Characterization of Tailoring Steps of Nargenicin A1 Biosynthesis Reveals a Novel Analogue with Anticancer Activities

Abstract

Nargenicin A1­(1) is an antibacterial macrolide with effective activity against various Gram-positive bacteria, including methicillin-resistant Staphylococcus aureus. Due to the promising properties of this compound in inhibiting cell proliferation, immunomodulation, and the cell protective effect, there has been significant interest in this molecule. Recently, the biosynthetic gene cluster (BGC) of 1 was reported from Nocardia argentinesis and Nocardia arthritidis. In addition, two crucial enzymes involved in the formation of the core decalin moiety and postmodification of the decalin moiety by an ether bridge were characterized. This study reports on the BGC of 1 from Nocardia sp. CS682. In addition, the direct capture and heterologous expression of nar BGC from Nocardia sp. CS682 in Streptomyces venezuelae led to the production of 1. Further metabolic profiling of wild type, Nocardia sp. CS682 in optimized media (DD media) resulted in the isolation of two acetylated derivatives, 18-O-acetyl-nodusmicin and 18-O-acetyl-nargenicin. The post-PKS modification pathway in biosynthesis of 1 was also deciphered by identifying intermediates and/or in vitro enzymatic reactions of NgnP1, NgnM, and NgnO3. Different novel analogues of 1, such as compound 6, compound 7, 23-demethyl 8,13-deoxy-nodusmicin (8), 23-demethyl 8,13-deoxynargenicin (9), 8,13-deoxynodusmicin (10), and 8,13-deoxynargenicin (11), were also characterized, which extended our understanding of key post-PKS modification steps during the biosynthesis of 1. In addition, the antimicrobial and anticancer activities of selected analogues were also evaluated, whereas compound 9 was shown to exhibit potent antitumor activity by induction of G2/M cell cycle arrest, apoptosis, and autophagy.

Novel Nargenicin A1 Analog Inhibits Angiogenesis by Downregulating the Endothelial VEGF/VEGFR2 Signaling and Tumoral HIF-1α/VEGF Pathway

“…In a recent study, we isolated a novel nargenicin A1 analog, termed compound 9, in an attempt to characterize the key biosynthetic genes involved in post-polyketide synthase (PKS) tailoring of nargenicin A1 in Nocardia sp. CS682 [15]. Although compound 9 did not show any effective antibacterial activity as observed nargenicin A1, it exhibited potential anticancer properties, unlike the parent compound.…”

“…Among the tested cancer cell lines, compound 9 most sensitively suppressed the growth of AGS gastric cancer cells without cytotoxic effects against normal cell lines. Notably, compound 9 inhibited the growth of AGS cells by inducing G2/M cell cycle arrest, ROS-and caspase-mediated apoptosis, and autophagy via the downregulation of the PI3K/AKT/mTOR pathway [15]. Furthermore, while compound 9 significantly decreased the migration and invasion of AGS cells, nargenicin A1 did not.…”

“…Nargenicin A1 has been previously found to act as an antibacterial macrolide against various Gram-positive bacteria isolated from Nocardia species, inhibiting DnaE involved in bacterial DNA replication [12,15,26]. In addition, nargenicin A1 has shown several other activities, including the activation of acute myeloid leukemia (AML) cell differentiation, anti-inflammation, and protection against oxidative stress [27][28][29].…”

“…Compound 9 was isolated from the culture extract of the constructed Nocardia sp. CS682 mutant, as shown in our previous report [15] and prepared at a concentration of 100 mM using dimethyl sulfoxide (DMSO). Endothelial growth medium-2 (EGM-2) and antibiotics were purchased from Lonza (Walkersville, MD, USA), and fetal bovine serum (FBS) and RPMI-1640 medium were purchased from Invitrogen (Grand Island, NY, USA).…”

“…Our previous study demonstrated that compound 9 exerts effective anticancer activity against AGS gastric cancer cells by inducing G2/M cell cycle arrest, apoptosis, and autophagy [15]. To further evaluate whether compound 9 affects tumor cell-induced angiogenesis, we investigated the effect of compound 9 on the invasion and tube formation of HUVECs stimulated by AGS cells.…”