Combating antimicrobial resistance
Antibiotics
Antibiotics are a mainstay of modern medicine, but drug-resistant pathogens are increasingly threatening their effectiveness. While preventing the misuse of antibiotics in humans and animals worldwide is necessary to combat what is now a very real but overlooked pandemic, there is also a need for new antibiotics that overcome existing resistance mechanisms.
Using structural biology and high-throughput functional characterization, we are revisiting the mechanisms of action of ribosome-targeting antibiotics, focusing on the defined functional states they target. In addition, we are reimagining antibiotic discovery as a process of directed evolution in which the bacterial ribosome is used both as a peptide production tool and as a target. We are particularly interested in molecules that target new sites on the ribosome, as they could be used as scaffolds to design drugs that bypass known resistance mechanisms.
Highlights
The ribosome is a major target for antibiotics
We believe that the ability of current antibiotics to treat drug-resistant infections can be improved through a structure-based approach. To do this, however, we must first know the detailed mechanisms of action of the drugs to be optimized.
The bacterial ribosome is a target for several major classes of antibiotics, including molecules that block peptide bond formation (chloramphenicol, oxazolidinones), impede the synthesis and movement of nascent proteins through the nascent polypeptide exit tunnel (macrolides), interfere with the decoding of messenger RNA (aminoglycosides) or prevent the translocation of tRNAs during the elongation step of protein synthesis (tuberactinomycins).​
Bacterial translation is the target for numerous classes of antibiotics (listed in red).
Like many antibiotics, ribosome-targeting drugs are derived from natural products extracted from soil microbes during the golden age of antibiotic discovery (1950s-1960s), and the search for improved molecules mainly involved screening large libraries of compounds on the basis of their minimum inhibitory concentration (MIC). In contrast, a structure-based optimization approach has the potential to limit the number of compounds to be screened. Many ribosome-targeting antibiotics, however, are poor candidates for a structure-based approach due to incompletely understood mechanisms of action.
iTP-Seq determination of the context specificity of the antibiotic Tetracenomycin X (TcmX), and cryo-EM structure of an E. coli 70S ribosome stalled during translation of a problematic QK motif in the presence of TcmX (PDB 7ZTA).
Context-dependent translation inhibition
Our limited understanding of these mechanisms has become particularly evident in recent years with the realization that translation inhibition can be context-dependent.
Indeed, translating ribosomes encounter a variety of amino acids, tRNA and mRNA molecules, each with their own chemical and physical properties, such that the molecular context in which translation takes place changes as the ribosome progresses along an mRNA. We now know that an increasing number of antibiotics block translation in a manner dependent on the composition of the translational complex, and modern structural and biochemical approaches allow us to determine the molecular bases underlying this context dependence. A classical example is erythromycin, whose general mechanism of action we characterized as part of a collaborative study of the ermD leader peptide (Beckert et al. (2021) Nat Commun).
Another antibiotic that was suspected of inhibiting translation in a context-dependent manner is the aromatic polyketide antibiotic Tetracenomycin X (TcmX). Using iTP-seq to assess the ribosome stalling potential of a vast collection of amino acid sequences in response to TcmX, we discovered that this antibiotic preferentially blocks the translation of peptides harboring a Gln-Lys (QK) motif (Leroy et al. (2023) Nat Chem Biol). Using high-resolution cryo-EM, we could show that TcmX inhibits translation at QK motifs through an unusual mechanism involving the sequestration of the 3’ end of peptidyl-tRNA into the nascent polypeptide exit tunnel. By revealing the mechanism of action of TcmX, our work suggested a path forward for making improved derivatives of this drug with greater affinity and specificity for the bacterial ribosome.
​We continue to systematically assess the context dependence of ribosome-targeting antibiotics to obtain the detailed mechanistic insights needed for structure-based drug development.
Are peptides the future of antibiotics?
While current antibiotic development tends to focus on improving existing compounds or mining the natural world for antimicrobials, we propose a different approach, inspired by the observation that many existing ribosome-targeting antibiotics are in fact peptides. Peptides offer unique advantages over conventional small molecule drugs: they can be genetically encoded and produced by the ribosome, are easily modifiable, and represent a vast but under-exploited chemical space for antimicrobial discovery.
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In order to prioritize the discovery of new classes of antibiotics that avoid cross-resistance to known drugs, we are rethinking the antibiotic discovery process as a directed evolution challenge, in which vast libraries of ribosome-produced peptides are screened for antimicrobial activity, enabling high-throughput identification of new compounds with therapeutic potential.
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Stay tuned as we move closer to this goal!