Synthetic macrolides overcoming MLSBK-resistant pathogens

Abstract

Conventional macrolide-lincosamide-streptogramin B-ketolide (MLS_BK) antibiotics are unable to counter the growing challenge of antibiotic resistance that is conferred by the constitutive methylation of rRNA base A2058 or its G2058 mutation, while the presence of unmodified A2058 is crucial for high selectivity of traditional MLS_BK in targeting pathogens over human cells. The absence of effective modes of action reinforces the prevailing belief that constitutively antibiotic-resistant Staphylococcus aureus remains impervious to existing macrolides including telithromycin. Here, we report the design and synthesis of a novel series of macrolides, featuring the strategic fusion of ketolide and quinolone moieties. Our effort led to the discovery of two potent compounds, MCX-219 and MCX-190, demonstrating enhanced antibacterial efficacy against a broad spectrum of formidable pathogens, including A2058-methylated Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes, and notably, the clinical Mycoplasma pneumoniae isolates harboring A2058G mutations which are implicated in the recent pneumonia outbreak in China. Mechanistic studies reveal that the modified quinolone moiety of MCX-190 establishes a distinctive secondary binding site within the nascent peptide exit tunnel. Structure-activity relationship analysis underscores the importance of this secondary binding, maintained by a sandwich-like π-π stacking interaction and a water-magnesium bridge, for effective engagement with A2058-methylated ribosomes rather than topoisomerases targeted by quinolone antibiotics. Our findings not only highlight MCX-219 and MCX-190 as promising candidates for next-generation MLS_BK antibiotics to combat antibiotic resistance, but also pave the way for the future rational design of the class of MLS_BK antibiotics, offering a strategic framework to overcome the challenges posed by escalating antibiotic resistance.

Fig. 1. The SARs of new hybrids of macrolides and quinolones.

a Structures of the first-, second- and third-generation marketed erythromycins. b Structures of the representative macrolones MCX-219 and MCX-190. c MICs (μg/mL) of the selected synthetic macrolides. Ery^S erythromycin sensitive, AZM^S azithromycin sensitive, TEL telithromycin, CLA clarithromycin, CIP ciprofloxacin. The strains 07P390, PU09, 12-206, 15B196, and PU32 are erythromycin-resistant clinical isolates encoding various resistant genes.

Fig. 2. MCX-219 and MCX-190 are active against a variety of resistant clinical isolates and highly accumulative inside cells.

a MICs of MCX-219 and MCX-190 against S. aureus, M. pneumoniae, S. pyogenes, S. pneumoniae, E. faecium, E. faecalis, H. influenzae and M. catarrhalis. b MIC₅₀ and MIC₉₀ of erythromycin (ERY), telithromycin (TEL), MCX-219, and MCX-190 against 23 clinical isolates of S. aureus from hospitals in China, 2021. c Cellular accumulation in S. aureus 15B196 for MCX-219, MCX-190, ERY, TEL and ciprofloxacin (CIP). Data are expressed as mean ± SD and analyzed by using one-way ANOVA plus Dunnett. Statistical significance is reported as follows: *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs ERY.

Fig. 3. Cryo-EM reconstruction of the complex of MCX-190 and WT ribosomes of S. aureus.

a The overall and detail of MCX-190 bound to the NPET. The distance of the hydrogen bond between the 2’-OH of desosamine and N1 positions of A2058 (A2085) is 2.93 Å. b The density map of MCX-190 and cation Mg²⁺ in the WT ribosome complex. c Cation Mg²⁺ is chelated by the quinolone element and the other four H₂O molecules. d A cyclopropyl group at the N1 position of quinolone could create steric clashes with U2609 (U2636).

Fig. 4. Comparison of MCX-190 and WT/15B196 S. aureus ribosome complexes.

a The results of in vitro translation inhibition assays showed that 15B196 ribosomes were sensitive to MCX-219 and MCX-190. IC₅₀ Values are shown as mean ± SD of at least three independent experiments. b Q-TOF mass spectrometry analysis of S. aureus rRNA 2052–2064. In the triply deprotonated ion of the molecule, the fragment GGACGGAAAGACC runs at 1433.54 m/z (when unmethylated). Monomethylation and dimethylation at A2058 result in mass shifts to 1438.21 m/z and 1442.89 m/z, respectively. The unmethylated fragment with a Na ion is observed at 1440.87 m/z. c The density maps of A2058 (A2085) in the 15B196/WT S. aureus ribosome complex. Both of two density maps of are contoured at 7.5σ. d The density of the desosamine in the complex of 15B196 ribosome complex was significantly decreased, compared to the WT ribosome complex. e The π–π interaction of quinolone ring with A2062 (A2089) and C1782–C2586 (C1809–C2613), was not affected by whether A2058 (A2085) was methylated.

Fig. 5. Cytotoxicity and inhibition of eukaryotic translation by MCX-219 and MCX-190.

a Cytotoxicity of the optimal compounds at a concentration of 5 μM with an incubation for 48 h. b, c Cell viability of HepG2 (b) and HEK293T (c) cells in the presence of increasing concentrations of MCX-219 or MCX-190. MCX-219 showed a calculated cytotoxicity concentration at 26 μM that inhibits 50% viability (CC₅₀), while TcmX reportedly had a CC₅₀ of 2 μM (c). d In vitro translation of Fluc mRNA in HEK293F whole-cell lysate in increasing concentrations of MCX-219 or MCX-190. CHX cycloheximide. Values are shown as the mean ± SD of at least three independent experiments.