Scull Erin M, Bandari Chandrasekhar, Johnson Bryce P, Gardner Eric D, Tonelli Marco, You Jianlan, Cichewicz Robert H, Singh Shanteri
Department of Chemistry and Biochemistry, Stephenson Life Sciences Research Center, University of Oklahoma, 101 Stephenson Parkway, Norman, OK, 73019, USA.
National Magnetic Resonance Facility at Madison, University of Wisconsin-Madison, 411 Babcock Drive, Madison, WI, 45005, USA.
Appl Microbiol Biotechnol. 2020 Sep;104(18):7853-7865. doi: 10.1007/s00253-020-10790-x. Epub 2020 Jul 28.
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.
达托霉素是治疗由多种革兰氏阳性菌引起的感染的最后一道抗生素防线，这些细菌包括耐万古霉素肠球菌（VRE）以及耐甲氧西林和耐万古霉素金黄色葡萄球菌（MRSA和VRSA）。然而，近年来金黄色葡萄球菌和肠球菌的耐达托霉素菌株的出现，重新引发了人们对合成达托霉素类似物以克服耐药机制的兴趣。在此背景下，已证明三种芳香族异戊烯基转移酶可将达托霉素作为底物，并且所得的异戊烯基化类似物对革兰氏阳性菌的活性比母体化合物更强。因此，利用异戊烯基转移酶对达托霉素进行衍生化，为传统合成方法提供了一种有吸引力的替代方案，特别是考虑到该分子的结构复杂性。在此，我们报告利用异戊烯基转移酶CdpNPT与合成的非天然烷基焦磷酸文库相结合，合成烷基多样化的达托霉素类似物的能力。结果表明，CdpNPT可以使用相应的烷基焦磷酸将多种烷基转移到达托霉素的色氨酸残基上，而随后的放大反应表明该酶可以使吲哚环的N1、C2、C5和C6位烷基化。使用16种达托霉素类似物进行的体外抗菌活性测定表明，其中一些类似物对MRSA、VRE以及耐达托霉素的金黄色葡萄球菌和粪肠球菌菌株的活性提高了2至80倍。因此，连同新的强效类似物一起，这些发现表明色氨酸残基上烷基取代的区域化学可以调节达托霉素的活性。通过额外的蛋白质工程来提高区域选择性，所描述的方法有可能成为使复杂的含吲哚分子多样化的有力工具。要点：• CdpNPT以达托霉素作为受体时表现出令人印象深刻的供体混杂性。• CdpNPT催化达托霉素色氨酸残基上的N1、C2、C5和C6烷基化。• 达托霉素色氨酸残基的不同烷基化调节其活性。
