New antibiotics with a dual strategy against multidrug-resistant bacteria

By Austėja Čiulkinytė

During bacterial infection, innate immune cells ingest the bacterial cell, digest it and present pieces of the bacterium on their surface. This triggers adaptive immune cells, including T cells, to kill any remaining bacteria. To speed up this process and to prevent the immune system from getting overwhelmed, antibiotics are commonly used to directly suppress or kill invading bacteria.

However, bacteria of the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa and Enterobacter species), as well as the tuberculosis pathogen Mycobacterium tuberculosis and malaria parasite Plasmodium falciparum, can evade being captured and digested by innate immune cells. As a result, these pathogens cause severe infections as well as harbouring resistance to common antibiotics, paving the way to a global health crisis.

Recently, Singh et al. (2020) described a novel IspH inhibitor class of antibiotics with a dual mechanism of action. The first mechanism targets the bacteria directly. It involves inhibition of IspH, an enzyme crucial for cell wall integrity, protein synthesis, and respiration. Inhibition of IspH causes its substrate, (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP), to build up. This molecule is recognised by a specific type of T cells triggering the second mechanism, which involves activation of the host’s immune system to clear infection.

Drug design

Since HMBPP must bind to IspH to be metabolised, a potential inhibitor of IspH would bind to the same site and compete with HMBPP. The authors used a crystal structure of IspH to model the binding site of HMBPP. They then fitted a library of over 9 million drug compounds to the same site and found 24 compounds with the best fit. In vitro studies of these compounds revealed three of them as potent inhibitors of IspH. Finally, multiple chemical analogues of each of these compounds were designed. Five of them were confirmed as the most potent inhibitors, two of which bind to IspH with greater affinity than the natural substrate HMBPP.

Because these compounds cannot enter bacteria, they were chemically linked to 6‑hydroxyhexyl triphenylphosphonium bromide (TPP), another compound that can easily pass into the bacterium. However, TPP exits the bacteria just as readily, therefore the chemical linker was designed so that it would be cleaved once inside the bacterium, releasing the original inhibitor compound. The final prodrug compounds were shown to be effective at killing bacteria in vitro, at lower drug concentrations than current best-in-class antibiotics.

Direct antibacterial activity and specificity

Bacteria treated with these prodrugs were observed to lose membrane and cell wall integrity, decrease respiration rate, release reactive oxygen radicals, and had a typical dying cell morphology. Additionally, in presence of the prodrugs, genes involved in electron transfer chain, protein, and lipid synthesis, were downregulated. All of these signify impaired cell wall synthesis, impaired respiration, and eventual cell death.

Previously, TPP has been associated with muscle cell toxicity, mitochondrial damage, and calcium channel blockage. Although the authors used a methylated version of TPP, they still tested the prodrugs and their individual components on isolated mammalian cells. No toxicity or mitochondrial damage was observed. In addition, these prodrugs were found to be less effective at binding calcium channels than a current calcium channel blocker medication. Overall, these results suggest that IspH inhibitors cause significant damage to bacteria but are safe to mammals.

Indirect activation of the immune system

Finally, authors investigated involvement of the adaptive immune system. They observed that in vitro, bacteria eventually develop resistance to the prodrug unless human immune cells are also present in culture. Meanwhile, in vivo the prodrugs are more effective in clearing bacteria and improving survival of mice when they have been injected with human γδ T cells. These particular T cells are activated by HMBPP, the substrate that accumulates when IspH is inhibited. In a final experiment, the authors found that treating mice with the prodrug, but not any of its components, nor a current best-in-class antibiotic, is able to clear multidrug-resistant Enterobacter aerogenes infection. This suggests a lot of potential for IspH inhibitors as treatment against severe bacterial infections.

Outlook

Overall, the prodrugs described in this paper offer a very clever and promising antibacterial strategy. They are more effective than current best-in-class antibiotics, specific to bacteria, and non-toxic to mammals. Their dual mechanism of action and lack of natural analogues reduces likelihood of resistance development.

However, more studies are needed to investigate other potential mechanisms of resistance. In addition, other immune cells besides γδ T cells may be involved in the immune activating mechanism of these drugs. The authors of this paper focused on the entire class of inhibitors, but no single compound is proposed as a potential new antibiotic. Therefore, there is still a lot of developmental potential in this area.

References

Singh, K.S., Sharma, R., Reddy, P.A.N. et al. IspH inhibitors kill Gram-negative bacteria and mobilize immune clearance. Nature 589, 597–602 (2021). https://doi.org/10.1038/s41586-020-03074-x

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