Abstract:Therapeutic options to combat Gram-negative bacterial pathogens are dwindling with increasing antibiotic resistance. This study presents a proof of concept for the heterologous-expression approach to expand on the novel antibiotic class of darobactins and to generate analogs with different activities and pharmacokinetic properties.
“…4 ). This observation suggests that DarBCD do not play an essential role in darobactin biosynthesis, which is consistent with the recent heterologous studies 28 – 30 . Fig.…”
Darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP), which possesses potent activity against various Gram-negative bacteria. Darobactin features a highly unique bicyclic scaffold, consisting of an ether crosslink between two Trp residues and a C–C crosslink between a Lys and a Trp. Here we report in vivo and in vitro activity of darobactin synthase DarE. We show DarE is a radical S-adenosylmethionine (rSAM) enzyme and is solely responsible for forming the bicyclic scaffold of darobactin. DarE mainly produced the ether-crosslinked product in vitro, and when the assay was performed in H218O, apparent 18O incorporation was observed into the ether-crosslinked product. These observations suggested an rSAM-dependent process in darobactin biosynthesis, involving a highly unusual oxygen insertion step from a water molecule and subsequent O–H and C–H activations. Genome mining analysis demonstrates the diversity of darobactin-like biosynthetic gene clusters, a subclade of which likely encode monocyclic products with only an ether linkage. We propose the name daropeptide for this growing family of ether-containing RiPPs produced by DarE enzymes.
“…4 ). This observation suggests that DarBCD do not play an essential role in darobactin biosynthesis, which is consistent with the recent heterologous studies 28 – 30 . Fig.…”
Darobactin is a ribosomally synthesized and post-translationally modified peptide (RiPP), which possesses potent activity against various Gram-negative bacteria. Darobactin features a highly unique bicyclic scaffold, consisting of an ether crosslink between two Trp residues and a C–C crosslink between a Lys and a Trp. Here we report in vivo and in vitro activity of darobactin synthase DarE. We show DarE is a radical S-adenosylmethionine (rSAM) enzyme and is solely responsible for forming the bicyclic scaffold of darobactin. DarE mainly produced the ether-crosslinked product in vitro, and when the assay was performed in H218O, apparent 18O incorporation was observed into the ether-crosslinked product. These observations suggested an rSAM-dependent process in darobactin biosynthesis, involving a highly unusual oxygen insertion step from a water molecule and subsequent O–H and C–H activations. Genome mining analysis demonstrates the diversity of darobactin-like biosynthetic gene clusters, a subclade of which likely encode monocyclic products with only an ether linkage. We propose the name daropeptide for this growing family of ether-containing RiPPs produced by DarE enzymes.
“…For the design of new non-native Darobactin derivatives, we considered current available data at this time, including relevant interactions of D9 (W 1 S 2 W 3 S 4 K 5 S 6 W 7 ) (Figure 1) and Darobactin A (W 1 S 2 W 3 S 4 K 5 S 6 F 7 ) with the BAM complex and the bioactivity ranking of Darobactin-containing extracts (Darobactin A to E and 1 to 21), as well as the pure Darobactins A, B and 9. [10,19,21] To ensure proper ligand-receptor interaction (Figure 1 g, h), positions 1, 2 and 3 of Darobactin remained unaltered. Furthermore, the binding behaviour of Darobactin A and 9 on E. coli BAM complex revealed position 4, 6 and 7 as the most flexible positions for core sequence engineering, without deteriorating target binding (Figure 1 g, h).…”
Section: Resultsmentioning
confidence: 99%
“…Additionally, increased activity for Darobactin B and Darobactin 4 (WNWTKSF) compared to Darobactin A (WNWSKSF) motivated the exchange of L-serine to L-threonine at position 4. [19,21] The previous breakthrough in terms of increased activity by replacing the terminal L-phenylalanine with an L-tryptophan led to the design of more derivatives derived from D9, while varying positions 4 and 6. Consequently, derivatives 22 to 25 and 36 to 38 carrying L-arginine, L-histidine, L-serine, L-alanine, L-lysine or L-threonine on position 6 and L-threonine on position 4, as present in the more active Darobactin B [21] were altered to study the influence of each position.…”
Section: Resultsmentioning
confidence: 99%
“…[19,21] The previous breakthrough in terms of increased activity by replacing the terminal L-phenylalanine with an L-tryptophan led to the design of more derivatives derived from D9, while varying positions 4 and 6. Consequently, derivatives 22 to 25 and 36 to 38 carrying L-arginine, L-histidine, L-serine, L-alanine, L-lysine or L-threonine on position 6 and L-threonine on position 4, as present in the more active Darobactin B [21] were altered to study the influence of each position. Changing L-serine at position 4 to L-alanine, seems to have less impact on activity, as observed in D14 (WNWAKSF), [19] probably caused in no direct side-chain interaction of position 4 (Figure 1 g, h).…”
Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. Darobactins, a recently discovered class of peptide antibiotics, belong to the most promising drug candidates, binding to a new target, the outer membrane protein BamA. Previously, we reported that biosynthetic engineering in a heterologous host allowed for the production of non-natural Darobactins with significantly enhanced antibacterial activity. We here utilize an optimized purification procedure and present cryo-EM structures of the Bam-complex with Darobactin 9 (D9), motivating the biotechnological generation of twenty novel Darobactins including halogenated analogues via structure-based design. The newly engineered Darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, while toxic effects were not observed in human cells and zebrafish embryos up to 500 µg/mL.
“…The rigid structure of darobactin A is likely responsible for its activity, based on crystallographic studies. 2 The fact that all natural analogs retain the unique core skeleton [3][4] , which acts as a 𝛽-strand mimetic 2 , supports this notion. Architecturally ornate peptide-based macrocycles have a rich history of acting as promising antibacterial agents from the clinically validated vancomycins [5][6][7][8] to the more recently pursued arylomycins.…”
A concise, modular synthesis of the novel antibiotic darobactin A is disclosed. The synthesis successfully forges the hallmark strained macrocyclic ring systems in a sequential fashion. Key transformations include two atroposelective Larock-macrocylizations, one of which proceeds with exquisite regioselectivity despite bearing an unprotected alkyne. The synthesis is designed with medicinal chemistry considerations in mind, appending key portions of the molecule at a late-stage. Requisite unnatural amino acid building blocks are easily prepared in enantiopure form using C–H activation and decarboxylative cross-coupling tactics.
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