Discovery of nostatin A, an azole-containing proteusin with prominent cytostatic and pro-apoptotic activity

Abstract

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are intriguing compounds with potential pharmacological applications. While many RiPPs are known as antimicrobial agents, a limited number of RiPPs with anti-proliferative effects in cancer cells are available. Here we report the discovery of nostatin A (NosA), a highly modified RiPP belonging among nitrile hydratase-like leader peptide RiPPs (proteusins), isolated from a terrestrial cyanobacterium Nostoc sp. Its structure was established based on the core peptide sequence encoded in the biosynthetic gene cluster recovered from the producing strain and subsequent detailed nuclear magnetic resonance and high-resolution mass spectrometry analyses. NosA, composed of a 30 amino-acid peptide core, features a unique combination of moieties previously not reported in RiPPs: the simultaneous presence of oxazole/thiazole heterocycles, dehydrobutyrine/dehydroalanine residues, and a sactionine bond. NosA includes an isobutyl-modified proline residue, highly unusual in natural products. NosA inhibits proliferation of multiple cancer cell lines at low nanomolar concentration while showing no hemolysis. It induces cell cycle arrest in S-phase followed by mitochondrial apoptosis employing a mechanism different from known tubulin binding and DNA damaging compounds. NosA also inhibits Staphylococcus strains while it exhibits no effect in other tested bacteria or yeasts. Due to its novel structure and selective bioactivity, NosA represents an excellent candidate for combinatorial chemistry approaches leading to development of novel NosA-based lead compounds.

Fig. 1. NosA is encoded by a gene cluster similar to thiazole/oxazole modified microcins (TOMMs). (A) Gene map and functional annotation of the biosynthetic gene cluster of NosA compared to known TOMMs. The precursor/core peptide sequence and a schematic representation of the final NosA product including the internal sactionine bridge and sequential methylation of the proline residue are shown below the gene map. Red – precursor peptides, blue – enzymes involved in thiazole/oxazole formation, green – Lan dehydratases, pink – putative SAM-dependent methyltransferase, brown – epimerase, grey – transporters and accessory enzymes. (B) Alignment of the core peptides of selected TOMMs with similar amino acid sequences. Blue – amino acid residues modified to thiazoles/oxazoles, green – dehydrated residues, pink – modified proline. (C) Primary sequences of the selected TOMMs are color-coded as above. Typical structural building blocks, each consisting of three amino acids, are highlighted in boxes.

Fig. 2. Schematic representation of the structure of NosA showing the integrated results obtained from MS and NMR spectroscopic analyses. (A) Structure of Nos A – individual residues are labeled consecutively from the N-terminus. Main HRMS fragments F1–F12 are indicated with grey boxes. Arrows show the most relevant ¹H-/¹³C and ¹H-/¹⁵N HMBC correlations; COSY correlations are depicted as bolded bonds. (B) Schematic representation of the most informative fragments of NosA (top) and mass spectrum (bottom). Individual y- and b-ions are labeled in red and blue, respectively. The interpretation of the most intense MS fragments which were not used for establishing of the peptide sequence are given in Fig. S16.

Fig. 3. ATP-based viability assay on human cervical cancer cell line HeLa WT (black bars) and primary GK cells (grey bars) and hemolytic assay on sheep red blood cells. The cells were treated with graded doses of NosA and cell viability was evaluated after (A) 48 h, (B) 72 h, and (C) 96 h. Hemolytic activity was evaluated after 2 h exposure (D).

Fig. 4. DNA content analysis of NosA-treated HeLa cells by flow cytometry. HeLa WT and Bax/Bak DKO cells were synchronized by a double thymidine block, released into the media with NosA (100 nM) or to control media, stained by propidium iodide (PI), and cell cycle profiles (subG1, G1, S, G2/M) were analyzed.

Fig. 5. NosA induces mitochondrial apoptosis, while not causing DNA damage or tubulin network alteration. (A) The cytostatic effect shown in HeLa WT cells treated by 100 nM NosA. Red arrows show apoptotic cell blebbing. (STS – stauroporine positive control, NC – negative control). (B) The immunolabeling of tubulin (green) in combination with nucleus staining (blue) in HeLa WT cells. (C) The caspase 3/7 activity of HeLa WT and Bax/Bak DKO cells treated by NosA and STS normalized to control. (D and E) PARP and γH2AX immunoblot of HeLa WT and Bax/Bak DKO cells after treatment with DMOS (Un.) or 100 nM NosA. (F) The viability of HeLa WT and Bax/Bak DKO after NosA treatment. For panels (C and F), the values are expressed as mean ± SD, n = 3. The full images of immunoblots are depicted in Fig. S38.

Fig. 6. Metabolomic analysis of HeLa WT cells treated with NosA for 24 h visualized with a volcano plot. Individual metabolites are presented by dots referring to their log2 fold change and significance value (log10 p-value). Upregulated/downregulated metabolites are highlighted by red/green color, respectively.