Targeted and Logical Discovery of Piperazic Acid-Bearing Natural Products Based on Genomic and Spectroscopic Signatures

• Published in: Journal of the American Chemical Society Vol. 145; no. 36; pp. 19676 - 19690
• Main Authors: Shin, Daniel, Byun, Woong Sub, Kang, Sangwook, Kang, Ilnam, Bae, Eun Seo, An, Joon Soo, Im, Ji Hyeon, Park, Jiyoon, Kim, Eunji, Ko, Keebeom, Hwang, Sunghoon, Lee, Honghui, Kwon, Yun, Ko, Yoon-Joo, Hong, Suckchang, Nam, Sang-Jip, Kim, Seung Bum, Fenical, William, Yoon, Yeo Joon, Cho, Jang-Cheon, Lee, Sang Kook, Oh, Dong-Chan
• Format: Journal Article
• Language: English
• Published: American Chemical Society 13.09.2023
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/jacs.3c04699

A targeted and logical discovery method was devised for natural products containing piperazic acid (Piz), which is biosynthesized from ornithine by l-ornithine N-hydroxylase (KtzI) and N–N bond formation enzyme (KtzT). Genomic signature-based screening of a bacterial DNA library (2020 strains) using polymerase chain reaction (PCR) primers targeting ktzT identified 62 strains (3.1%). The PCR amplicons of KtzT-encoding genes were phylogenetically analyzed to classify the 23 clades into two monophyletic groups, I and II. Cultivating hit strains in media supplemented with 15NH4Cl and applying 1H–15N heteronuclear multiple bond correlation (HMBC) along with 1H–15N heteronuclear single quantum coherence (HSQC) and 1H–15N HSQC-total correlation spectroscopy (HSQC-TOCSY) NMR experiments detected the spectroscopic signatures of Piz and modified Piz. Chemical investigation of the hit strains prioritized by genomic and spectroscopic signatures led to the identification of a new azinothricin congener, polyoxyperuin B seco acid (1), previously reported chloptosin (2) in group I, depsidomycin D (3) incorporating two dehydropiperazic acids (Dpz), and lenziamides A and B (4 and 5), structurally novel 31-membered cyclic decapeptides in group II. By consolidating the phylogenetic and chemical analyses, clade–structure relationships were elucidated for 19 of the 23 clades. Lenziamide A (4) inhibited STAT3 activation and induced G2/M cell cycle arrest, apoptotic cell death, and tumor growth suppression in human colorectal cancer cells. Moreover, lenziamide A (4) resensitized 5-fluorouracil (5-FU) activity in both in vitro cell cultures and the in vivo 5-FU-resistant tumor xenograft mouse model. This work demonstrates that the genomic and spectroscopic signature-based searches provide an efficient and general strategy for new bioactive natural products containing specific structural motifs.