Publisher’s summary Lipid A biosynthesis is essential for the formation of the outer membrane of most Gram-negative bacteria and has been considered a potential target for antibiotic therapy. Zhao et al. have now characterized an inhibitor (LPC-233) of the UDP-3- O -( R -3-hydroxyacyl)- N -acetylglucosamine deacetylase LpxC, which can specifically inhibit lipid A synthesis. Previous attempts to make antibiotics targeting LpxC were limited by unfavorable cardiovascular toxicity. In contrast, preclinical evaluation of LPC-233 revealed promising safety profiles in vitro and in vivo , tight binding to LpxC with picomolar affinity, oral bioavailability, and bactericidal activity against a wide variety of Gram-negative pathogens. These results support the further development of antibiotic therapies targeting LpxC. —Christiana Fogg |
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A decades-long scientific journey at Duke University has found a new antibiotic strategy to defeat gram-negative bacteria such as Salmonella, Pseudomonas and E. coli, the culprits of many urinary tract infections (UTIs). The synthetic molecule works fast and is durable in animal tests.
It works by interfering with a bacteria’s ability to produce its outer lipid layer.
"If you disrupt the synthesis of the bacterial outer membrane, the bacteria can’t survive without it," said lead researcher Pei Zhou, a professor of biochemistry at Duke Medical School. "Our compound is very good and very powerful."
The compound, called LPC-233, is a small molecule that has been shown to be effective in destroying outer membrane lipid biosynthesis in all of the gram-negative bacteria it was tested against. Co-authors from the University of Lille in France tested it against a collection of 285 bacterial strains, including some that were highly resistant to commercial antibiotics, and it was effective. And it works fast. "LPC-233 can reduce bacterial viability by 100,000 times in four hours," Zhou said.
The compound is also tenacious enough to survive into the urinary tract after oral administration, which may make it a vital tool against persistent urinary tract infections (UTIs).
Tests conducted at high concentrations of the compound showed "extremely low rates of spontaneous resistance mutations in these bacteria," according to a paper describing the findings, appearing August 9 in Science Translational Medicine .
In animal studies , the compound was successful when administered orally and intravenously or injected into the abdomen. In one experiment, mice given what should have been a fatal dose of multidrug-resistant bacteria were rescued by the new compound.
The search for this compound took decades due to the specificity and safety required of the synthetic molecule.
Zhou credits his late colleague, former Duke biochemistry chair Christian Raetz, for starting the search decades ago. "He spent his entire career working on this path," Zhou said. "Dr. Raetz proposed a conceptual model for this pathway in the 1980s, and it took him more than two decades to identify all the players," Zhou said.
The target of the new drug is an enzyme called LpxC, which is the second enzyme in the "Raetz pathway" and is essential for producing lipids in the outer membrane of gram-negative bacteria.
Raetz joined Duke as chair of biochemistry in 1993 after his work in this pathway at Merck & Co. failed to produce a successful clinical candidate. Merck’s antibiotic worked, but only against E. coli, so it was not commercially viable and the pharmaceutical company abandoned it. "He actually recruited me to Duke to work on this enzyme, initially just from a structural biology perspective," said Zhou, who came to Duke in 2001.
Zhou and Raetz had solved the structure of the LpxC enzyme and revealed molecular details of some potential inhibitors. "We realized we could modify the compound to make it better," Zhou said. Since then, Zhou has been working with his colleague, Duke chemistry professor Eric Toone, to make more potent LpxC inhibitors.
The first human trial of LpxC inhibitors failed due to cardiovascular toxicity. The focus of the Duke group’s subsequent work was to avoid cardiovascular effects while maintaining the potency of the compound.
They worked on more than 200 different versions of the enzyme inhibitor, always seeking greater safety and more potency. Other compounds worked to varying degrees, but compound number 233 was the winner.
LPC-233 fits into a binding site on the LpxC enzyme and prevents it from doing its job. "It’s tuned in the right way to inhibit lipid formation," Zhou said. "We are blocking the system." In addition to its durability, the composite works through a remarkable two-step process, Zhou said. After initial binding to LpxC, the enzyme-inhibitor complex changes its shape somewhat to become an even more stable complex.
The inhibitor binding lifetime in this more stable complex is longer than the lifetime of the bacteria. "We think that contributes to the potency because it has a semi-permanent effect on the enzyme," he said. "Even after the body metabolizes the unbound drug, the enzyme is still inhibited due to the extremely slow inhibitor dissociation process," Zhou said.
Multiple patents are being filed on the series of compounds, and Toone and Zhou have co-founded a company called Valanbio Therapeutics, Inc. that will seek partners to take LPC-233 through Phase 1 clinical trials to evaluate safety and efficacy in humans . . "All of these studies were done in animals ," Zhou said. "Ultimately, cardiovascular safety needs to be tested in humans."
The large-scale synthesis of LPC-233 was first performed by David Gooden at the Duke Small Molecule Synthesis Facility. Vance Fowler and Joshua Thaden (Duke Medical School), Ziqiang Guan (Biochemistry), and Ivan Spasojevic (Duke PK/PD Core) assisted with in vivo studies, mass spectrometry, and pharmacokinetic analyses.
This work was supported by grants from the National Institutes of Health (R01 GM115355, AI094475, AI152896, AI148366), the North Carolina Biotechnology Center (2016-TEG-1501), and a core grant from the National Institute’s Comprehensive Cancer Center. of Cancer (P30CA014236).
