Cytotoxic properties, glycolytic effects and high-resolution respirometry mitochondrial activities of Eriocephalus racemosus against MDA-MB 231 triple-negative breast cancer.

INTRODUCTION: Triple-negative breast cancer (TNBC) represents a significant global health crisis due to its resistance to conventional therapies and lack of specific molecular targets. This study explored the potential of Eriocephalus racemosus (E. racemosus) as an alternative treatment for TNBC. The cytotoxic properties and high-resolution respirometry mitochondrial activities of E. racemosus against the MDA-MB 231 TNBC cell line were evaluated. METHODS: Hexane solvent and bioactive fraction extractions of E. racemosus were performed, while mass spectrometry-based metabolite profiling was used to identify the phytochemical constituents of the extracts. The extracts were further tested against MDA-MB 231 TNBC cells to determine their cytotoxicity. The mode of cell death was determined using flow cytometry. The activities of caspases 3, 8, and 9 were assessed using a multiplex activity assay kit. Glycolytic activity and High-resolution respirometry measurements of mitochondrial function in the MDA-MB 231 cell line were conducted using the Seahorse XFp and Oroboros O2K. RESULTS: Metabolite profiling of E. racemosus plant crude extracts identified the presence of coumarins, flavonoids, sesquiterpenoids, triterpenoids, and unknown compounds. The extracts demonstrated promising cytotoxic activities, with a half maximal inhibitory concentration (IC50) of 12.84 µg/mL for the crude hexane extract and 15.49 µg/mL for the bioactive fraction. Further, the crude hexane and bioactive fraction extracts induced apoptosis in the MDA-MB-231 TNBC cells, like the reference drug cisplatin (17.44%, 17.26% and 20.25%, respectively) compared to untreated cells. Caspase 3 activities confirmed the induction of apoptosis in both cisplatin and the plant crude extracts, while caspase 8 and 9 activities confirmed the activation of both the intrinsic and extrinsic apoptosis pathways. Increased levels of glycolytic activity were observed in the hexane crude extract. High-resolution respiratory measurements showed elevated mitochondrial activities in all mitochondrial states except for complex-IV activity. CONCLUSION: These findings support further exploration of E. racemosus as a potential therapeutic agent for TNBC, offering a promising avenue for the development of targeted treatments with minimal adverse effects.

Molecular docking, molecular dynamics simulations and binding free energy studies of interactions between Mycobacterium tuberculosis Pks13, PknG and bioactive constituents of extremophilic bacteria.

Mycobacterial pathogens present a significant challenge to disease control efforts globally due to their inherent resistance to multiple antibiotics. The rise of drug-resistant strains of Mycobacterium tuberculosis has prompted an urgent need for innovative therapeutic solutions. One promising way to discover new tuberculosis drugs is by utilizing natural products from the vast biochemical space. Multidisciplinary methods can used to harness the bioactivity of these natural products. This study aimed to evaluate the antimycobacterial efficacy of functional crude extracts from bacteria isolated from gold mine tailings in South Africa. Bacterial strains were identified using 16S rRNA sequencing. The crude extracts obtained from the bacteria were tested against Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, and Mycobacterium aurum A+. Untargeted HPLC-qTOF and molecular networking were used to identify the functional constituents present in extracts that exhibited inhibitory activity. A virtual screening workflow (VSW) was used to filter compounds that were strong binders to Mycobacterium tuberculosis Pks13 and PknG. The ligands returned from the VSW were subjected to optimization using density functional theory (DFT) at M06-2X/6-311++ (d,p) level of theory and basis set implemented in Gaussian16 Rev.C01. The optimized ligands were re-docked against Mycobacterium tuberculosis Pks13 and PknG. Molecular dynamics simulation and molecular mechanics generalized born surface area were used to evaluate the stability of the protein-ligand complexes formed by the identified hits. The hit that showed promising binding characteristics was virtually modified through multiple synthetic routes using reaction-driven enumeration. Three bacterial isolates showed significant activity against the two strains of Mycobacterium, while only two, Bacillus subtilis and Bacillus licheniformis, exhibited activity against both Mycobacterium tuberculosis H37Rv, Mycobacterium smegmatis mc2155, and Mycobacterium aurum A+. The tentatively identified compounds from the bacterial crude extracts belonged to various classes of natural compounds associated with antimicrobial activity. Two compounds, cyclo-(L-Pro-4-OH-L-Leu) and vazabitide A, showed strong binding against PknG and Pks13, with pre-MD MM-GBSA values of - 42.8 kcal/mol and - 47.6 kcal/mol, respectively. The DFT-optimized compounds exhibited the same docking scores as the ligands optimized using the OPSL-4 force field. After modifying vazabitide A, its affinity to the Pks13 binding site increased to - 85.8 kcal/mol, as revealed by the post-MD MM-GBSA analysis. This study highlights the potential of bacteria isolates from gold mine tailings as a source of new scaffolds for designing and optimizing anti-Mycobacterium agents. These agents synthesized in-silico can be further tested in-vitro to evaluate their efficacy.

In-silico and in-vitro assessments of some fabaceae, rhamnaceae, apocynaceae, and anacardiaceae species against Mycobacterium tuberculosis H37Rv and triple-negative breast cancer cells.

Medicinal plants play a huge role in the treatment of various diseases in the Limpopo province (South Africa). Traditionally, concoctions used for treating tuberculosis and cancer are sometimes prepared from plant parts naturally occurring in the region, these include (but not limited to) Schotia brachypetala, Rauvolfia caffra, Schinus molle, Ziziphus mucronate, and Senna petersiana. In this study, the aim was to evaluate the potential antimycobacterial activity of the five medicinal plants against Mycobacterium smegmatis mc2155, Mycobacterium aurum A + , and Mycobacterium tuberculosis H37Rv, and cytotoxic activity against MDA-MB 231 triple-negative breast cancer cells. Phytochemical constituents present in R. caffra and S. molle were tentatively identified by LC-QTOF-MS/MS as these extracts showed antimycobacterial and cytotoxic activity. A rigorous Virtual Screening Workflow (VSW) of the tentatively identified phytocompounds was then employed to identify potential inhibitor/s of M. tuberculosis pantothenate kinase (PanK). Molecular dynamics simulations and post-MM-GBSA free energy calculations were used to determine the potential mode of action and selectivity of selected phytocompounds. The results showed that plant crude extracts generally exhibited poor antimycobacterial activity, except for R. caffra and S. molle which exhibited average efficacy against M. tuberculosis H37Rv with minimum inhibitory concentrations between 0.25-0.125 mg/mL. Only one compound with a favourable ADME profile, namely, norajmaline was returned from the VSW. Norajmaline exhibited a docking score of -7.47 kcal/mol, while, pre-MM-GBSA calculation revealed binding free energy to be -37.64 kcal/mol. All plant extracts exhibited a 50% inhibitory concentration (IC50) of < 30 µg/mL against MDA-MB 231 cells. Flow cytometry analysis of treated MDA-MB 231 cells showed that the dichloromethane extracts from S. petersiana, Z. mucronate, and ethyl acetate extracts from R. caffra and S. molle induced higher levels of apoptosis than cisplatin. It was concluded that norajmaline could emerge as a potential antimycobacterial lead compound. Validation of the antimycobacterial activity of norajmaline will need to be performed in vitro and in vivo before chemical modifications to enhance potency and efficacy are done. S. petersiana, Z. mucronate, R.caffra and S. molle possess strong potential as key contributors in developing new and effective treatments for triple-negative breast cancer in light of the urgent requirement for innovative therapeutic solutions.

Antimycobacterial activity and molecular docking of methanolic extracts and compounds of marine fungi from Saldanha and False Bays, South Africa.

The number and diversity of drugs in the tuberculosis (TB) drug development process has increased over the years, yet the attrition rate remains very high, signaling the need for continued research in drug discovery. In this study, crude secondary metabolites from marine fungi associated with ascidians collected from Saldanha and False Bays (South Africa) were investigated for antimycobacterial activity. Isolation of fungi was performed by sectioning thin inner-tissues of ascidians and spreading them over potato dextrose agar (PDA). Solid state fermentation of fungal isolates on PDA was then performed for 28 days to allow production of secondary metabolites. Afterwards, PDA cultures were dried and solid-liquid extraction using methanol was performed to extract fungal metabolites. Profiling of metabolites was performed using untargeted liquid chromatography quadrupole time-of-flight tandem mass spectrometry (LC-QTOF-MS/MS). The broth microdilution method was used to determine antimycobacterial activity against Mycobacterium smegmatis mc2155 and Mycobacterium tuberculosis H37Rv, while in silico flexible docking was performed on selected target proteins from M. tuberculosis. A total of 16 ascidians were sampled and 46 fungi were isolated. Only 32 fungal isolates were sequenced, and their sequences submitted to GenBank to obtain accession numbers. Metabolite profiling of 6 selected fungal extracts resulted in the identification of 65 metabolites. The most interesting extract was that of Clonostachys rogersoniana MGK33 which inhibited Mycobacterium smegmatis mc2155 and Mycobacterium tuberculosis H37Rv growth with minimum inhibitory concentrations (MICs) of 0.125 and 0.2 mg/mL, respectively. These results were in accordance with those from in silico molecular docking studies which showed that bionectin F produced by C. rogersoniana MGK33 is a potential inhibitor of M. tuberculosis ß-ketoacyl-acyl carrier protein reductase (MabA, PDB ID = 1UZN), with the docking score observed as -11.17 kcal/mol. These findings provided evidence to conclude that metabolites from marine-derived fungi are potential sources of bioactive metabolites with antimycobacterial activity. Even though in silico studies showed that bionectin F is a potent inhibitor of an essential enzyme, MabA, the results should be validated by performing purification of bionectin F from C. rogersoniana MGK33 and in vitro assays against MabA and whole cells (M. tuberculosis).