Ribosome-targeting antibiotics and resistance via ribosomal RNA methylation

Abstract

The rise of multidrug-resistant bacterial infections is a cause of global concern. There is an urgent need to both revitalize antibacterial agents that are ineffective due to resistance while concurrently developing new antibiotics with novel targets and mechanisms of action. Pathogen associated resistance-conferring ribosomal RNA (rRNA) methyltransferases are a growing threat that, as a group, collectively render a total of seven clinically-relevant ribosome-targeting antibiotic classes ineffective. Increasing frequency of identification and their growing prevalence relative to other resistance mechanisms suggests that these resistance determinants are rapidly spreading among human pathogens and could contribute significantly to the increased likelihood of a post-antibiotic era. Herein, with a view toward stimulating future studies to counter the effects of these rRNA methyltransferases, we summarize their prevalence, the fitness cost(s) to bacteria of their acquisition and expression, and current efforts toward targeting clinically relevant enzymes of this class.

Fig. 1. Approaches for discovery of antibiotics with novel mechanisms of action and improving activity of existing drugs. (A) The odilorhabdin (NOSO-95C) and darobactin were both isolated from nematode symbionts. Search for biosynthetic gene clusters similar to that of darobactin led to another inhibitor, dynobactin A. (B) Non-traditional culturing methods (e.g. iChip) have also enabled the culturing of bacterial species with currently untapped natural product repositories. (C) Implementation of physicochemical guidelines (eNTRY rules) has expanded the spectrum of originally Gram-positive-only antibiotics. Created with https://BioRender.com.

Fig. 2. Aminoglycoside interaction networks with h44 residues in the decoding center. (A) The 4,6-DOS aminoglycoside amikacin (PDB: 6YPU) and (B) 4,5-DOS aminoglycoside paromomycin (PDB: 7K00) bind in the A site on the 30S ribosomal subunit, and they both interact directly with conserved decoding nucleotides (A1492 and A1493) and the targets of the aminoglycoside-resistance 16S rRNA methyltransferases (G1405 and A1408). Interaction with G1405 is different for the two DOS aminoglycoside groups, whereas A1408 is close to the neamine core for both. For clarity, only the noted residues of interest and their direct interactions (dashed lines) with the aminoglycosides (yellow) are shown. All structure images were prepared using PyMol.

Fig. 3. Interactions of antibiotics with the 50S ribosome. (A) The macrolide desosamine ring directly interacts with A2058 (N1) and forms water-bridged interactions with the indicated NPET residues (PDB: 6XHX). A2058 (N6) is the target site of macrolide-resistance 23S rRNA methyltransferases. For clarity, only residues of interest are shown; erythromycin is shown as yellow sticks. (B) Chemical structure of the 16-member macrolide tylosin. (C) Chemical structures of other PTC targeting antibiotics noted in the main text. (D) Chemical structure of streptogramin antibiotics. (E) Superposition of PTC binding antibiotics clindamycin (blue, PDB: 1JZX), chloramphenicol (yellow, PDB: 1K01), tiamulin (grey, PDB: 1XBP), linezolid (orange, PDB: 3CPW) and dalfopristin (light blue, PDB: 1SM1) from the respective ribosome-antibiotic cocrystal structures.

Fig. 4. Mechanisms of resistance against ribosome-targeting antibiotics. (A) To prevent antibiotic entry, drug-resistant bacteria can alter the expression level or pore structure of porins. (B) The expression of efflux pumps facilitates drug extrusion from the cell thereby reducing intracellular drug concentrations. (C) As antibiotics reach the cytoplasm, they may be deactivated by drug-modifying enzymes or (D) degraded in their native form or after modification to enhance degradation. (E) Through the use of ribosome protection proteins (RPP), bacteria can resist inhibition by dislodging antibiotics from their ribosomal binding site. Resistant bacteria can also alter the drug target site via (F) mutation of residues critical for drug-binding or (G) chemical modification of target residues. Drug or target site chemical modification reduces binding affinity due to steric clash and/or electrostatic repulsion. Created with https://BioRender.com.

Fig. 5. Methyl transfer mechanisms and modified residues. (A) In the S_N2 mechanism, the nucleophilic nitrogen from the nucleotide base attacks the electrophilic methyl group of a positively charged SAM. (B) Radical mechanism mediated by an iron–sulfur cluster that reductively cleaves the second SAM (SAM2) to methionine and a 5′-dA radical. The radical abstracts hydrogen from a methylcysteine intermediate formed via an S_N2 mechanism between SAM1 and a cysteine residue. The resulting radical then reacts with the target adenine base and the conserved active site disulfide bridge reforms to complete the transfer followed by an enamine/imine tautomerization mediated by the undefined base B- and acid/conjugate base HB. (C) Select structures of methylated rRNA residues: m¹A, m⁷G, m⁶₂G, and m²m⁶A.

Fig. 6. Phylogenetic and structural comparison of antibiotic-resistance methyltransferase families. (A) Neighbor joining phylogenetic tree created with 100 steps of bootstrapping (left) and views of the RmtB structure (PDB 3FRH) as an example of the m⁷G1405 methyltransferase family. Structure views (left to right) highlight: (i) the seven β-strand core with the SAM binding pocket, (ii) appended region with critical determinants for rRNA interaction, and (iii) the same orientation but shown as an electrostatic surface. (B) and (C) As for panel A, but for the aminoglycoside-resistance m¹A1408 (with example of NpmA; PDB 3MTE) and macrolide-resistance Erm (with example structure of ErmC′; PDB 1QAO) and related methyltransferases, respectively. (D) Phylogentic analysis of Cfr and the related RlmN enzymes. Example RlnM structure (4PL1) highlights (left to right): (i) the radical SAM core fold with bound SAM and [Fe4–S4] cluster, (ii) appended N-terminal domain, and (iii) the same orientation but shown as an electrostatic surface. Species abbreviations: Ba, Bacillus anthracis; Bc, Bacillus clausii; Bf, Bacteroides fragillis; Bs, Bacillus subtilis; Ca, Catenulisporales acidiphilia; Cb, Clostridium botulinum; Cd, Clostridioides difficile; Ec, Escherichia coli; Ef, Enterococcus faecium; Ka, Klebsiella aerogenes; Kp, Klebsiella pneumoniae; Mb, Mycobacterium bovis; Mg, Micromonospora griseorubida; Mt, Mycobacterium tuberculosis; Mz, Micromonospora zionesis; Pa, Pseudomonas aeruginosa; Ps, Pseudomonas stutzeri; Sa, Staphylococcus aureus; Sc, Sorangium cellulosum So ce56; Sf, Streptomyces fradiae; Sk, Streptomyces kanamyceticus; Sm, Saccharothrix mutabilis subsp. capreolus; Sp, Streptococcus pyogenes; St, Streptoalloteichus tenebrarius.

Fig. 7. Base flipping positions A1408 for N1-methylation by NpmA. During methylation, A1408 is flipped from its helix by NpmA and the adenosine nucleotide is stabilized by π–π stacking interactions with two tryptophan residues. The backbone carbonyl of F105 forms a hydrogen bond (red dashed line) with N6 of A1408, positioning the N1 for methylation (PDB: 4OX9). Structure image was prepared using PyMol.

Fig. 8. Factors that dictate the fitness cost of bacteria hosting resistance rRNA methyltransferase genes. (A) Locations of E. coli housekeeping methylations in h44 impacted by adjacent resistance modifications at G1405 and A1408. (B) The exiting nascent peptide does not interact with either A2062 or A2503 when the tunnel is clear. The A2503 base changes conformation to relay nascent peptide information from the NPET to the PTC when the channel is blocked by erythromycin (Ery). (C) Ribosome-erythromycin-ErmCL complex stalling at ermCL triggers the mRNA to release a second ribosome binding sequence (SD2). Created with https://BioRender.com.

Fig. 9. Antibiotic analogs that are active against bacteria with resistance rRNA methyltransferase genes. (A) Chemical structure of apramycin and 5-furanosyl appended apralogs. Studies have explored different substituents at R¹, R², and R³. (B) Chemical structure of iboxamycin and the structural basis for its binding in an Erm-modified ribosome (PDB: 7RQ9). (C) Chemical structures of tedizolid and radezolid and structure of radezolid (yellow) bound to Cfr-modified ribosome (PDB: 7S1K). Structure images were prepared using PyMol.

Fig. 10. Chemical structures of Erm methyltransferase inhibitors. Erm inhibitors identified using (A) high throughput screening (HTS), (B) NMR based screening followed by an extensive SAR campaign, (C) in silico docking on ErmC′ and in vitro assays using COMT, (D) virtual screening using ErmC′ 3D structure, and (E) virtual screening then SAR of the lead compound after in vitro studies.