Argyrin B

Inhibitors of the proteasome have found broad therapeutic applications; however, they show severe toxicity due to the abundance of proteasomes in healthy cells. In contrast, inhibitors of the immunoproteasome, which is upregulated during disease states, are less toxic and have increased therapeutic potential including against autoimmune disorders. In this project, we report argyrin B, a natural product cyclic peptide to be a reversible, non-competitive inhibitor of the immunoproteasome. Argyrin B showed selective inhibition of the β5i and β1i sites of the immunoproteasome over the β5c and β1c sites of the constitutive proteasome with nearly 20-fold selective inhibition of β1i over the homologous β1c. Molecular modelling attributes the β1i over β1c selectivity to the small hydrophobic S1 pocket of β1i and β5i over β5c to site-specific amino acid variations that enable additional bonding interactions and stabilization of the binding conformation. These findings facilitate the design of immunoproteasome selective and reversible inhibitors that may have a greater therapeutic potential and lower toxicity.

Argyrins are natural products with antibacterial activity against Gram-negative pathogens, such as Pseudomonas aeruginosa, Burkholderia multivorans, and Stenotrophomonas maltophilia We previously showed that argyrin B targets elongation factor G (FusA). Here, we show that argyrin B activity against P. aeruginosa PAO1 (MIC = 8 μg/ml) was not affected by deletion of the MexAB-OprM, MexXY-OprM, MexCD-OprJ, or MexEF-OprN efflux pump. However, argyrin B induced expression of MexXY, causing slight but reproducible antagonism with the MexXY substrate antibiotic ciprofloxacin. Argyrin B activity against Escherichia coli increased in a strain with nine tolC efflux pump partner genes deleted. Complementation experiments showed that argyrin was effluxed by AcrAB, AcrEF, and MdtFX. Argyrin B was inactive against Acinetobacter baumannii Differences between A. baumannii and P. aeruginosa FusA proteins at key residues for argyrin B interaction implied that natural target sequence variation impacted antibacterial activity. Consistent with this, expression of the sensitive P. aeruginosa FusA1 protein in A. baumannii conferred argyrin susceptibility, whereas resistant variants did not. Argyrin B was active against S. maltophilia (MIC = 4 μg/ml). Spontaneous resistance occurred at high frequency in the bacterium (circa 10-7), mediated by mutational inactivation of fusA1 rather than by amino acid substitutions in the target binding region. This strongly suggested that resistance occurred at high frequency through loss of the sensitive FusA1, leaving an alternate argyrin-insensitive elongation factor. Supporting this, an additional fusA-like gene (fusA2) is present in S. maltophilia that was strongly upregulated in response to mutational loss of fusA1.

Argyrins are a family of naturally produced octapeptides that display promising antimicrobial activity against Pseudomonas aeruginosa. Argyrin B (ArgB) has been shown to interact with an elongated form of the translation elongation factor G (EF-G), leading to the suggestion that argyrins inhibit protein synthesis by interfering with EF-G binding to the ribosome. Here, using a combination of cryo-electron microscopy (cryo-EM) and single-molecule fluorescence resonance energy transfer (smFRET), we demonstrate that rather than interfering with ribosome binding, ArgB rapidly and specifically binds EF-G on the ribosome to inhibit intermediate steps of the translocation mechanism. Our data support that ArgB inhibits conformational changes within EF-G after GTP hydrolysis required for translocation and factor dissociation, analogous to the mechanism of fusidic acid, a chemically distinct antibiotic that binds a different region of EF-G. These findings shed light on the mechanism of action of the argyrin-class antibiotics on protein synthesis as well as the nature and importance of rate-limiting, intramolecular conformational events within the EF-G-bound ribosome during late-steps of translocation.