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Published in final edited form as: Science. 2024 Feb 15;383(6684):721–726. doi: 10.1126/science.adk8013

An antibiotic preorganized for ribosomal binding overcomes antimicrobial resistance

Kelvin J Y Wu 1, Ben I C Tresco 1, Antonio Ramkissoon 1, Elena V Aleksandrova 2, Egor A Syroegin 2, Dominic N Y See 1, Priscilla Liow 1, Georgia A Dittemore 1, Meiyi Yu 1, Giambattista Testolin 1, Matthew J Mitcheltree 1, Richard Y Liu 1, Maxim S Svetlov 3,4, Yury S Polikanov 2,3,4,*, Andrew G Myers 1,*
PMCID: PMC11665821  NIHMSID: NIHMS2025232  PMID: 38359125

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 DFT-calculated, solution-state, solid-state, and (wild-type) ribosome-bound structures, which all align identically within the macrobicyclic subunits. Finally, we report two additional X-ray crystal structures of CRM in complex with bacterial ribosomes modified by the rRNA methylases, Cfr and Erm, respectively, revealing concessive adjustments by the target and antibiotic that permit CRM to maintain binding where other antibiotics fail.

One-Sentence Summary:

A synthetic molecule preorganized for bacterial ribosome binding is highly effective against multidrug-resistant bacteria.

Main Text:

The emergence and widespread distribution of bacteria resistant to most or all approved antibiotics portends a global public health crisis. Estimates attribute 1.27 million deaths worldwide to antimicrobial resistance (AMR) in 2019 alone (1), and a 2016 forecast projected as many as 10 million deaths by 2050 absent effective countermeasures (2). It is evident that the pace of discovery and development of antibiotics effective against AMR has not matched the rate of its global dissemination (3). The reasons for this are complex, but contributing factors include antibiotics misuse, a failure of the marketplace to provide effective economic incentives for antibiotics development (4), and the limitations of semi-synthesis as an engine for antibiotics discovery (5). Our laboratory uses component-based chemical synthesis to explore and broadly extend natural products families that have evolved over millennia as antibacterial agents. Representatives of these families were often developed as drugs during the golden age of antibiotics discovery (ca. 1960–1980), but decades of use in the community and the clinic have since fostered the emergence of multiple forms of AMR. Lincosamide antibiotics, which arrest bacterial protein synthesis by binding to the peptidyl transferase center (PTC) of the bacterial ribosome (6), are exemplary, for their clinical utility (and that of other ribosome-targeting antibiotics) is now limited by multiple, evolutionarily distinct mechanisms of AMR. Two mechanisms of increasing prevalence preclude antibiotic binding by post-transcriptional methylation of ribosomal RNA (rRNA) nucleobases near the catalytic PTC. Erythromycin resistance rRNA methylases (Erm) confer resistance to macrolide, lincosamide, and streptogramin B (collectively termed MLSB) antibiotics by N6-dimethylation of A2058 in 23S rRNA (7–9), while chloramphenicol-florfenicol resistance (Cfr) methylase confers resistance to phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A (PhLOPSA) antibiotics by C8-methylation of A2503, also in 23S rRNA (10, 11). Lincosamide nucleotidyltransferases (Lin/Lnu) O-adenylate the 3′-hydroxyl group of lincosamides, whose product precludes ribosomal binding (12, 13), and ATP-Binding Cassette F (ABC-F) proteins confer resistance to phenicols, lincosamides, oxazolidinones, pleuromutilins, macrolides, and streptogramins by recognizing and displacing these antibiotics from stalled ribosomes (14–16). We describe here the design conception, chemical synthesis, and in vitro and in vivo antimicrobial evaluation of the conformationally restricted antibiotic cresomycin, which overcomes each of these evolutionarily diverse AMR mechanisms and simultaneously extends the spectrum of antibacterial activity beyond that of any lincosamide.

Design hypothesis and synthesis of a bridged macrobicyclic antibiotic preorganized for ribosomal binding

For both descriptive purposes and practical retrosynthetic disconnection, the lincosamides can be structurally separated into two halves by hydrolysis of the central amide bond to form a “northern” aminooctose fragment and an elaborated proline residue as a “southern” fragment (Fig. S1). We and others (17–19) have shown that coupling of diverse fully synthetic southern scaffolds with the northern aminosugar component of clindamycin (7-chloro-1-methylthiolincosamine, 7-Cl-MTL, Fig. S1) can, after deprotection, produce novel lincosamide antibiotics with improved efficacy against antibiotic-resistant bacterial strains, as well as an expanded spectrum of activity against Gram-negative pathogens. Specifically, we reported the discovery by chemical synthesis of iboxamycin (IBX, Fig. 1A), containing a bicyclic oxepanoprolinamide southern scaffold, which engages a key ribosomal binding pocket near the PTC and thereby overcomes a broad range of mechanisms of AMR (19). Lincosamide analogs prepared by semisynthetic transformations of the northern aminooctose residue, though largely restricted to modifications of positions 1 or 7 (20, 21), have also shown promise as candidates with efficacy against certain forms of AMR, but more extensive explorations of the northern residue have been limited thus far.

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

Fig. 1.

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(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.

There are three X-ray crystal structures and one cryo-EM structure of clindamycin bound to ribosomes of the diverse bacterial species Deinococcus radiodurans (6) and Escherichia coli (22, 23), as well as the archaeon Haloarcula marismortui (24), the latter bearing a G2099A (equivalent to A2058 in E. coli) mutation that permits lincosamide binding. In addition, there are two X-ray crystal structures of iboxamycin bound, respectively, to wild-type (WT) and Erm-methylated ribosomes of Thermus thermophilus (19). We noted that in each of these structures the antibiotics adopt conformations that place the C1 S-methyl group gauche to the endocyclic C–O bond, subtending a mean O–C1–S–CH3 dihedral angle of 66° ± 6° (mean ± SD, Fig. S1, labeled therein as θ). This conformation is one of two that are stabilized by the exo-anomeric effect (S lone pair with C–O σ* orbital overlap). We also recognized that in each published structure the antibiotic adopts a conformation with the 7-chloro substituent gauche to the vicinal C–N bond of the amide residue, subtending a mean N–C6–C7–Cl dihedral angle of –63° ± 6° (Fig. S1, labeled therein as φ), and with the hydrogen atoms located on C5 and C6 antiperiplanar to each other, subtending a mean H–C5–C6–H dihedral angle of 174° ± 6° (Fig. S1, labeled therein as α). We imagined a molecule preorganized (25) for ribosomal binding through conformational restriction, achieved by linking positions C1 and C7 within the bridging macrocyclic (Z)-alkene illustrated in Fig. 1A (cresomycin, CRM). We speculated that placement of the metabolically labile sulfur atom within the bridged macrocyclic ring of CRM might additionally impede its degradation by hepatic sulfoxidation (26), an expectation borne out in in vitro experiments (vide infra).

We applied density-functional theory (ωB97XD/def2-TZVP) to map the conformational landscape of both CRM and the non-macrobicyclic antibiotic iboxamycin, replacing the oxepanoprolinamide southern fragment of each with acetamide to simplify the computations. We found that the calculated global minimum-energy conformation of CRM (vide infra, Fig. 4A) essentially overlaid with the three-dimensional structure of our design hypothesis (Fig. 1A). Calculations revealed that rotations about C1–S, C5–C6, and C6–C7 are greatly restricted for CRM: the mean dihedral angles θ, φ, and α (Fig. S2), respectively, across the five lowest minimum-energy conformations (up to +3.9 kcal·mol−1) are 71° ± 1°, –69° ± 7°, and 177° ± 5°. The first local minimum-energy conformation of CRM that deviates significantly within the macrobicyclic substructure (θ = –21°) lies +9.2 kcal·mol−1 above the global minimum-energy conformation (Figs. S3A–B). In contrast, the first local minimum-energy conformation of iboxamycin with a significant deviation about the C1–S bond (θ = 149°) is nearly isoenergetic (+0.1 kcal·mol−1) with the global minimum-energy conformation (Figs. S3A, S3C). The calculations make clear that the accessible conformations of CRM are significantly reduced in number relative to those accessible to iboxamycin (6 vs. 28 conformations < 9.2 kcal·mol−1, respectively; Fig. S3A) and overlap essentially identically within the macrobicyclic substructure (Figs. S2, S3B).

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

Fig. 4.

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(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.

To synthesize bridged macrobicyclic oxepanoprolinamide antibiotics, we employed a diversifiable route, illustrated in Fig. 1B for the specific instance of CRM. In its generalized form, the route employs asymmetric additions of diverse allylic organozinc reagents to an Ellman sulfinimine (intermediate 3, Fig. 1B) (27), followed by macrocyclization using the Grubbs ring-closing metathesis reaction (28). In all, more than 60 diverse macrobicyclic analogs of different ring sizes (9-, 10-, 11- and 12-membered) were prepared using these two powerful transformations in combination. The known α-thiogalactoside 1 (29) is accessible from D-galactose on 100-gram scale and served as starting material. S-alkylation of 1 with 4-bromo-1-butene followed by selective deprotection of the primary benzoyl group using an organostannane catalyst (30) afforded alcohol 2. Oxidation of 2 with the Dess-Martin periodinane followed by condensation of the resultant aldehyde with (R)-t-butylsulfinamide (31) afforded the Ellman sulfinimine 3. Zinc-mediated crotylation of 3 at −108 °C proceeded with complete diastereoselectivity at C6 and a diastereoselectivity of 85:15 at C7, favoring the desired (7R)-stereoisomer. It proved expedient to conduct ring-closing metathesis directly upon the diastereomeric mixture, using the Grubbs II catalyst in refluxing toluene for 15 min (32). After purification of the product mixture by flash column chromatography (with a single repurification of mixed fractions), the desired (Z)-macrobicycle 4 was isolated in ≥ 95% purity and in 51% yield (3.6 g) for the two-step sequence. Global benzoyl and sulfinamide deprotection of 4 then afforded the macrobicyclic amine 5 (1-g amount). Amide coupling of 5 with the N-Boc-protected oxepanoproline derivative 6 (33) using HATU for carboxyl activation (34), followed by N-Boc removal, provided CRM in amounts sufficient for in vivo studies (320 mg on largest scale).

In vitro and in vivo antibacterial activity of CRM

In broth microdilution antimicrobial analyses, CRM was found to potently inhibit the growth of both Gram-positive and Gram-negative bacteria, including high-priority multidrug-resistant (MDR) ESKAPE pathogens (35, 36) (Fig. 2). Against clinical isolates of MDR Gram-positive bacteria, CRM was broadly superior to iboxamycin in inhibiting the growth of staphylococci (90% Minimum Inhibitory Concentration, MIC90 = 2 vs. 8 μg·mL−1, n = 31), streptococci (MIC90 ≤ 0.06 vs. 0.25 μg·mL−1, n = 13), enterococci (MIC90 = 0.25 vs. 2 μg·mL−1, n = 37), and Clostridioides difficile (MIC90 = 0.125 vs. 16 μg·mL−1, n = 10) (Data S1). Among these isolates, certain were strains expressing at least two mechanistically distinct forms of AMR that protect bacteria from lincosamides and other ribosome-targeting antibiotics. For example, CRM arrested the growth of an E. faecalis isolate known to encode both Erm and LsaA (37) resistance proteins (Fig. 2, AR-0671, MIC ≤ 0.06 vs. 0.5 μg·mL−1), as well as a C. difficile isolate encoding both Erm and CplR (38) resistance proteins (Fig. 2, AR-1072, MIC = 2 vs. 16 μg·mL−1). Against MDR Gram-negative pathogens, CRM was broadly superior to iboxamycin in inhibiting the growth of Escherichia coli (MIC90 = 2 vs. 16 μg·mL−1, n = 22), Klebsiella pneumoniae (MIC90 = 8 vs. >32 μg·mL−1, n = 16), Acinetobacter baumannii (MIC90 = 8 vs. 32 μg·mL−1, n = 21), Neisseria gonorrhoeae (MIC90 = 0.125 vs. 0.5 μg·mL−1, n = 15), Moraxella catarrhalis (MICs ≤ 0.125 vs. ≤ 0.25 μg·mL−1, n = 4), and Haemophilus influenzae (MIC ≤ 0.06 μg·mL−1 for both CRM and iboxamycin, n = 1) (Data S1). The in vitro efficacy of CRM against the Gram-negative bacterium Pseudomonas aeruginosa depended upon the conditions under which the bacteria were grown. For example, while CRM was only weakly active against the MDR clinical isolate AR-0236 grown in conventional cation-adjusted Mueller-Hinton Broth (ca-MHB), its activity was significantly potentiated when the same strain was grown in iron-depleted ca-MHB (MICs: 32 vs. 2 μg·mL−1, respectively, Data S1) (39, 40).

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

Fig. 2.

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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.

Time-kill studies against a strain of cfr-expressing S. aureus (Fig 2, dark gray) revealed that CRM was bacteriostatic at concentrations of 1 ×, 2 ×, 4 ×, and 10 × MIC for up to 24 hours (Fig. S4A). Separate incubations of S. aureus (ATCC 29213, 1 × 109 CFU) in media containing CRM at concentrations of 4 × MIC and 10 × MIC for 48 hours at 37 °C failed to produce any observable resistance colonies, consistent with a spontaneous resistance frequency of <10–9 in this strain (Fig. S4B). In vitro safety profiling experiments showed that CRM displayed low cytotoxicity against all primary and immortalized human cell lines tested (human lung fibroblasts, human umbilical vein endothelial cells, A375 cells, and HepG2 cells), did not induce hemolysis of human erythrocytes, and did not exhibit mitochondrial toxicity in HepG2 cells at concentrations of up to 125 μM (Fig. S4D). When incubated with human hepatocytes in vitro (Fig. S4C), the half-lives of CRM, iboxamycin, and clindamycin were 55.6 min, 42.3 min, and 14.6 min, respectively.

In murine sepsis models, CRM rescued mice from systemic infections with a 90% lethal dose (LD90) of cfr-expressing S. aureus (Fig. 3A). Following one day of subcutaneous treatment with CRM at 25 mg·kg−1 q.i.d. (four times a day), 10 of 10 mice survived for seven days post-infection. In contrast, 9 of 10 mice receiving vehicle died within two days of infection. In separate murine neutropenic thigh-infection models, intraperitoneal administration of CRM reduced the bacterial burden of cfr-expressing S. aureus, ermA-expressing S. aureus (AR-0693), carbapenem-resistant E. coli (AR-0137), and carbapenem-resistant P. aeruginosa (AR-0236) by –4.6, –2.2, –2.6, and –2.7 log10CFUs, respectively, relative to untreated controls (Figs. 3B–C).

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

Fig. 3.

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(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.

Structures of CRM in solution, in the solid state, and bound to bacterial ribosomes

The solution-state structure of CRM in methanol-d4 was inferred from proton nuclear Overhauser effect spectroscopy (1H-NOESY) analysis (Fig. 4B). Strong NOEs correlating H1 with H9, H7 with H10, and H5 with H12 revealed that the northern macrobicyclic thiolincosamine of CRM adopts a conformation that aligns with the global minimum-energy conformation predicted by DFT calculations (Fig. 4A). The solid-state structure of CRM was determined by single-crystal X-ray diffraction analysis at 0.84-Å resolution (Figs. 1, 4C). When aligned, the global minimum-energy conformation predicted by DFT calculations (Fig. 4A) and the macrobicyclic solid-state substructure differed by a minimized root-mean-square distance of just 0.22 Å. The crystal structure of CRM bound to WT T. thermophilus 70S ribosomes was determined in complex with mRNA, non-hydrolysable aminoacyl-tRNA (fMet-NH-tRNAiMet) in the P site, and deacylated tRNAPhe in the A and E sites, at 2.70-Å resolution (Figs. 4D, S5A–B, S6). The macrobicyclic substructures in ribosome-bound CRM and its conformations in solution and in the solid state were essentially identical (Figs. 4B–D). Together, these results reveal that CRM assumes a single macrobicyclic conformation that is highly preorganized for binding to bacterial ribosomes.

To assess, albeit indirectly, the thermodynamics of target engagement, we separately measured the binding of CRM and iboxamycin to immobilized E. coli ribosomes that had been pre-incubated with [14C]-labeled erythromycin (Fig. S7). CRM displaced 50% of bound radiolabeled erythromycin (IC50) at a concentration of ≤ 8.2 nM, the lower limit of detection of the assay, while iboxamycin required a four-fold higher concentration (35 nM) to achieve the same level of displacement of the macrolide. Clindamycin had previously been shown to bind E. coli ribosomes with ~70-fold lower affinity than iboxamycin using a similar radio-displacement assay (41). These data support the proposal that preorganization by transannular macrocyclization enhances the antibacterial activity of CRM against Gram-positive and Gram-negative bacteria by improving its binding to the bacterial ribosome.

Structural basis for the antibacterial activity of CRM against Cfr- and Erm-expressing bacteria

To understand how CRM so effectively inhibits the growth of bacterial strains expressing the ribosomal methylase genes cfr and erm, which passivate all other PTC-targeting antibiotics, we determined at 2.55-Å and 2.60-Å resolution, respectively, X-ray crystal structures of CRM bound to methylated 70S ribosomes isolated from cfr-expressing (42) and erm-expressing (43) T. thermophilus strains (Figs. S5C–F). Both structures incorporated bound mRNA, a non-hydrolyzable aminoacyl-tRNA (fMet-NH-tRNAiMet) in the P site, and deacylated tRNAPhe in both the A and E sites. The structure of CRM bound to Cfr-modified ribosomes (Figs. 5A–B) revealed that nucleobase A2503 was dimethylated (m2m8A2503) as expected, but its position was shifted by ~0.6 Å relative to its positions in both the antibiotic-free methylated ribosome (Fig. S8) and the CRM-bound WT ribosome (Fig. 5A). The movement of the A2503 nucleobase is reminiscent of its displacement previously observed in a cryo-EM structure of radezolid, a second-generation oxazolidinone antibiotic, bound to the Cfr-modified E. coli ribosome (44). In addition, we observed that within CRM itself the C7–C6–N–CO dihedral angle was rotated by −14° relative to the CRM-WT ribosome structure (84° vs. 98°, respectively, Fig. 5B), placing the oxygen atom of the amide carbonyl within Van der Waals (VDW) contact of the C7-methyl group (Fig. 5B) and moving it away from within the sphere of contact of the methylated nucleobase (0.8-Å displacement). A priori, neither the movement of the nucleobase nor the rotation of the amide residue of CRM could be anticipated. Together, the two movements suggest small concessive adjustments by both the target and the antibiotic to maintain binding in Cfr-methylated ribosomes.

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

Fig. 5.

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(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.

In Erm-modified ribosomes (Figs. 5C–D), we found that CRM, like iboxamycin (19), displaces the Erm-dimethylated m62A2058 nucleobase by approximately 2.0 Å, effectively restructuring the ribosomal binding pocket to allow CRM to retain the same position and conformation it adopts within the WT ribosome. In all structures (WT, Cfr-, and Erm-modified ribosomes), the newly introduced atoms within the macrobicyclic fragment of CRM make no additional contacts with 23S rRNA when compared to iboxamycin (Figs. 5, S6B–C).

Discussion

Using practical synthetic chemistry and guided by principles of rational structure-based design, we have found that conformational restriction of the northern aminooctose residue of “lincosamides” through transannular macrocyclization produces a new antibiotic of unprecedented efficacy against multiple, evolutionarily divergent forms of AMR. The broad efficacy of CRM against clinical isolates of MDR Gram-negative pathogens differentiates the synthetic macrobicyclic class from traditional lincosamides. This is a phenomenon we first observed with the semisynthetic oxepanoprolinamide iboxamycin (19), and it is evident that preorganization of the northern fragment of CRM by macrobicyclization has substantially reinforced this distinction (see comparison with iboxamycin; Data S1). The progression of increased Gram-positive and Gram-negative activity alongside increased efficacy against diverse independent resistance mechanisms in the transition from clindamycin to iboxamycin to, now, CRM bears one identifiable common feature — enhanced engagement of the ribosomal target, achieved largely through conformational restriction and preorganization. We do not suggest that CRM is fully optimized for inhibition of the bacterial ribosome, for in view of the innumerable macrobicyclic structural and substitutional variants that can be conceived, but have not yet been explored, that would be statistically improbable. Though perhaps a daunting consideration, we believe that this portends favorably for the future discovery of antibacterial agents broadly effective against AMR.

Supplementary Material

Supplementary Information
Supplementary Data S1
Supplementary Data S2

Acknowledgments:

We gratefully acknowledge and thank S.L. Zheng for the collection of X-ray crystallographic data for CRM under the support of the Major Research Instrumentation (MRI) Program of the National Science Foundation (NSF) award 2216066; K. Chatman and J. X. Wang for the collection of mass spectrometry data; M. D. Cameron for the collection of liver microsomal stability data; D. R. Andes, M. Brönstrup, L. Burrows, S. Chiang, P. Courvalin, T. J. Dougherty, R. Duggal, M. S. Gilmore, C. Grillot-Courvalin, V. Hauryliuk, C. Keith, S. Lahiri, F. H. Lebreton, A. S. Mankin, H. E. Moser, D. P. Nicolau, K. M. Otte, A. Pisipati, S. Projan, N. C. Vazquez-Laslop, D. N. Wilson, and B. Wohl for their invaluable insights over the course of our research. G.T. is presently affiliated with Nuvisan ICB GmbH. M.J.M. is presently an employee and shareholder of Merck & Co., Inc.

Funding:

This work was supported by the National Institute of Allergy and Infectious Diseases (NIAID) of the NIH, grant R01-AI168228 (A.G.M.); NIAID NIH grant R21-AI163466 (Y.S.P.); National Institute of General Medical Sciences (NIGMS) NIH grant R01-GM132302 (Y.S.P.); NIGMS NIH grant T32-GM139775 (G.A.D.); NSF grant MCB-1907273 (Y.S.P.); Illinois State Startup Funds (Y.S.P.); the Agency for Science, Technology and Research National Science Scholarship (K.J.Y.W.); and the Deutsche Forschungsgemeinschaft Postdoctoral Research Fellowship TE-1311–1-1 (G.T.).

This work is based upon research conducted at the Northeastern Collaborative Access Team (NE-CAT) beamlines, which are funded by the National Institute of General Medical Sciences from the NIH [P30-GM124165 to NE-CAT]. The Eiger 16M detector on the 24-ID-E beamline is funded by an NIH-ORIP HEI grant [S10-OD021527 to NE-CAT]. This research used resources of the Advanced Photon Source, a US Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Argonne National Laboratory under contract no. DE-AC02-06CH11357.

Footnotes

Competing interests: K.J.Y.W., B.I.C.T., and A.G.M. are inventors in a provisional patent application submitted by the President and Fellows of Harvard College covering macrobicyclic antibiotics of the type described in this work. A.G.M., G.T., and M.J.M. have filed international patent application WO/2021/236911 ‘Lincosamide Antibiotics and Uses Thereof’. A.G.M. and M.J.M. have filed the following international patent applications: WO/2019/032936 ‘Lincosamide Antibiotics and Uses Thereof’ and WO/2019/032956 ‘Lincosamide Antibiotics and Uses Thereof’. All other authors declare no competing financial or non-financial interests.

Data and materials availability:

Coordinates and structure factors were deposited in the RCSB Protein Data Bank with accession codes: 8UD6 for the wild-type T. thermophilus 70S ribosome in complex with mRNA, deacylated A-site tRNAPhe, aminoacylated P-site fMet-tRNAiMet, deacylated E-site tRNAPhe, and cresomycin; 8UD7 for the A2058-N6-dimethylated T. thermophilus 70S ribosome in complex with mRNA, deacylated A-site tRNAPhe, aminoacylated P-site fMettRNAiMet, deacylated E-site tRNAPhe, and cresomycin; 8UD8 for the A2503-C2,C8-dimethylated T. thermophilus 70S ribosome in complex with mRNA, deacylated A-site tRNAPhe, aminoacylated P-site fMet-tRNAiMet, deacylated E-site tRNAPhe, and cresomycin. Single-crystal X-ray crystallographic data for cresomycin are deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition number 2298060. All other data are available in the main text or the supplementary materials.

References and Notes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Supplementary Information
NIHMS2025232-supplement-Supplementary_Information.pdf (13.9MB, pdf)
Supplementary Data S1
NIHMS2025232-supplement-Supplementary_Data_S1.xlsx (65.6KB, xlsx)
Supplementary Data S2
NIHMS2025232-supplement-Supplementary_Data_S2.xlsx (87.2KB, xlsx)

Data Availability Statement

Coordinates and structure factors were deposited in the RCSB Protein Data Bank with accession codes: 8UD6 for the wild-type T. thermophilus 70S ribosome in complex with mRNA, deacylated A-site tRNAPhe, aminoacylated P-site fMet-tRNAiMet, deacylated E-site tRNAPhe, and cresomycin; 8UD7 for the A2058-N6-dimethylated T. thermophilus 70S ribosome in complex with mRNA, deacylated A-site tRNAPhe, aminoacylated P-site fMettRNAiMet, deacylated E-site tRNAPhe, and cresomycin; 8UD8 for the A2503-C2,C8-dimethylated T. thermophilus 70S ribosome in complex with mRNA, deacylated A-site tRNAPhe, aminoacylated P-site fMet-tRNAiMet, deacylated E-site tRNAPhe, and cresomycin. Single-crystal X-ray crystallographic data for cresomycin are deposited at the Cambridge Crystallographic Data Centre (CCDC) under deposition number 2298060. All other data are available in the main text or the supplementary materials.

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