Proteomics Guided Discovery of Flavopeptins: Anti-proliferative Aldehydes Synthesized by a Reductase Domain-Containing Non-ribosomal Peptide Synthetase

Abstract

Due to the importance of proteases in regulating cellular processes, the development of protease inhibitors has garnered great attention. Peptide-based aldehydes are a class of compounds that exhibit inhibitory activities against various proteases and proteasomes in the context of anti-proliferative treatments for cancer and other diseases. More than a dozen peptide-based natural products containing aldehydes have been discovered such as chymostatin, leupeptin, and fellutamide; however, the biosynthetic origin of the aldehyde functionality has yet to be elucidated. Herein we describe the discovery of a new group of lipopeptide aldehydes, the flavopeptins, and the corresponding biosynthetic pathway arising from an orphan gene cluster in Streptomyces sp. NRRL-F6652, a close relative of Streptomyces flavogriseus ATCC 33331. This research was initiated using a proteomics approach that screens for expressed enzymes involved in secondary metabolism in microorganisms. Flavopeptins are synthesized through a nonribosomal peptide synthetase containing a terminal NAD(P)H dependent reductase domain likely for the reductive release of the peptide with a C-terminal aldehyde. Solid phase peptide synthesis of several flavopeptin species and derivatives enabled structural verification and subsequent screening of biological activity. Flavopeptins exhibited submicromolar inhibition activities against cysteine proteases such as papain and calpain as well as the human 20S proteasome. They also showed anti-proliferative activities against multiple myeloma and lymphoma cell lines.

Targeting Reactive Carbonyls for Identifying Natural Products and Their Biosynthetic Origins

“…Of the remaining NRPS genes, anpC solely contains a condensation (C) domain while anpG contains thiolation and peptide carrier protein (PCP) and C domains, as well as a putative NAD-binding reductive (R) domain (instead of a traditional thioesterase) that is likely responsible for release of the product to yield the C-terminal aldehyde. 24 Outside of the NRPS genes, anpA encodes a putative hydrolase that could potentially play a role in Cit formation, either pre- or post-installation of Arg. The most similar protein to AnpA occurs in Streptomyces exfoliatus (81% aa sequence identity, 88% similarity).…”

Strain-specific proteogenomics accelerates the discovery of natural products via their biosynthetic pathways

“…By focusing on a characteristic ion associated with the phosphopantetheine post-translational modification that is essential for the function of thiotemplated systems, active site peptides could be identified, enabling the sequencing and characterization of whole biosynthetic clusters [2]. This approach was successfully employed in the discovery of several new natural products including koranimine [12], flavopeptin [6], and gobichelins A and B [8]. …”

Omics-based natural product discovery and the lexicon of genome mining

“…Despite these significant unknowns, there are numerous examples where orphan BGCs have yielded new compounds[34,40,62–64], thus supporting the concept that genome mining and associated omic approaches hold considerable promise for NP discovery. The continued development and application of these technologies will help fill the knowledge gaps discussed above and allow for the better exploitation of NP resources.…”

“…Early reports describe the detection of proteins associated with six orphan BGCs in M. xanthus [38]. More recently, the Kelleher group has pioneered the application of proteomics in NP discovery by capitalizing on the properties and size of non-ribosomal peptide synthetases and polyketide synthases (often >200 kDa) and their unique marker ions derived from phosphopantetheinyl cofactors[39,40]. The PrISM (Proteomic Investigation of Secondary Metabolism) platform (not to be confused with the more recent PRISM genome mining tool[11]) was developed to exploit this concept and represents a ‘protein-first’ strategy that is complementary to bioassay-guided and genomic approaches[41].…”