Abstract
Daptomycin is a last resort antibiotic for the treatment of infections caused by many Gram-positive bacterial strains, including vancomycin-resistant Enterococcus (VRE) and methicillin- and vancomycin-resistant Staphylococcus aureus (MRSA and VRSA). However, the emergence of daptomycin-resistant strains of S. aureus and Enterococcus in recent years has renewed interest in synthesizing daptomycin analogs to overcome resistance mechanisms. Within this context, three aromatic prenyltransferases have been shown to accept daptomycin as a substrate, and the resulting prenylated analog was shown to be more potent against Gram-positive strains than the parent compound. Consequently, utilizing prenyltransferases to derivatize daptomycin offered an attractive alternative to traditional synthetic approaches, especially given the molecule’s structural complexity. Herein, we report exploiting the ability of prenyltransferase CdpNPT to synthesize alkyl-diversified daptomycin analogs in combination with a library of synthetic non-native alkyl-pyrophosphates. The results revealed that CdpNPT can transfer a variety of alkyl groups onto daptomycin’s tryptophan residue using the corresponding alkyl-pyrophosphates, while subsequent scaled-up reactions suggested that the enzyme can alkylate the N1, C2, C5, and C6 positions of the indole ring. In vitro antibacterial activity assays using 16 daptomycin analogs revealed that some of the analogs displayed 2–80-fold improvements in potency against MRSA, VRE, and daptomycin-resistant strains of S. aureus and Enterococcus faecalis. Thus, along with the new potent analogs, these findings have established that the regio-chemistry of alkyl substitution on the tryptophan residue can modulate daptomycin’s potency. With additional protein engineering to improve the regio-selectivity, the described method has the potential to become a powerful tool for diversifying complex indole-containing molecules.Key points• CdpNPT displays impressive donor promiscuity with daptomycin as the acceptor.• CdpNPT catalyzes N1-, C2-, C5-, and C6-alkylation on daptomycin’s tryptophan residue.• Differential alkylation of daptomycin’s tryptophan residue modulates its activity.
Highlights
Daptomycin (Dap, Fig. 1a) belongs to a family of calciumdependent lipodepsipeptide antibiotics produced by Streptomyces roseosporus (Debono et al 1987; Miao et al 2005)
It is active against several resistant strains of Grampositive bacteria, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA), vancomycin-resistant Staphylococcus aureus (VRSA), and vancomycin-resistant Enterococci (VRE) (Steenbergen et al 2005; Tally and Debruin 2000)
The assessment of CdpNPT-catalyzed alkylation of Dap was carried out using a library of 38 non-native alkyl-PPs synthesized in-house as described previously (1–4, 6–39, Fig. 3) (Bandari et al 2019; Bandari et al 2017; Johnson et al 2020)
Summary
Daptomycin (Dap, Fig. 1a) belongs to a family of calciumdependent lipodepsipeptide antibiotics produced by Streptomyces roseosporus (Debono et al 1987; Miao et al 2005). Dap is one of the most promising drugs currently under evaluation for the treatment of pneumococcal meningitis (Cottagnoud et al 2004; Muri et al 2018), and while it is active against Streptococcus pneumoniae, it does not meet non-inferiority criteria for the treatment of community-acquired pneumonia due to inhibition by lung surfactants (Silverman et al 2005) These potent antibacterial activities have been traced back to Dap’s complex structure (Fig. 1a) leading to its unique mechanism of action (MOA), which has recently been shown to involve binding to regions of increased fluidity (RIFs) within the Gram-positive cell membrane (Müller et al 2016). De-anchoring of the lipid II synthase MurG and the phospholipid synthase PlsX was proposed to hinder synthesis of the cell wall and outer membrane, respectively, resulting in Dap’s bactericidal effect (Gray and Wenzel 2020; Müller et al 2016)
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