KSEF RDE: Discovery and development of a non-toxic actinomycin antibiotic

Actinomycin D, also known as dactinomycin, was the first natural product antibiotic shown to have potent anticancer activity. A new actinomycin (actinomycin Y5) containing a unique N-aryl-L-Thr bridged macrocycle was recently discovered and shown to be have excellent antibiotic activity against Gram-positive bacteria but no apparent cytotoxicity against several human cell lines. The primary objective of this proposal is to define the biosynthetic mechanism by which the novel macrocycle is installed and develop technologies to achieve a tractable platform to access actinomycin derivatives with alternative macrocycles and to establish new enzyme/protein functions that are integral to the biosynthesis of nonribosomally-derived peptides. Three specific aims will be addressed: (1) establishing a unifying biosynthetic mechanism by cloning and characterization the gene clusters for the Y-type actinomycins, Z-type actinomycins, and pronactin (hemi-actionomycin) (2) define the metabolic origin of actinomycin Y5 via feeding and isotopic enrichment experiments, and (3) identify and characterize the enzymes involved in macrocycle formation using an in vivo and in vitro approach. We expect that the completion of the specific aims will provide critical insight into how antibiotic peptides such as actinomycin are assembled, enable further investigation into the structure-activity relationship of actinomycins by establishing important cell lines and tools to prepare advanced precursors for semisynthetic applications, and be the first step toward advancing an actinomycin-inspired antibiotic toward clinical use.