Bifunctional Nitrone-Conjugated Secondary Metabolite Targeting the Ribosome

Abstract

Many microorganisms possess the capacity for producing multiple antibiotic secondary metabolites. In a few notable cases, combinations of secondary metabolites produced by the same organism are used in important combination therapies for treatment of drug-resistant bacterial infections. However, examples of conjoined roles of bioactive metabolites produced by the same organism remain uncommon. During our genetic functional analysis of oxidase-encoding genes in the everninomicin producer Micromonospora carbonacea var. aurantiaca, we discovered previously uncharacterized antibiotics everninomicin N and O, comprised of an everninomicin fragment conjugated to the macrolide rosamicin via a rare nitrone moiety. These metabolites were determined to be hydrolysis products of everninomicin P, a nitrone-linked conjugate likely the result of nonenzymatic condensation of the rosamicin aldehyde and the octasaccharide everninomicin F, possessing a hydroxylamino sugar moiety. Rosamicin binds the erythromycin macrolide binding site approximately 60 Å from the orthosomycin binding site of everninomicins. However, while individual ribosomal binding sites for each functional half of everninomicin P are too distant for bidentate binding, ligand displacement studies demonstrated that everninomicin P competes with rosamicin for ribosomal binding. Chemical protection studies and structural analysis of everninomicin P revealed that everninomicin P occupies both the macrolide- and orthosomycin-binding sites on the 70S ribosome. Moreover, resistance mutations within each binding site were overcome by the inhibition of the opposite functional antibiotic moiety binding site. These data together demonstrate a strategy for coupling orthogonal antibiotic pharmacophores, a surprising tolerance for substantial covalent modification of each antibiotic, and a potential beneficial strategy to combat antibiotic resistance.

Ribosome‐targeting antibacterial agents: Advances, challenges, and opportunities

“…In vitro translation assay showed that, for E. coli ribosome with A2059G mutation which causes resistance to macrolides, the IC 50 values of rosamicin, everninomicin H (an analog of everninomicin F) and everninomicin P were >100, 0.1, and 0.1 μM, respectively. For E. coli ribosome with A2471C mutation which causes resistance to everninomicins, the IC 50 values of everninomicin H, rosamicin, and everninomicin P were >50, 5, and 10 μM, respectively 66 …”

“…A study showed that for S. aureus with G2576T mutations, the MIC range of linezolid was 16–32 μg/ml, while that of tedizolid was 1–2 μg/ml 65 . Limbrick et al 66 identified a nitrone‐conjugated secondary metabolite everninomicin P from mutated Micromonospora carbonacea var. aurantiaca 66 .…”

“…Limbrick et al 66 identified a nitrone‐conjugated secondary metabolite everninomicin P from mutated Micromonospora carbonacea var. aurantiaca 66 . Cryo‐EM experiments showed that this metabolite can be naturally dissociated into rosaramicin and everninomicin F (Figure 4D).…”

Microbial Oligosaccharides with Biomedical Applications

“…It is generally acknowledged that the rarely observed orthoester linkage is necessary for the antibiotic properties of the orthosomycins. Although the orthosomycin family includes hygromycin B, everninomicin, avilamycin, flambamycin, curamycin, etc., the former three members have been extensively studied, especially in the aspect of their bioactivity and biosynthesis [ 58 , 59 , 60 , 61 , 62 , 63 , 64 , 65 , 66 , 67 , 68 , 69 , 70 , 71 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Herein, restricted by the space, everninomicin (EVN) and avilamycin (AVI) will be mainly discussed in this review.…”

“…Until now, twenty EVN ( 43 – 62 ) ( Figure 8 ) have been purified from wide- or mutant-type M. carbonacea var. africana [ 59 , 60 , 61 , 62 , 63 , 71 ], while thirty-seven AVI ( 63 – 99 ) ( Figure 9 ) have been isolated from the wide- or mutant-type Streptomyces viridochromogenes Tü57 [ 59 , 64 , 72 , 73 , 74 , 75 , 76 , 77 , 78 , 79 , 80 , 81 , 82 ]. Structurally, EVN and AVI share the same seven-sugar core (rings B to H) and characteristic substituted partner including two orthoester linkages located between rings C and D and rings G and H and a methylenedioxy bridge attached to ring H. The major differences between EVN and AVI are attributed to the presence (or absence) of the A-ring nitrosugar and the orsellinic acid in EVN (or in AVI).…”