Stress Driven Discovery of Natural Products From Actinobacteria with Anti-Oxidant and Cytotoxic Activities Including Docking and ADMET Properties

Abstract

Elicitation through abiotic stress, including chemical elicitors like heavy metals, is a new technique for drug discovery. In this research, the effect of heavy metals on actinobacteria Streptomyces sp. SH-1312 for secondary metabolite production, with strong pharmacological activity, along with pharmacokinetics profile, was firstly investigated. The optimum metal stress conditions consisted of actinobacteria strain Streptomyces sp. SH-1312 with addition of mix metals (Co²⁺ + Zn²⁺) ions at 0.5 mM in Gause's medium. Under these conditions, the stress metabolite anhydromevalonolactone (MVL) was produced, which was absent in the normal culture of strain and other metals combinations. Furthermore, the stress metabolite was also evaluated for its anti-oxidant and cytotoxic activities. The compound exhibited remarkable anti-oxidant activities, recording the IC₅₀ value of 19.65 ± 5.7 µg/mL in DPPH, IC₅₀ of 15.49 ± 4.8 against NO free radicals, the IC₅₀ value of 19.65 ± 5.22 µg/mL against scavenging ability, and IC₅₀ value of 19.38 ± 7.11 µg/mL for iron chelation capacity and the cytotoxic activities against PC3 cell lines were recorded with IC₅₀ values of 35.81 ± 4.2 µg/mL after 24 h, 23.29 ± 3.8 µg/mL at 48 h, and 16.25 ± 6.5 µg/mL after 72 h. Further mechanistic studies have revealed that the compound MVL has shown its pharmacological efficacy by upregulation of P53 and BAX while downregulation of BCL-2 expression, indicating that MVL is following apoptosis in varying degrees. To better understand the pharmacological properties of MVL, in this work, the absorption, distribution, metabolism, excretion, and toxicity (ADMET) were also evaluated. During ADMET predictions, MVL has displayed a safer profile in case of hepatotoxicity, cytochrome inhibition and also displayed as non-cardiotoxic. The compound MVL showed good binding energy in the molecular docking studies, and the results revealed that MVL bind in the active region of the target protein of P53 and BAX. This work triumphantly announced a prodigious effect of heavy metals on actinobacteria with fringe benefits as a key tool of MVL production with a strong pharmacological and pharmacokinetic profile.

Keywords: ADMET; actinobacteria; anti-cancer; anti-oxidant; natural products; stress-technique.

Figure 1

HPLC profile of metal treated and untreated SH-1312 strain with several mixed metals (Co²⁺ + Zn²⁺) ions. The red circle indicates the new peak in the HPLC chromatogram.

Figure 2

Chemical structure of anhydromevalonolactone.

Figure 3

In vitro antioxidant activities of MVL. (A) DPPH radical scavenging activity, (B) NO scavenging activity, (C) OH● scavenging activity, and (D) iron-chelating % inhibition. Each value represents, Mean ± SD (n = 3).

Figure 4

Effect of MVL on the viability of prostate cancer cells. MTT assay was used to determine the viability of cancer cells after 24, 48, and 72 h treatment of PC3 cells. Data is mean ± SEM of % cell viability (n = 3) at ***: p < 0.001 and ****: p < 0.0001.

Figure 5

In vitro scratch assay on MVL treated prostate cancer cells. PC3 cells were plated in 6-well plates and scratched at the full confluence. Migration of cells to heal the area of the scratch was observed at 0 h, 12 h, and 24 h after treatment. Reduction in the area of the scratch was Photographed using Olympus CKX41 microscope and measured using ImageJ software. Data is mean ± SEM percent area of scratch in triplicate at ***: p < 0.001 and ****: p < 0.0001.

Figure 6

Western blot analysis of proteins associated with MVL induced apoptosis. Prostate cancer cells, PC3 were treated with MVL at a concentration of 0, 5, and 10 µg/mL for 48 h. Data shows increased expression of pro-apoptotic P53 and BAX while decreased expression of anti-apoptotic BCL2. GAPDH was used as a loading control. Fold change in P-53, BAX, and BCL-2 expression after treating for 48 h with different concentrations of MVL. Means with different superscripts (^a–c) indicate significant (p < 0.001) difference in fold change between groups.

Figure 7

Genotoxicity evaluation of MVL on blood lymphocytes. “H” represents Head and “T” represents Tail. (A) Vehicle control (1% DMSO) (B) Ethyl methane sulfonate (20 µg/mL) (C) MVL (10 µg/mL) (D) MVL (20 µg/mL).

Figure 8

(A). Bioavailability radar chart for MVL. The pink zone represents the physicochemical space for oral bioavailability, and the red line represents the oral bioavailability properties. (B) Predicted BOILED-Egg plot from swiss ADME online web tool for MVL.

Figure 9

Map of Cardiac toxicity of MVL obtained from pred-hERG.

Figure 10

Binding analysis of MVL with BAX.

Figure 11

Binding analysis of P53 with MVL.