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

A targeted and logical discovery method was devised for natural products containing piperazic acid (Piz), which is biosynthesized from ornithine by l-ornithine -hydroxylase (KtzI) and - bond formation enzyme (KtzT). Genomic signature-based screening of a bacterial DNA library (2020 strains) using polymerase chain reaction (PCR) primers targeting 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 NHCl and applying H-N heteronuclear multiple bond correlation (HMBC) along with H-N heteronuclear single quantum coherence (HSQC) and H-N 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 (), previously reported chloptosin () in group I, depsidomycin D () incorporating two dehydropiperazic acids (Dpz), and lenziamides A and B ( and ), 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 () inhibited STAT3 activation and induced G/M cell cycle arrest, apoptotic cell death, and tumor growth suppression in human colorectal cancer cells. Moreover, lenziamide A () 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.