Muraymycin Nucleoside Antibiotics: Structure-Activity Relationship for Variations in the Nucleoside Unit

Muraymycins are a subclass of naturally occurring nucleoside antibiotics with promising antibacterial activity. They inhibit the bacterial enzyme translocase I (MraY), a clinically yet unexploited target mediating an essential intracellular step of bacterial peptidoglycan biosynthesis. Several structurally simplified muraymycin analogues have already been synthesized for structure–activity relationship (SAR) studies. We now report on novel derivatives with unprecedented variations in the nucleoside unit. For the synthesis of these new muraymycin analogues, we employed a bipartite approach facilitating the introduction of different nucleosyl amino acid motifs. This also included thymidine- and 5-fluorouridine-derived nucleoside core structures. Using an in vitro assay for MraY activity, it was found that the introduction of substituents in the 5-position of the pyrimidine nucleobase led to a significant loss of inhibitory activity towards MraY. The loss of nucleobase aromaticity (by reduction of the uracil C5-C6 double bond) resulted in a ca. tenfold decrease in inhibitory potency. In contrast, removal of the 20 -hydroxy group furnished retained activity, thus demonstrating that modifications of the ribose moiety might be well-tolerated. Overall, these new SAR insights will guide the future design of novel muraymycin analogues for their potential development towards antibacterial drug candidates

Synthesis of an Antimicrobial Enterobactin-Muraymycin Conjugate for Improved Activity Against Gram-Negative Bacteria

Overcoming increasing antibiotic resistance requires the development of novel antibacterial agents that address new targets in bacterial cells. Naturally occurring nucleoside antibiotics (such as muraymycins) inhibit the bacterial membrane protein MraY, a clinically unexploited essential enzyme in peptidoglycan (cell wall) biosynthesis. Even though a range of synthetic muraymycin analogues has already been reported, they generally suffer from limited cellular uptake and a lack of activity against Gram-negative bacteria. We herein report an approach to overcome these hurdles: a synthetic muraymycin analogue has been conjugated to a siderophore, i. e. the enterobactin derivative EntKL, to increase the cellular uptake into Gram-negative bacteria. The resultant conjugate showed significantly improved antibacterial activity against an efflux-deficient E. coli strain, thus providing a proof-ofconcept of this novel approach and a starting point for the future optimisation of such conjugates towards potent agents against Gram-negative pathogens

Self-Resistance During Muraymycin Biosynthesis: A Complementary Nucleotidyltransferase and Phosphotransferase with Identical Modification Sites and Distinct Temporal Order

Muraymycins are antibacterial natural products from Streptomyces spp. that inhibit translocase I (MraY), which is involved in cell wall biosynthesis. Structurally, muraymycins consist of a 5′-C-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 in vitro 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

Muraymycin nucleoside antibiotics : novel SAR Insights and synthetic approaches

Muraymycins, a class of structurally complex nucleoside-peptide natural products, inhibit MraY, an enzyme involved in bacterial cell wall biosynthesis. The present work comprises a detailed structure-activity relationship study on the peptide chain of muraymycins and its contribution to inhibitory activity. Several systematically truncated and simplified analogues were synthesised via a modular approach. Evaluation of these compounds in in vitro assays revealed that the full-length muraymycin backbone is a key feature for inhibitory potency. Moreover, a novel synthetic approach was developed that allowed the preparation of full-length muraymycin analogues on solid support via solid phase peptide synthesis. By that, a protocol preparing peptide aldehydes on the solid phase combined with late-stage coupling to the nucleoside building block was identified as most efficient access to new analogues. The advantages of this method were demonstrated with an alanine scan of the peptide backbone. The obtained analogues showed a surprisingly pronounced influence of the leucine residue of the muraymycin peptide chain on activity. Put together, these results provided extended elucidation of the SAR of muraymycins. Promising strategies for further developments could be deduced.Muraymycine sind eine Klasse strukturell komplexer Nucleosid-Peptid-Naturstoffe und hemmen MraY, ein Enzym der bakteriellen Zellwand-Biosynthese. Die vorliegende Arbeit untersucht detailliert die Struktur-Aktivitäts-Beziehungen der Peptidkette der Muraymycine. Dazu wurden mehrere systematisch verkürzte und vereinfachte Analoga mit Hilfe eines modularen Ansatzes dargestellt und in einem in vitro-Aktivitäts-Assay evaluiert. Dabei konnte gezeigt werden, dass das vollständige Grundgerüst der Muraymycine eine Schlüsselrolle für eine effiziente Hemmung des Enzyms einnimmt. Zudem wurde ein neuer Syntheseansatz entwickelt, in dem Muraymycin-Derivate an einem festen Träger nach dem Prinzip der Festphasenpeptidsynthese dargestellt werden können. Als besonders effektiv erwies sich dabei die Darstellung von Peptidaldehyden, die erst in einem späten Schritt mit dem Nucleosid-Baustein verknüpft werden. Die Vorteile dieser Methode wurden in einem Alanin-Scan des Peptidgerüsts demonstriert. Bei der biologischen Evaluierung zeigte sich ein unerwartet großer Einfluss der Leucin-Einheit der Peptidkette auf die inhibitorische Aktivität dieser Muraymycin-Analoga. Zusammengefasst ermöglichen diese Ergebnisse tiefgehende Einblicke in die Struktur-Aktivitäts- Beziehung der peptidischen Teilstruktur von Muraymycinen und ihrer Analoga. Auf dieser Basis konnten vielversprechende Ansatzpunkte für weitergehende Modifikationen der Muraymycin- Leitstruktur identifiziert werden

Aminoribosylated Analogues of Muraymycin Nucleoside Antibiotics

Nucleoside antibiotics are uridine-derived natural products that inhibit the bacterial membrane protein MraY. MraY is a key enzyme in the membrane-associated intracellular stages of peptidoglycan biosynthesis and therefore considered to be a promising, yet unexploited target for novel antibacterial agents. Muraymycins are one subclass of such naturally occurring MraY inhibitors. As part of structure-activity relationship (SAR) studies on muraymycins and their analogues, we now report on novel derivatives with different attachment of one characteristic structural motif, i.e., the aminoribose moiety normally linked to the muraymycin glycyluridine core unit. Based on considerations derived from an X-ray co-crystal structure, we designed and synthesised muraymycin analogues having the aminoribose attached (via a linker) to either the glycyluridine amino group or to the uracil nucleobase. Reference compounds bearing the non-aminoribosylated linker units were also prepared. It was found that the novel aminoribosylated analogues were inactive as MraY inhibitors in vitro, but that the glycyluridine-modified reference compound retained most of the inhibitory potency relative to the unmodified parent muraymycin analogue. These results point to 6′-N-alkylated muraymycin analogues as a potential novel variation of the muraymycin scaffold for future SAR optimisatio

Unexpected Seven-Membered Ring Formation for Muraymycin-Type Nucleoside-Peptide Antibiotics

Naturally occurring nucleoside-peptide antibiotics such as muraymycins or caprazamycins are of major interest for the development of novel antibacterial agents. However, the synthesis of new analogues of these natural products for structure–activity relationship (SAR) studies is challenging. In our synthetic efforts towards a muraymycin-derived nucleoside building block suitable for attachment to a solid support, we came across an interesting side product. This compound resulted from an undesired Fmoc deprotection with subsequent cyclization, thus furnishing a remarkable caprazamycin-like seven-membered diazepanone ring

DISCOVERY OF NOVEL MURAYMYCIN ANTIBIOTICS AND INSIGHT INTO THE BIOSYNTHETIC PATHWAY

New antibiotics with novel targets or mechanisms of action are needed to counter the steady emergence of bacterial pathogens that are resistant to antibiotics used in the clinic. MraY, a promising novel target for antibiotic development, initiates the lipid cycle for the biosynthesis of peptidoglycan cell wall, which is essential for the survival of most, if-not-all, bacteria. MraY is an enzyme that catalyzes the transfer and attachment of phospho-MurNAc-pentapeptide to a lipid carrier, undecaprenylphosphate. Muraymycins are recently discovered lipopeptidyl nucleoside antibiotics that exhibit remarkable antibiotic activity against Gram-positive as well as Gram-negative bacteria by inhibiting MraY. We conducted a thorough examination of the metabolic profile of Streptomyces sp. strain NRRL 30473, a known producer of muraymycins. Eight muraymycins were isolated and characterized by a suite of spectroscopic methods, including three new members of muraymycin family named B8, B9 and C5. Muraymycins B8 and B9, which differ from other muraymycins by having an elongated fatty acid side chain, showed potent antibacterial activity against Escherichia coli ∆tolC mutant and pM IC50 against Staphylococcus aureus MraY. Muraymycin C5, which is characterized by an N-acetyl modification of the disaccharide’s primary amine, greatly reduced its antibacterial activity, which possibly indicates this modification is used for self-resistance. In addition to the discovery of new muraymycins, eleven enzymes from the biosynthetic pathway were functionally assigned and characterized in vitro. Six enzymes involved in the biosynthesis of amino ribofuranosylated uronic acid moiety of muraymycin were characterized: Mur16, a non-heme, Fe(II)-dependent α-ketoglutarate: UMP dioxygenase; Mur17, an L-threonine: uridine-5′-aldehyde transaldolase; Mur20, an L-methionine:1-aminotransferase; Mur26, a low specificity pyrimidine nucleoside phosphorylase; Mur18, a primary amine-requiring nucleotidylyltransferase; Mur19, a 5-amino-5-deoxyribosyltransferase. A one-pot enzyme reaction was utilized to produce this disaccharide moiety and its 2′′-deoxy analogue. Two muraymycin-modifying enzymes that confer self-resistance were functionally assigned and characterized: Mur28, a TmrB-like ATP-dependent muraymycin phosphotransferase, and Mur29, a muraymycin nucleotidyltransferase. Notably, Mur28 preferentially phosphorylates the intermediate, aminoribofuranosylated uronic acid, in the muraymycin biosynthetic pathway to produce a cryptic phosphorylated-dissacharide intermediate. Mur23 and Mur24 were assigned as two enzymes that modify the cryptic phosphorylated intermediate by attachment of an aminopropyl group. Mur24 catalyzes the incorporation of butyric acid into the phosphorylated-disaccharide. Following the incorporation, Mur23 catalyzes a PLP-dependent decarboxylation. Finally, Mur15, which belongs to the cupin family, is functionally assigned as a non-heme, Fe(II)-dependent α-ketoglutarate dioxygenase that catalyzes the β-hydroxylation of a leucine moiety in muraymycin D1 to form muraymycin C1. Mur15 can also hydroxylate the γ-position of leucine moiety to muraymycins with fatty acid chain in β-position

Solid Phase-Supported Synthesis of Muraymycin Analogues

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

Mechanism of action of nucleoside antibacterial natural product antibiotics

This article reviews the structures and biological activities of several classes of uridine-containing nucleoside antibiotics (tunicamycins, mureidomycins/pacidamycins/sansanmycins, liposidomycins/caprazamycins, muraymycins, capuramycins) that target translocase MraY on the peptidoglycan biosynthetic pathway. In particular, recent advances in structure-function studies, and recent X-ray crystal structures of translocase MraY complexed with muraymycin D2 and tunicamycin are described. The inhibition of other phospho-nucleotide transferase enzymes related to MraY by nucleoside antibiotics and analogues is also reviewed

Studies on the Selectivity of Nucleoside Antibiotics

Das Ziel der vorliegenden Arbeit war die Synthese und biologische Evaluierung von Derivaten der Nucleosid-Antibiotika als MraY-Inhibitoren mit antibakterieller Aktivität. In Teil A wurden zehn vereinfachte Muraymycin-Analoga als Teil einer Struktur-Aktivitätsbeziehungs-(SAR)-Studie erfolgreich synthetisiert. Unter sechs antibakteriell aktiven Verbindungen, inklusive einer literaturbekannten Referenz, konnte eine neuartige Leitstruktur identifiziert werden, welche nennenswerte Aktivitäten gegen P. aeruginosa, S. aureus und E. coli aufwies. Zusätzlich konnte eine gewisse Selektivität bezüglich der Toxizität gegenüber Bakterienzellen im Vergleich zu humanen HepG2-Zellen gezeigt werden. In Teil B wurde ein Hybrid-Antibiotikum, welches die Peptidstruktur von Sansanmycin B mit der 5'-defunktionalisierten Nucleosid-Einheit der Muraymycine verbindet, dargestellt. Zusätzlich wurden drei Fragmente, welche Schlüsselintermediate der Syntheseroute repräsentieren, synthetisiert. Alle vier Verbindungen wurden als MraY-Inhibitoren identifiziert, wodurch das Konzept solcher Hybrid-Strukturen bestätigt werden konnte. Die in-vitro-Aktivität der Fragmente korrelierte sowohl zu ihrer Struktur als auch zueinander, wodurch der Weg für weitere SAR-Studien geebnet wurde. Das Hybrid-Antibiotikum zeigte keine antibakterielle Aktivität, inhibierte MraY jedoch im hohen nanomolaren Bereich und stellt damit eine geeignete Leitstruktur für weitere Studien dar.This PhD project focused on the synthesis and biological evaluation of analogues of nucleoside antibiotics as MraY inhibitors with enhanced antibacterial activity. In part A, ten simplified muraymycin analogues were synthesized as part of a detailed structure-activity relationship study via a modular approach. Among six antimicrobially active target compounds, including a literature-known reference, a novel lead structure was identified which exhibited noteworthy activity against P. aeruginosa, S. aureus and E .coli, which furthermore displayed a certain selectivity of toxicity towards bacteria over human HepG2 cells. In part B, a hybrid antibiotic combining the peptide chain of sansanmycin B with the nucleoside moiety of 5'-defunctionalized muraymycins has been synthesized along with three fragments, which represent key intermediates of the synthetic route. All four target compounds were found to be MraY inhibitors, providing a general proof of concept for such hybrid-type structures. In addition, the in vitro inhibitory activity of the fragments corresponded well to each other and to their structure, paving the way for further SAR studies based on these results. The hybrid antibiotic itself did not show antibacterial activity against the tested bacterial strains, but inhibited MraY in vitro in the high nanomolar range. It was therefore found to be a suitable lead for further optimization and SAR studies