Molecular Networking and Whole-Genome Analysis Aid Discovery of an Angucycline That Inactivates mTORC1/C2 and Induces Programmed Cell Death.

Rediscovery of known compounds and time consumed in identification, especially high molecular weight compounds with complex structure, have let down interest in drug discovery. In this study, whole-genome analysis of microbe and Global Natural Products Social (GNPS) molecular networking helped in initial understanding of possible compounds produced by the microbe. Genome data revealed 10 biosythethic gene clusters that encode for secondary metabolites with anticancer potential. NMR analysis of the pure compound revealed the presence of a four-ringed benz[a]anthracene, thus confirming angucycline; molecular networking further confirmed production of this class of compounds. The type II polyketide synthase gene identified in the microbial genome was matched with the urdamycin cluster by BLAST analysis. This information led to ease in identification of urdamycin E and a novel natural derivative, urdamycin V, purified from Streptomyces sp. OA293. Urdamycin E (Urd E) induced apoptosis and autophagy in cancer cell lines. Urd E exerted anticancer action through inactivation of the mTOR complex by preventing phosphorylation at Ser 2448 and Ser 2481 of mTORC1 and mTORC2, respectively. Significant reduction in phosphorylation of the major downstream regulators of both mTORC1 (p70s6k and 4e-bp1) and mTORC2 (Akt) were observed, thus further confirming complete inhibition of the mTOR pathway. Urd E presents itself as a novel mTOR inhibitor that employs a novel mechanism in mTOR pathway inhibition.

Bioactivities and molecular networking-based elucidation of metabolites of potent actinobacterial strains isolated from the Unkeshwar geothermal springs in India.

The bioactive potential of Actinobacteria endemic to hot springs has rarely been investigated. This study highlights the cultivable diversity and bioactivities of Actinobacteria associated with the Unkeshwar hot springs, India. Potent strains were evaluated for their biosynthetic potentials and metabolite analysis was performed using effective dereplication molecular networking tools. A total of 86 actinobacterial strains were isolated and grouped into 21 distinct genera, based on 16S rRNA gene sequence analysis. These strains included rare members such as Micromonospora, Marmoricola, Actinomadura, Cellulomonas, Cellulosimicrobium, Janibacter, Rothia, Barrentisimonas, Dietzia and Glycomyces. In antimicrobial screening, Micromonospora sp. strain GH99 and Streptomyces sp. strain GH176 were found to be potent antimicrobial strains. The metabolic extracts of these strains exhibited strong antimicrobial activity against Staphylococcus epidermidis (NCIM 2493), Shigella flexneri (NCIM 5265), Klebsiella pneumonia (NCIM 2098), and Salmonella abony (NCIM 2257). The extracts also displayed strong anti-biofilm and anticancer activities against Pseudomonas aeruginosa (NCIM 5029), Acinetobacter junii (NCIM 5188) and breast cancer cell line MCF7, respectively. Both strains also tested positive for the presence of the PKS biosynthetic gene cluster in their genomes. To effectively delineate the secondary metabolites, the extracts were subjected to MS/MS-guided molecular networking analysis. Structurally diverse compounds including the polyketides 22-dehydroxymethyl-kijanolide (GH99 strain) and Abyssomicin I (GH176 strain) were detected in the extracts. Interestingly, Brevianamide F was detected in the extract of Micromonospora, which has previously been mostly found in fungal species. Other compounds such as cyclic tripeptides, Cyclo(l-Pro-d-Ile) and Cyclo(d-Pro-l-Phe), were also identified in this strain. In summary, for the first time, we explored the diversity of Actinobacteria and evaluated their bioactive potential from the Unkeshwar hot springs. The potent strains isolated in the study could be useful in drug discovery programs.

Streptomyces sp metabolite(s) promotes Bax mediated intrinsic apoptosis and autophagy involving inhibition of mTOR pathway in cervical cancer cell lines.

In cervical cancer, the association between HPV infection and dysregulation of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTOR pathway) places mTOR as an attractive therapeutic target. The failure of current treatment modalities in advanced stages of this cancer and drawbacks of already available mTOR inhibitors demand for novel drug candidates. In the present study we identified the presence of a mTOR inhibitor in an active fraction of the ethyl acetate extract of Streptomyces sp OA293. The metabolites(s) in the active fraction completely inhibited mTORC1 and thereby suppressed activation of both of its downstream targets, 4E-BP1 and P70S6k, in cervical cancer cells. In addition, it also stalled Akt activation via inhibition of mTORC2. The mechanism of mTOR inhibition detailed in our study overcomes significant drawbacks of well known mTOR inhibitors such as rapamycin and rapalogs. The active fraction induced autophagy and Bax mediated apoptosis suggesting that mTOR inhibition resulted in programmed cell death of cancer cells. The molecular weight determination of the components in active fraction confirmed the absence of any previously known natural mTOR inhibitor. This is the first report of complete mTOR complex inhibition by a product derived from microbial source.