SummaryMuraymycins are a promising class of antimicrobial natural products. These uridine-derived nucleoside-peptide antibiotics inhibit the bacterial membrane protein translocase I (MraY), a key enzyme in the intracellular part of peptidoglycan biosynthesis. This review describes the structures of naturally occurring muraymycins, their mode of action, synthetic access to muraymycins and their analogues, some structure–activity relationship (SAR) studies and first insights into muraymycin biosynthesis. It therefore provides an overview on the current state of research, as well as an outlook on possible future developments in this field.

Muraymycins are a subclass of antimicrobially active uridine-derived natural products. Biological data on several muraymycin analogues have been reported, including some inhibitory in vitro activities toward their target protein, the bacterial membrane enzyme MraY. However, a structure-activity relationship (SAR) study on naturally occurring muraymycins based on such in vitro data has been missing so far. In this work, we report a detailed SAR investigation on representatives of the four muraymycin subgroups A-D using a fluorescence-based in vitro MraY assay. For some muraymycins, inhibition of MraY with IC values in the low-picomolar range was observed. These inhibitory potencies were compared with antibacterial activities and were correlated to modelling data derived from a previously reported X-ray crystal structure of MraY in complex with a muraymycin inhibitor. Overall, these results will pave the way for the development of muraymycin analogues with optimized properties as antibacterial drug candidates.

Nucleoside analogues have found widespread application as antiviral and antitumor agents, but not yet as antibacterials. Naturally occurring uridine-derived ‘nucleoside antibiotics’ target the bacterial membrane protein MraY, an enzyme involved in peptidoglycan biosynthesis and a promising target for the development of novel antibacterial agents. Muraymycins represent a nucleoside-peptide subgroup of such MraY-inhibiting natural products. As part of detailed structure-activity relationship (SAR) studies on muraymycins and their analogues, we now report novel insights into the effects of stereochemical variations in the nucleoside core structure. Using a simplified version of the muraymycin scaffold, it was shown that some formal inversions of stereochemistry led to about one order of magnitude loss in inhibitory potency towards the target enzyme MraY. In contrast, epimers of the core motif with retained inhibitory activity were also identified. These 5′,6′-anti-configured analogues might serve as novel chemically tractable variations of the muraymycin scaffold for the future development of uridine-derived drug candidates.

Muraymycins are nucleoside antibiotics isolated from Streptomyces sp. NRRL 30471 and several mutant strains thereof that were generated by random, chemical mutagenesis. Reinvestigation of two mutant strains using new media conditions led to the isolation of three new muraymycin congeners, named B8, B9, and C6 (1-3), as well as a known muraymycin, C1. Structures of the compounds were elucidated by HRMS and 1D and 2D NMR spectroscopic analyses. Complete 2D NMR assignments for the known muraymycin C1 are also provided for the first time. Compounds 1 and 2, which differ from other muraymycins by having an elongated, terminally branched fatty acid side chain, had picomolar IC values against Staphylococcus aureus and Aquifex aeolicus MraY and showed good antibacterial activity against S. aureus (MIC = 2 and 6 μg/mL, respectively) and Escherichia coli Δ tolC (MIC = 4 and 2 μg/mL, respectively). Compound 3, which is characterized by an N-acetyl modification of the primary amine of the dissacharide core that is shared among nearly all of the reported muraymycin congeners, greatly reduced its inhibitory and antibacterial activity compared to nonacylated muraymycin C1, which possibly indicates this modification is used for self-resistance.

The membrane protein translocase I (MraY) is a key enzyme in bacterial peptidoglycan biosynthesis. It is therefore frequently discussed as a target for the development of novel antibiotics. The screening of compound libraries for the identification of MraY inhibitors is enabled by an established fluorescence-based MraY assay. However, this assay requires a dansylated derivative of the bacterial biosynthetic intermediate Park's nucleotide as the MraY substrate. Isolation of Park's nucleotide from bacteria and subsequent dansylation only furnishes limited amounts of this substrate, thus hampering the high-throughput screening for MraY inhibitors. Accordingly, the efficient provision of dansylated Park's nucleotide is a major bottleneck in the exploration of this promising drug target. In this work, we present the first total synthesis of dansylated Park's nucleotide, affording an unprecedented amount of the target compound for high-throughput MraY assays.

Muraymycins are antibacterial natural products from spp. that inhibit translocase I (MraY), which is involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5'--glycyluridine (GlyU) appended to a 5″-amino-5″-deoxyribose (ADR), forming a disaccharide core that is found in several peptidyl nucleoside inhibitors of MraY. For muraymycins, the GlyU-ADR disaccharide is further modified with an aminopropyl-linked peptide to generate the simplest structures, annotated as the muraymycin D series. Two enzymes encoded in the muraymycin biosynthetic gene cluster, Mur29 and Mur28, were functionally assigned as a Mg·ATP-dependent nucleotidyltransferase and a Mg·ATP-dependent phosphotransferase, respectively, both modifying the 3″-OH of the disaccharide. Biochemical characterization revealed that both enzymes can utilize several nucleotide donors as cosubstrates and the acceptor substrate muraymycin also behaves as an inhibitor. Single-substrate kinetic analyses revealed that Mur28 preferentially phosphorylates a synthetic GlyU-ADR disaccharide, a hypothetical biosynthetic precursor of muraymycins, while Mur29 preferentially adenylates the D series of muraymycins. The adenylated or phosphorylated products have significantly reduced (170-fold and 51-fold, respectively) MraY inhibitory activities and reduced antibacterial activities, compared with the respective unmodified muraymycins. The results are consistent with Mur29-catalyzed adenylation and Mur28-catalyzed phosphorylation serving as complementary self-resistance mechanisms, with a distinct temporal order during muraymycin biosynthesis.

Naturally occurring muraymycin nucleoside antibiotics represent a promising class of novel antimicrobials as they inhibit MraY, an enzyme involved in bacterial cell wall biosynthesis. The synthesis of muraymycins and their analogues is challenging as it involves multi‐step routes, thus hampering detailed structure‐activity relationship (SAR) studies. In this work, we report a novel solid phase‐based synthetic strategy for accessing muraymycin analogues via a modular approach, thereby enabling a more efficient access to structural variations, particularly of the muraymycin peptide moiety. The efficiency of this new method was exemplified in an alanine scan of the peptide unit. The inhibitory in vitro activities of the resultant analogues towards MraY provided novel SAR insights. Overall, this new synthetic method for the preparation of muraymycin analogues might support the development of these antibacterial agents towards potential drug candidates.

No abstract