A synthetic antibiotic class overcoming bacterial multidrug resistance

Abstract

The dearth of new medicines effective against antibiotic-resistant bacteria presents a growing global public health concern¹. For more than five decades, the search for new antibiotics has relied heavily on the chemical modification of natural products (semisynthesis), a method ill-equipped to combat rapidly evolving resistance threats. Semisynthetic modifications are typically of limited scope within polyfunctional antibiotics, usually increase molecular weight, and seldom permit modifications of the underlying scaffold. When properly designed, fully synthetic routes can easily address these shortcomings². Here we report the structure-guided design and component-based synthesis of a rigid oxepanoproline scaffold which, when linked to the aminooctose residue of clindamycin, produces an antibiotic of exceptional potency and spectrum of activity, which we name iboxamycin. Iboxamycin is effective against ESKAPE pathogens including strains expressing Erm and Cfr ribosomal RNA methyltransferase enzymes, products of genes that confer resistance to all clinically relevant antibiotics targeting the large ribosomal subunit, namely macrolides, lincosamides, phenicols, oxazolidinones, pleuromutilins and streptogramins. X-ray crystallographic studies of iboxamycin in complex with the native bacterial ribosome, as well as with the Erm-methylated ribosome, uncover the structural basis for this enhanced activity, including a displacement of the [Formula: see text] nucleotide upon antibiotic binding. Iboxamycin is orally bioavailable, safe and effective in treating both Gram-positive and Gram-negative bacterial infections in mice, attesting to the capacity for chemical synthesis to provide new antibiotics in an era of increasing resistance.

Fig. 1. Evolution of a novel antibiotic scaffold.

a, Chemical structures of lincomycin and clindamycin, and a preclinical antibiotic discovered by Vicuron scientists. b, The design progression of oxepanoprolinamide antibiotics. Conformational constraint of the C4′ substituent within a bicyclic oxepanoprolyl scaffold, together with presentation of a lipophilic group at its 7′ position provided improved activity. c, Synthesis of oxepanoprolinamides OPP-1, OPP-2 and IBX (OPP-3). Reagents and conditions are as follows: (i) LiHMDS, LiCl; (ii) NaOH, >99% recovery of (R,R)-pseudoephenamine; (iii) Boc₂O; (iv) MeI, Cs₂CO₃; (v) allyl ethyl carbonate, Pd(PPh₃)₄; (vi) Hoveyda–Grubbs catalyst I; (vii) LiOH; (viii) 7-Cl-MTL, HATU, EtⁱPr₂N; (ix) TFA; (x) Pd(OAc)₂, benzoquinone, HBF₄ (aq.); (xi) DAST; (xii) LiHMDS, Comins’ reagent, 51% (plus, separately, 31% Δ6′ regioisomer); (xiii) ⁱBuMgCl, Fe(acac)₃; (xiv) H₂, Pd(OH)₂/C, 1:1 dr; (xv) BSTFA, TMSI, 17% over 2 steps (plus, separately, the 7′R epimer; 17% over 2 steps).

Fig. 2. In vitro and in vivo antibacterial activity of the broad-spectrum antibiotic IBX.

a–c, MICs in μg ml⁻¹ of IBX in standard and multidrug-resistant bacterial strains. a, IBX displays a broad antibacterial spectrum of activity, and overcomes erm, cfr and lsaA resistance. B. fragilis, Bacteroides fragilis; H. influenzae, Haemophilus influenzae; K. oxytoca, Klebsiella oxytoca; S. epidermidis, Staphylococcus epidermis; S. haemolyticus, Staphylococcus haemolyticus. Comparisons of IBX with standard antibiotics against clinical Gram-positive (b) and Gram-negative (c) isolates illustrate the differentiated activity of the oxepanoprolinamide class. CLI, clindamycin; CTR, ceftriaxone; LEVO, levofloxacin; AZM, azithromycin; LNZ; linezolid; VAN, vancomycin; GEN, gentamicin. d, Efficacy of IBX in a neutropenic mouse thigh infection model; colours correspond to strains highlighted in a–c. Mice received vehicle or IBX intraperitoneally, and bacterial counts were determined 12 h after treatment. Data are mean ± s.d.; n = 8 thighs from 4 mice examined over 2 experiments; two-tailed unpaired Welch’s t-test. CFU, colony-forming units. e, Survival of mice receiving IBX or vehicle following systemic infection with S. pyogenes. 3GC-R, third-generation cephalosporin-resistant; CRAB, carbapenem-resistant A. baumannii; CRE, carbapenem-resistant Enterobacterales; ESBL, extended-spectrum beta-lactamase; FQ-R, fluoroquinolone-resistant; LNZ-R, linezolid-resistant; MDR, multidrug-resistant; MEC-R, methicillin-resistant; MRSA, methicillin-resistant S. aureus; VAN-R, vancomycin-resistant; VRE, vancomycin-resistant Enterococcus. Source data.

Fig. 3. Structure of IBX in complex with the 70S ribosome, mRNA and tRNAs.

a, Overview of the IBX binding site in the T. thermophilus 70S ribosome viewed as a cross- section through the NPET. Indicated are the 30S subunit (light yellow), the 50S subunit (grey), mRNA (magenta), A-site tRNA (green), P-site tRNA (dark blue) and E-site tRNA (orange). b, c, Detail views of IBX bound in the PTC, highlighting hydrogen-bond interactions (dashed lines) and the positioning of the 7′-isobutyl substituent within the A-site cleft formed by 23S rRNA residues A2451 and C2452. d, Models of A- and P-site tRNAs from the IBX–ribosome complex (green and blue, respectively) superimposed with those found in the drug-free ribosome (PDB entry 6XHW).

Fig. 4. Structure of iboxamycin (IBX) bound to the Erm-methylated 70S ribosome.

a, b, Electron density map (blue mesh), contoured at 1.0σ, of IBX (teal) in complex with the Erm-modified T. thermophilus 70S ribosome-containing N⁶-dimethylated A2058 residue in the 23S rRNA (a), highlighting the interaction with the methyl groups of ${\text{m}}_2^6\text{A}2058$ (orange) (b). c, d, Superposition of IBX (yellow) in complex with the WT 70S ribosome containing an unmodified residue A2058 (light blue), and the structure of IBX (teal) in complex with the Erm-modified 70S ribosome containing an ${\text{m}}_2^6\text{A}2058$ residue (medium blue) (c), highlighting hydrogen bonding (dashed lines) (d). Note that the position of IBX is almost identical in the two structures, whereas IBX binding to the Erm-modified ribosome causes substantial movement of ${\text{m}}_2^6\text{A}2058$ from its canonical position (red arrow). WT, wild type.

Extended Data Fig. 1. Structure-based design of 7′-substituted oxepanoprolinamides.

a, Superposition of the X-ray crystal structure of ribosome-bound lincosamide antibiotic clindamycin (2, blue, PDB entry 4V7V) with the energy-minimized structure of OPP-1 (green). Note that in this configuration, the C7′-atom of OPP-1 contacts the lipophilic surface of the A-site cleft. b, The same structure, overlaid with the X-ray crystal structure of iboxamycin (IBX, yellow) bound to the bacterial ribosome.

Extended Data Fig. 2. MICs (μg ml⁻¹) of antibiotics containing a bicyclic aminoacyl residue.

a, Effects of of 7′ substitution, ring size, and saturation on antibacterial activity. b, Effects of 7′-alkyl substituent chain length on antibacterial activity, including against MLS_B-resistant S. aureus and strains of E. coli engineered to lack key efflux or outer-membrane assembly machinery. c-ermA/B, constitutively expressed erythromycin ribosome methylase A/B gene.

Extended Data Fig. 3. Effects of iboxamycin on mammalian cells.

a, Normalized hemolysis (mean ± s.d.) of human erythrocytes by IBX (n = 3 replicates) and clindamycin (n = 5 replicates) relative to Triton X-100 (n = 45 replicates) measured over one independent experiment. b, c, Mitochondrial ToxGlo data showing effects of IBX on HepG2 cellular membrane integrity and ATP production relative to vehicle-treated control (antimycin serves as a positive control for mitotoxicity). Data are mean ± s.d.; n = 3 technical replicates from one independent experiment. d–f, Comparison of effects of IBX, clindamycin, doxycycline, and azithromycin on cell viability (CellTiter-Blue). Data are the mean ± s.d. of n = 3 independent experiments performed in technical quintuplicate. Where applicable, GI₅₀ values (µM) are reported beside the dose-response curves.

Extended Data Fig. 4. Efficacy of iboxamycin (IBX) in mouse models of infection. Bacterial counts were quantified in the thighs of neutropenic mice infected with S. pyogenes ATCC 19615.

(a), S. aureus MRSA HAV017 (b), A. baumannii ATCC 19616 (c) or E. coli MDR HAV504 (d) treated with IBX, vehicle, or comparator antibiotic at the listed time points post-treatment. Data are mean ± s.d.; n = 8 thighs from 4 mice examined over 2 experiments, with the exception the sub-set of part c where mice received IBX via intravenous administration (n = 4 thighs from 2 mice examined over a single experiment). e, Mouse survival in an an S. pyogenes systemic infection model. Abbreviations: AZM, azithromycin; CLI, clindamycin; CPFX, ciprofloxacin; IP, intraperitoneal administration; IV, intravenous administration.. Source data.

Extended Data Fig. 6. Iboxamycin (IBX) efficiently arrests translation at the start codon.

Toeprinting analysis showing sites of IBX-induced translation arrest of ErmBL and ErmDL leader peptides. Because all reactions contained mupirocin, an inhibitor of Ile-tRNA synthetase, the ribosomes that escape inhibition by ribosome-targeting antibiotics are trapped at the codon preceding Ile (black arrowheads). Red arrowheads mark translation arrest at the start codon, while cyan arrowheads denote known erythromycin-induced arrest sites D10 (ermBL) and L7 (ermDL). Each gel is representative of two independent experiments, for source data see Supplementary Figure 1. ERY, erythromycin; CLI, clindamycin.

Extended Data Fig. 5. Time-kill kinetics, post-antibiotic effect, and post-antibiotic sub-MIC effect data of iboxamycin (IBX) against susceptible strains.

a, Arrayed growth curves for three susceptible strains showing concentration effects on growth inhibition (time-kill), growth kinetics following exposure to antibiotic at 4×MIC (PAE), and growth kinetics under sub-MIC concentrations following exposure to antibiotic at 4×MIC (PA-SME). Points represent mean values from n = 2 biologically independent experiments. b, Tabulated PAE and PA-SME durations (determined as the difference in time required for bacterial counts to rise 10× between experimental and untreated control arms). Abbreviations: CLI, clindamycin; LNZ, linezolid.

Extended Data Fig. 7. High-resolution electron density maps of iboxamycin (IBX) bound to the bacterial ribosome.

a, b, Unbiased F_o-F_c and 2F_o-F_c electron density maps of IBX in complex with the T. thermophilus 70S ribosome (green and blue mesh, respectively). The refined model of IBX is displayed in its electron density before (a) and after (b) the refinement contoured at 3.0σ and 1.5σ, respectively c, Superposition of ribosome-bound IBX (yellow) with prior structures of clindamycin bound to the tRNA-free 70S ribosome from eubacterium E. coli (blue, PDB entry 4V7V) or to the 50S ribosomal subunit from archaeon H. marismortui harboring the 23S rRNA mutation G2099A (green, PDB entry 1YJN). All structures were aligned based on domain V of the 23S rRNA. d, 2F_o-F_c electron density map (blue mesh) corresponding to ribosome-bound IBX (yellow), deacylated A-site tRNA^Phe (green) and aminoacylated initiator P-site fMet-NH-tRNA_i^Met (dark blue). The refined models of tRNAs are displayed in their respective electron-density maps contoured at 1.0σ. In d, the entire bodies of the A- and P-site tRNAs are viewed from the back of the 50S subunit, as indicated by the inset. Ribosome subunits are omitted for clarity. Note that IBX binding to the ribosome prevents accommodation of the aminoacyl-bearing CCA-end of the A-site tRNA. e, Close-up view of the P-site tRNA CCA-end bearing a formyl-methionyl (cyan) residue. f, Detailed arrangement of the hydrogen bonds formed between the aminooctose component of IBX with 23S rRNA residues A2058 and G2505 (light blue).