Marcos Pires

Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

by Nisha Mahey, Rushikesh Tambat, Ritu Kalia, Rajnita Ingavale, Akriti Kodesia, Nishtha Chandal, Srajan Kapoor, Dipesh Kumar Verma, Krishan Gopal Thakur, Sanjay Jachak, Hemraj Nandanwar

Efflux pumps of the resistance-nodulation-cell division (RND) superfamily, particularly the AcrAB-TolC, and MexAB-OprM, besides mediating intrinsic and acquired resistance, also intervene in bacterial pathogenicity. Inhibitors of such pumps could restore the activities of antibiotics and curb bacterial virulence. Here, we identify pyrrole-based compounds that boost antibiotic activity in Escherichia coli and Pseudomonas aeruginosa by inhibiting their archetype RND transporters. Molecular docking and biophysical studies revealed that the EPIs bind to AcrB. The identified efflux pump inhibitors (EPIs) inhibit the efflux of fluorescent probes, attenuate persister formation, extend post-antibiotic effect, and diminish resistant mutant development. The bacterial membranes remained intact upon exposure to the EPIs. EPIs also possess an anti-pathogenic potential and attenuate P. aeruginosa virulence in vivo. The intracellular invasion of E. coli and P. aeruginosa inside the macrophages was hampered upon treatment with the lead EPI. The excellent efficacy of the EPI-antibiotic combination was evidenced in animal lung infection and sepsis protection models. These findings indicate that EPIs discovered herein with negligible toxicity are potential antibiotic adjuvants to address life-threatening Gram-negative bacterial infections.

Nature, Published online: 03 January 2024; doi:10.1038/d41586-023-03988-2

Infections caused by drug-resistant strains of the bacterium Acinetobacter baumannii have been hard to treat in the clinic. A new class of antibiotics has been identified with the potential to tackle these microbes.

Nature, Published online: 03 January 2024; doi:10.1038/s41586-023-06873-0

A tethered macrocyclic peptide antibiotic class described here—which shows potent antibacterial activity against carbapenem-resistant Acinetobacter baumannii—blocks the transport of bacterial lipopolysaccharide from the inner membrane to its destination on the outer membrane through inhibition of the LptB2FGC complex.

Nature, Published online: 03 January 2024; doi:10.1038/s41586-023-06799-7

A mechanism of lipid transport inhibition has been identified for a class of peptide antibiotics effective against resistant Acinetobacter strains, which may have applications in the inhibition of other Gram-negative pathogens.

Nature Chemical Biology, Published online: 28 December 2023; doi:10.1038/s41589-023-01496-y

Cyclic peptides show promise for modulating difficult disease targets; however, they often cannot be administered orally. The authors developed a method to synthesize and screen large libraries of small cyclic peptides while enabling the simultaneous interrogation of activity and permeability. This approach was applied to the disease target thrombin to discover peptides with high affinity, stability and oral bioavailability of up to 18% in rats.

Nature Chemical Biology, Published online: 07 December 2023; doi:10.1038/s41589-023-01495-z

Macrocyclic peptides are promising scaffolds for chemical tools and potential therapeutics, but their synthesis is currently difficult. Here, the authors report the characterization of Ulm16, a peptide cyclase of the penicillin-binding protein (PBP)-type class of thioesterases, that catalyzes head-to-tail macrolactamization of nonribosmal peptides of 4–6 amino acids in length.

Staphylococcus aureus can persist within host immune cells, potentially evading immune responses and antibiotics. Investigating antibiotic permeability into phagocytic vacuoles, we developed a permeability assay. By analyzing antibiotic arrival in phagosomes of infected macrophages, we identified permeability differences, offering insights into antibiotic contribution to intracellular pathogens.

Abstract

Staphylococcus aureus (S. aureus) has evolved the ability to persist after uptake into host immune cells. This intracellular niche enables S. aureus to potentially escape host immune responses and survive the lethal actions of antibiotics. While the elevated tolerance of S. aureus to small-molecule antibiotics is likely to be multifactorial, we pose that there may be contributions related to permeation of antibiotics into phagocytic vacuoles, which would require translocation across two mammalian bilayers. To empirically test this, we adapted our recently developed permeability assay to determine the accumulation of FDA-approved antibiotics into phagocytic vacuoles of live macrophages. Bioorthogonal reactive handles were metabolically anchored within the surface of S. aureus, and complementary tags were chemically added to antibiotics. Following phagocytosis of tagged S. aureus cells, we were able to specifically analyze the arrival of antibiotics within the phagosomes of infected macrophages. Our findings enabled the determination of permeability differences between extra- and intracellular S. aureus, thus providing a roadmap to dissect the contribution of antibiotic permeability to intracellular pathogens.

Nature, Published online: 22 November 2023; doi:10.1038/s41586-023-06760-8

We use a whole-cell accumulation assay to assess the ability of non-antibiotic, structurally diverse small molecules to accumulate in Pseudomonas aeruginosa, with potential application in developing drugs to target this pathogen.

Nature Immunology, Published online: 12 October 2023; doi:10.1038/s41590-023-01645-4

Here, the authors show that deletion of Pglyrp1 promotes antitumor immunity owing to its inhibitory function in CD8+ T cells and that targeting it can inhibit development of autoimmune neuroinflammation. These findings indicate that PGLYRP1 might be a target for immunotherapy.

Clovibactin, a new antibiotic isolated from soil bacteria, blocks bacterial cell wall synthesis by targeting essential peptidoglycan precursors, allowing it to kill drug-resistant bacterial pathogens without detectable resistance.

Proceedings of the National Academy of Sciences, Volume 120, Issue 25, June 2023.

Nature, Published online: 17 May 2023; doi:10.1038/s41586-023-06081-w

Tumour-infiltrating lymphocytes from glioblastoma can recognize bacterial and gut microbial peptides.

Papadopoulou et al. target the two main barriers to successful antibiotic chemotherapy against biofilms: poor drug penetration and persister cells. Utilizing sonobactericide to improve penetration of an anti-persister drug combination greatly enhanced therapeutic clearance in a diabetic wound infection model.

Homo-dimeric BacPROTACs induce the self-degradation of essential Clp components of the mycobacterial proteostasis system, introducing a potent antibiotic strategy against M. tuberculosis.

A commensal bacterium exploits phase separation of a transcription termination factor to colonize the gut.