PMC:6218659 / 32307-34985 JSONTXT

{"project":"2_test","denotations":[{"id":"30388443-22717438-20779503","span":{"begin":523,"end":527},"obj":"22717438"},{"id":"30388443-29844049-20779504","span":{"begin":662,"end":666},"obj":"29844049"},{"id":"30388443-22123769-20779505","span":{"begin":1511,"end":1515},"obj":"22123769"}],"text":"Development of Non-toxic “TUN-X,X” Analogues of Tunicamycin\nThe potent “off-target” inhibitory effects of tunicamycin on DPAGT1 prevent its use as an antibiotic. We used structural data coupled with genetics to design analogues (TUN-X,X) that retained anti-microbial activity yet no longer inhibited DPAGT1. We previously cloned and sequenced the tunicamycin biosynthetic gene cluster (tun) from Streptomyces chartreusis and expressed it heterologously in Streptomyces coelicolor (Wyszynski et al., 2010, Wyszynski et al., 2012). The cluster contains 14 genes, tunA-N. In-frame deletion mutations in all of the cloned tun genes in S. coelicolor (Widdick et al., 2018) revealed new insights into tunicamycin biosynthesis. Interestingly, deletion of tunI and tunJ, encoding components of an ABC transporter conferring immunity to tunicamycin, could only be achieved with mutations elsewhere in the tun gene cluster. Sequencing of one of the ΔtunI mutants revealed a G-to-A missense suppressor mutation in tunC, resulting in a Gly70Asp substitution in the N-acyltransferase TunC that attaches the lipid of tunicamycin. This presumed loss of function mutation abolished antibacterial activity, consistent with a key role for the lipid chain in the biological activity of tunicamycin.\nWe therefore designed a semi-synthetic strategy to access systematically “lipid-altered” variants of tunicamycin based on tunicamine scaffold TUN (Figure 5A). Large-scale fermentation of S. chartreusis NRRL 3882 (Doroghazi et al., 2011) (Methods S1) allowed access to crude tunicamycin on a multi-gram scale. Degradative conversion (Ito et al., 1979) of tunicamycin gave unfunctionalized core scaffold TUN. Critically, since the nucleobase of tunicamycin is hydrolytically sensitive, the creation of mixed Boc-imides at positions 10ʹ and 2ʹʹ allowed mild, selective deamidation on a gram-scale (see Supplemental Information SI 2). Chemoselective carbodiimide- or uronate-mediated acylation allowed direct lipid-tuning in a systematic, divergent manner through modification at 10ʹ-N and/or 2ʹʹ-N, yielding a library of novel analogues, TUN-X,X varying in chain length by one carbon, from C7 to C12 (TUN-7,7 to TUN-12,12, Figure 5A) with a typical purity of \u003e99% (see Methods S1) as judged by NMR and/or HPLC.\nFigure 5 Semi-synthetic Synthesis and Antibacterial Effects of TUN-X,X Analogues\n(A) Semi-synthetic strategy for TUN mimics.\n(B–D) MIC obtained from micro-broth dilution antimicrobial susceptibility tests of (B) B. subtilis EC1524, (C) B. cereus ATCC11778, (D) Mtb H37Rv (ATCC27294) cultured in 7H9/ADC/Tw (black), or GAST/Fe (grey) media (n=3). For all panels data presented are means ± SD"}