Lewis explained.

So far, the pair has used iChip to identify around 80,000 previously uncultured bacterial strains and to isolate three dozen encouraging compounds. Two of these have proven to be credible leads, including Teixobactin, which is particularly remarkable because it’s the first antibiotic that is essentially free of resistance development. “This was a surprise, for sure,” Lewis said. “We’re just so used to the standard paradigm that bacteria are always going to develop resistance at some point.”

Teixobactin blocks several different targets in the cell wall synthesis pathway of Gram-negative bacteria. Four years after this discovery, no evidence has yet arisen suggesting that bacteria have developed resistance. Teixobactin is now in formal IND-enabling studies. Lewis anticipates it will enter Phase I trials in late 2020.