Ribosome-Targeting Antibiotics: Modes of Action, Mechanisms of Resistance, and Implications for Drug Design

Abstract

Genetic information is translated into proteins by the ribosome. Structural studies of the ribosome and of its complexes with factors and inhibitors have provided invaluable information on the mechanism of protein synthesis. Ribosome inhibitors are among the most successful antimicrobial drugs and constitute more than half of all medicines used to treat infections. However, bacterial infections are becoming increasingly difficult to treat because the microbes have developed resistance to the most effective antibiotics, creating a major public health care threat. This has spurred a renewed interest in structure-function studies of protein synthesis inhibitors, and in few cases, compounds have been developed into potent therapeutic agents against drug-resistant pathogens. In this review, we describe the modes of action of many ribosome-targeting antibiotics, highlight the major resistance mechanisms developed by pathogenic bacteria, and discuss recent advances in structure-assisted design of new molecules.

Keywords: antibiotic resistance; antibiotics; drug design; mechanisms of inhibition; protein synthesis; ribosome.

Figure 1.. Overview of antibiotic binding sites on the 30S and 50S subunits.

(a, b) Major antibiotic binding sites on the 30S and 50S subunit, respectively. All binding sites are shown as red spheres of variable sizes with the larger ones indicating more drugs discovered at that site. The names of representative antibiotics (or classes) bound to each site are listed. (c) A detailed view of the decoding center (DC) on the 30S subunit. (d) A detailed view of the peptidyl transferase center (PTC) and the nascent peptide exit tunnel (NPET) on the 50S subunit. E. coli numbering is used throughout the text and figures. Abbreviations: A-tRNA, A-site tRNA; mRNA, messenger RNA; PrAMPs, proline-rich antimicrobial peptides; P-tRNA, P-site tRNA.

Figure 2.. Antibiotics interacting with the decoding center.

All drugs are shown as spheres with their chemical structures provided on the bottom of each panel. Major drug-interacting elements on the ribosome are indicated. (a) Tetracycline (PDB identification code 4V9B) (35) collides with the A-site tRNA, thereby preventing binding of aa-tRNA to the A site. (b) Negamycin (PDB code 4W2I) (36) binds to an overlapping position with that of tetracycline, but stabilizes the A-site tRNA and inhibits translocation. (c) Streptomycin (PDB code 4DR5) (37) binds outside h44 and interacts with h18, h27, and the ribosomal protein uS12. (d, e) Paromomycin (PDB code 4V51) (28) and gentamicin C1a (PDB code 4V53) (39) bind inside h44 and induce a flipped-out conformation of nucleotides A1492 and A1493, thereby increasing miscoding. Gentamicin has two binding sites on the ribosome: one involves h44 (BS1) and the other involves H69 (BS2). (f) Hygromycin B (PDB code 4V64) (38) binds to the top of h44, preventing tRNA translocation by interacting and stabilizing mRNA. (g) Viomycin (PDB code 4V7C) (41), a drug from tuberactinomycins class, interacts with both h44 and H69 and stabilizes a ratcheted state of the ribosome. (h) Spectinomycin (PDB code 1FJG) (25) binds to h34 located at the 30S neck and interferes with the dynamics of the 30S head domain. (i) Thermorubin (PDB code 4V8A) (44) binds to h44 and extends to H69, causing C1914 to flip out and to collide with the A-site tRNA.

Figure 3.. Interactions between antibiotics, the P and E sites on the 30S subunit and ribosomal protein uS12.

(a) Kasugamycin (PDB identification code 2HHH) (46) binds against h24 and overlaps with mRNA, interfering with translation initiation. (b, c) Pactamycin (PDB code 4W2H) and amicoumacin A (PDB code 4W2F) (47) bind to the E site. While pactamycin displaces the mRNA from its usual path in the E site, amicoumacin A tethers the mRNA to the ribosome. (d) Edeine B1 (PDB code 1I95) (24) binds to the P site and prevents translation initiation. (e) GE81112 (PDB code 5IWA) (48) binds to the P site of the small subunit and interacts with the C-terminus of uS13. It stabilizes the anticodon-stem loop of fMet-tRNA_i^fMet in a distorted conformation, thereby interfering with translation initiation. (f) Dityromycin (PDB code 4WQU) (53) interacts solely with uS12 and prevents conformational changes in EF-G required for tRNA translocation. Abbreviations: ASL, anticodon-stem loop; clash, unnatural overlap of any nonbonding atoms in a structure; EF-G, elongation factor G; fMet-tRNA_i^fMet, initiator formyl-methionine transfer RNA; mRNA, messenger RNA; PDB, Protein Data Bank.

Figure 4.. Antibiotics interacting with the PTC.

(a, b, c) Chloramphenicol (PDB identification code 4V7W) (91), linezolid (PDB code 4WFA) (57), and clindamycin (PDB code 4V7V) (59) collide with the CCA-end of the A-site tRNA and prevent its accommodation. (d) Sparsomycin (PDB code 1VQ9) (62) collides with the CCA-end of the A-site tRNA and stabilizes the P-site tRNA. (e, f) A201A (PDB code 4Z3S) and hygromycin A (PDB code 5DOY) (67) induce an unusual conformation of the CCA-end of the A-site tRNA, which is not competent of peptide bond formation. (g) Lefamulin (PDB code 5HL7) (10) occupies the PTC and interferes with the binding of both the A- and P-site tRNAs. (h, i) Blasticidin S (PDB code 4V9Q) (77) and bactobolin A (PDB code 4WT8) (81) distort the 3’-terminus of the P-site tRNA, inhibiting peptidyl-tRNA hydrolysis mediated by release factors.

Figure 5.. Antibiotics interacting with the NPET.

(a, b, c) Erythromycin (PDB identification code 4V7X) (91), carbomycin A (PDB code 1K8A) (23), and telithromycin (PDB code 4V7Z) (91) are macrolides that bind to the NPET. Carbomycin A has a disaccharide extension that reaches into the PTC and collides with the A-site tRNA. Telithromycin has an alkyl-aryl group that extends further into the NPET, increasing its affinity for the ribosome. (d) Synercid is a combination of dalfopristin, a streptogramin A drug, and quinupristin, a streptogramin B drug, with both binding synergistically on the ribosome, occupying the PTC and the NPET. (e, f) Oncocin (PDB code 4Z8C) (115) and apidaecin (PDB code 5O2R) (116) are proline-rich antimicrobial peptides that bind to the 50S subunit. Oncocin prevents the accommodation of the A-site tRNA, whereas apidaecin interferes with translation termination by locking RF1 on the ribosome. (g) Klebsazolicin (PDB code 5W4K) (119) is a ribosomally-synthesized post-translationally modified peptide that obstructs the NPET. Abbreviations: A-tRNA, A-site tRNA; clash, unnatural overlap of any nonbonding atoms in a structure; PDB, Protein Data Bank; PTC, peptidyl transferase center; P-tRNA, P-site tRNA.

Figure 6.. Interactions of thiopeptides and orthosomycins with the 50S subunit.

(a) Thiostrepton (PDB identification code 3CF5) (120), a thiopeptide interacting with H43/H44 and ribosomal protein uL11, sterically clashes with domain V of EF-G and inhibits the EF-G-catalyzed tRNA translocation. (b) Evernimicin (PDB code 5KCS) (121) interacts with H89, H91 and protein uL16, and may interfere (clash) with the binding of IF2 during translation initiation.

Figure 7.. The most prominent mechanisms of resistance to tetracyclines.

Resistance to tetracyclines is mainly conferred by the acquisition of mobile genes expressing tetracycline-specific efflux pumps, ribosome protection proteins, or tetracycline degrading enzymes (tetracycline destructases).

Figure 8.. The widespread mechanisms of resistance to aminoglycosides and macrolides.

(a) Drug modification at the amino and hydroxyl groups is the main mechanism of resistance to aminoglycosides (AG). (b) Modifications of gentamicin C1a disrupt its binding to h44. (c) Resistance conferred by drug-target modification exemplified by the macrolide erythromycin (ERY). Dimethylation of A2058 at the N6 atom abolishes the binding of erythromycin. (d) Cfr-catalyzed methylation at the C8 position of A2503, which has an intrinsic methylation at the C2 position, confer resistance against a wide range of antibiotics and synergizes with the A2508 modification to extend resistance to macrolides. Abbreviations: AAC, acetyltransferase; ANT, nucleotidyltransferase; APH, phosphotransferase; Cfr, chloramphenicol florphenicol resistance; clash, unnatural overlap of any nonbonding atoms in a structure; m⁶2A, N6, N6-dimethyladenine; m²A and m⁸A, 2-methyladenine and 8-methyladenine, respectively.