An antibiotic preorganized for ribosomal binding overcomes antimicrobial resistance

Abstract

We report the design conception, chemical synthesis, and microbiological evaluation of the bridged macrobicyclic antibiotic cresomycin (CRM), which overcomes evolutionarily diverse forms of antimicrobial resistance that render modern antibiotics ineffective. CRM exhibits in vitro and in vivo efficacy against both Gram-positive and Gram-negative bacteria, including multidrug-resistant strains of Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. We show that CRM is highly preorganized for ribosomal binding by determining its density functional theory-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Lastly, we report two additional x-ray crystal structures of CRM in complex with bacterial ribosomes separately modified by the ribosomal RNA methylases, chloramphenicol-florfenicol resistance (Cfr) and erythromycin-resistance ribosomal RNA methylase (Erm), revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.

Fig. 1.. Design and synthesis of the conformationally restricted macrobicyclic antibiotic cresomycin (CRM).

(A) Conception of CRM as a molecule preorganized for binding to the bacterial ribosome achieved through conformational restriction of the aminooctose residue of iboxamycin (IBX). (B) Synthesis of CRM. (i) 4-bromo-1-butene, DBU; (ii) [t-Bu₂Sn(OH)Cl]₂, MeOH–THF; (iii) DMP; (iv) CuSO₄, (R)-(+)-t-butylsulfinamide; (v) Crotyl chloride, Zn, LiCl, −108 °C; (vi) Grubbs II catalyst, 110 °C; (vii) NaOMe, MeOH; (viii) HCl, Dioxane; (ix) HATU, DIPEA; (x) HCl, Dioxane–MeOH. Abbreviations: DBU, 1,8-Diazabicyclo[5.4.0]undec-7-ene; DMP, Dess-Martin periodinane; HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate; DIPEA, N,N-Diisopropylethylamine.

Fig. 2.. In vitro efficacy of CRM against MDR bacteria.

Minimum inhibitory concentrations (μg·mL⁻¹) of CRM in comparison with iboxamycin (IBX) and clinically approved antibiotics. Abbreviations: CLI, clindamycin; CRO, ceftriaxone; GEN, gentamicin; AZM, azithromycin; CIP, ciprofloxacin; LZD, linezolid; DOX, doxycycline; VAN, vancomycin; ATM, aztreonam; LVX, levofloxacin; IPM, imipenem; CST, colistin; cfr, chloramphenicol-florfenicol resistance methylase; c-ermA/B, constitutively-expressed erythromycin rRNA methylase A/B; vgaA, lsaA, and cplR are ABC-F proteins; lnuA, lincosamide nucleotidyltransferase A; mecA, methicillin-resistance gene; tet(L/M), tetracycline efflux protein L/M; vanA, vancomycin-resistance gene; LZD-R, linezolid-resistant; VRE, vancomycin-resistant Enterococcus; CRE, carbapenem-resistant Enterobacteriaceae; CRAB, carbapenem-resistant A. baumannii. Genes that are known to confer resistance to lincosamides are highlighted in red italics. Full strain descriptions are provided in Data S1. The four strains banded in dark gray, orange, green, and purple were selected for in vivo studies, summarized in Fig. 3. †Fastidious Gram-negative bacteria. *P. aeruginosa MICs were recorded in cation-adjusted Muller-Hinton broth treated with Chelex 100 ion-exchange resin (with subsequent re-supplementation of calcium, magnesium, and zinc) to remove iron. Experiments were performed in independent triplicates and the modal MICs are reported.

Fig. 3.. In vivo efficacy studies of CRM against MDR bacteria.

(A) Kaplan-Meier plot of a murine systemic infection model against cfr-expressing S. aureus administered at 90% lethal dose (LD₉₀). Ten infected mice were provided subcutaneous (SC) 25 mg·kg⁻¹ q.i.d. administration of either CRM or vehicle over one day, then monitored for six days thereafter. 9 of 10 mice receiving vehicle died within 2 days post-infection (brown line), while 10 of 10 mice receiving CRM survived for 7 days post-infection (green line). (B, C) Murine neutropenic thigh infection models against cfr-expressing S. aureus (gray), ermA-expressing S. aureus AR-0693 (orange), E. coli AR0137 (green), and P. aeruginosa AR-0236 (purple). Bacterial counts (log₁₀CFU per gram of thigh) were enumerated before treatment (circles) and after treatment with intraperitoneal (IP) administration of CRM (diamonds) or vehicle (triangles) at the stated dosing regimen for 24 h. Data are shown as mean ± SD, with n = 8 thighs from 4 mice for each treatment arm. Statistical significance was assessed by the log-rank test for panel A, and two-tailed unpaired Welch’s t-tests for panels B and C. In all panels, **** indicates a p-value of <0.0001.

Fig. 4.. Predicted and experimentally determined structures of CRM.

(A) Predicted global minimum-energy conformation of CRM, as determined by DFT calculations. (B) Solution-state conformation of CRM, as determined by ¹H NOESY NMR. (C) Solid-state conformation of CRM, as determined by X-ray diffraction (XRD). (D) Ribosome-bound conformation of CRM, as determined by XRD of CRM in complex with T. thermophilus 70S ribosomes.

Fig. 5.. Structures of CRM in complex with WT, Cfr-modified, and Erm-modified T. thermophilus 70S ribosomes.

(A) Superposition of CRM (yellow) in complex with the WT 70S ribosome (light blue) and CRM (green) in complex with Cfr-modified 70S ribosome (blue) containing the Cfr-methylated m²m⁸A2503 nucleobase (dark blue). To accommodate the methyl group introduced by Cfr (m⁸ of m²m⁸A2503, orange), the nucleobase is shifted by approximately 0.6 Å (red arrow) relative to the native nucleobase m²A2503 in the WT ribosome. (B) In the Cfr-modified ribosome, the C7–C6–N–CO dihedral angle of CRM is deflected by −14°, shifting the carbonyl oxygen atom by 0.8 Å and placing it within VDW contact of the C7-methyl group. (C) Comparison of CRM (yellow) in complex with the WT 70S ribosome (light blue) and CRM (teal) in complex with Erm-modified 70S ribosome (blue). The Erm-dimethylated nucleobase m⁶₂A2058 (dark blue) is shifted by approximately 2.0 Å (red arrow) relative to the WT structure. (D) The two methyl groups (orange) introduced by Erm in m⁶₂A2058 disrupt key hydrogen bonds (dotted lines) with CRM. Note that the position of ribosome-bound CRM remains nearly identical in all three structures, while the positions of m²m⁸A2503 and m⁶₂A2058 in Cfr- and Erm-methylated ribosomes, respectively, are displaced relative to their canonical positions in WT ribosomes.