Disarming Gram-Negative Bacteria in the Fight Against Antimicrobial Resistance

space in their study. The target of this work, LasB, is an example of a virulence factor, which are enzymes associated with microbe “infectivity” and inflicting damage on the host. Inhibiting virulence factors hinders the ability of the bacterium to infect the host, effectively “disarming” it, rendering it unable to establish an infection or to cause damage to the host. However, this inhibition does not kill the pathogen, and consequently less selection pressure is placed upon the pathogen to develop resistance against drugs targeting these proteins.

I n this issue of ACS Central Science, Hirsch and co-workers report an innovative strategy to treat infections caused by the Gram-negative bacterium Pseudomonas aeruginosa by targeting its main virulence factor, LasB. 1 P. aeruginosa is a multidrug resistant opportunistic bacterium that is listed among the World Health Organization's critical group of pathogens for which new antibiotics are urgently required. 2Antimicrobial resistance (AMR) is a significant and increasing threat to global public health that was associated with 4.95 million deaths in 2019. 3,4This figure could rise to over 10 million deaths per year by 2050 unless immediate action is taken. 5AMR has been exacerbated by the overuse and misuse of antibiotics, and new strategies for treating infections are being sought that will minimize the development of resistance.
P. aeruginosa causes either acute or chronic infection in people who are immunocompromised, including those with chronic obstructive pulmonary disease (COPD), cystic fibrosis, and cancer. 6As P. aeruginosa is a Gram-negative pathogen, it has an outer membrane, in addition to an inner cell wall.This makes targeting proteins in Gram-negative pathogens especially challenging, as drugs that function by interacting with targets inside the bacterium must first pass through the outer membrane and cell wall before they can become effective.To achieve this, medicinal chemists must develop drug molecules that possess an appropriate balance of lipophilicity, hydrophobicity, molecular weight, and stability.This challenge is one of the reasons that there is a lack of drugs to treat multidrug resistant P. aeruginosa-derived infections.
To sidestep this issue, Hirsch and co-workers have elected to target LasB, which is an extracellular enzyme, bypassing the need for the drugs to enter the bacterium.By removing the need for the compounds to enter the P. aeruginosa, the authors were free to explore a wider range of chemical space in their study.The target of this work, LasB, is an example of a virulence factor, which are enzymes associated with microbe "infectivity" and inflicting damage on the host.Inhibiting virulence factors hinders the ability of the bacterium to infect the host, effectively "disarming" it, rendering it unable to establish an infection or to cause damage to the host.However, this inhibition does not kill the pathogen, and consequently less selection pressure is placed upon the pathogen to develop resistance against drugs targeting these proteins.

Published: December 13, 2023
Inhibiting virulence factors hinders the ability of the bacterium to infect the host, effectively "disarming" it, rendering it unable to establish an infection or to cause damage to the host.However, this inhibition does not kill the pathogen, and consequently less selection pressure is placed upon the pathogen to develop resistance against drugs targeting these proteins.

FIRST REACTIONS
Targeting the Pseudomonas aeruginosa virulence factor LasB is an innovative strategy to treat infections caused by this Gram-negative bacterium while minimizing resistance developing.

Glen Brodie and Stuart J. Conway*
LasB is a Zn 2+ -dependent metalloprotease, and consequently its inhibitors contain a zinc-binding group (ZBG).The nature of this group can present challenges in terms of selectivity over other Zn 2+ -dependent enzymes and the physicochemical properties of the ZBG itself. 7Hirsch and co-workers had previously developed the thiol-based LasB inhibitor, 1 (Figure 1A). 8While this compound shows reasonably high potency against LasB, the low chemical stability of the thiol group means that this compound is not suitable for development as a therapeutic agent.Analysis of alternative ZBGs identified the hydroxamic acid 3g and the phosphonate derivatives 4b, 4h, and 4k, as highly potent LasB inhibitors.
To determine the optimum ZBG for further studies, ADMET (Absorption, Distribution, Metabolism, Excretion, and Toxicity) profiling was used to compare the phosphonates and hydroxamic acid.A key criterion was permeability across a Calu-3 monolayer, and unusually, the aim was to identify compounds that have low permeability in the Calu-3 assay, as this corresponds with high retention in the lung.This low permeability minimizes systemic circulation of the drug and is an important feature of anti-pseudomonal drugs targeting the lung.The authors noted that there was a strong correlation between measured permeabilities and chromatographic lipophilicities, with the hydroxamic acid 3g showing higher permeability than the phosphonate 4b.The hydroxamic acid has a higher logD 7.4 value than the phosphonate, which likely results from the differing pK a values of these motifs.Hydroxamic acids typically have a pK a value of around 9.3, the calculated value for 3g is 9.28, and so they are not substantially deprotonated at physiological pH. 9 Conversely, the pK a value of the first phosphonate deprotonation is typically 2.4, the calculated value for 4b is 1.60, and the pK a value of the second deprotonation is approximately 7.5, the calculated value for 4b is 8.12, and so phosphonates are at least monoanionic at pH 7.4 (Figure 1B). 10 In addition, the hydroxamic acid 3g caused some growth inhibition of the human A549 lung adenocarcinoma cell line through offtarget activity.Based on these data, the phosphonates were progressed for further studies.The authors obtained an X-ray crystal structure of compound 4b bound to LasB  (Figure 2; PDB ID 8CC4), demonstrating that the phosphonate acts as the ZBG, as expected.Overlaying the structure with that of an analog of compound 1 bound to LasB (Figure 2; PDB ID: 7OC7) shows that both compounds bind in a similar manner.Interestingly, the (R)-enantiomer of each compound is observed in the structures, suggesting that this enantiomer binds to LasB in preference to its antipode.While it is likely that the individual enantiomers of the phosphonates would not be stable in cells, addition of a fluoride or other nonproton group at the stereogenic center might enable separation and biological evaluation of the stereoisomers.
Studies on the phosphonates in 2D A549 cells, and 3D lung organoids, treated with P. aeruginosa showed that the cell viability was retained even at low concentrations of the compounds.These studies also demonstrated that the mechanism of action of these compounds derives predominantly from inhibition of LasB.Work in the Galleria mellonella larvae infection model demonstrated that phosphonate 4b does not have any antibacterial action itself, which is important to help avoid resistance developing, but significantly enhanced the action of the antibiotic tobramycin.Following pharmacokinetic studies in mice, a combination of the antibiotic levofloxacin and 4b was shown to significantly reduce the number of colony-forming units in mice lungs treated with P. aeruginosa DSM-1117.
Overall, this is a very interesting study that provides compelling evidence for targeting the LasB virulence factor as a method to aiding P. aeruginosa treatment.The extracellular location of this protein makes it an attractive target in this Gram-negative bacterium, where permeability is often a key challenge in antibiotic drug development.The use of phosphonate groups in drugs is often hampered by their poor permeability, and here Hirsch and co-workers elegantly use this characteristic to their advantage, minimizing the systemic exposure of the compounds and retaining it at the key site of action in the lung.The lead compounds reported here look well-suited for further development toward inhaled drugs as treatment options for infections in cystic fibrosis and non-cystic fibrosis bronchiectasis patients.
The use of phosphonate groups in drugs is often hampered by their poor permeability, and here Hirsch and co-workers elegantly use this characteristic to their advantage, minimizing the systemic exposure of the compounds and retaining it at the key site of action in the lung.
A key criterion was permeability across a Calu-3 monolayer, and unusually, the aim was to identify compounds that have low permeability in the Calu-3 assay, as this corresponds with high retention in the lung.

Figure 1 .
Figure 1.(A) The evaluation of the zinc-binding group (ZBG) to identify potent LasB inhibitors.(B) The pK a and logD 7.4 values for compounds 3g and 4b.The pK a values were calculated using Chemicalize.The logD 7.4 values are taken from Konstantinovićet al. 1