During the past three decades, humans have been confronted with different new coronavirus outbreaks. Since the end of the year 2019, COVID-19 threatens the world as a rapidly spreading infectious disease. For this work, we targeted the non-structural protein 16 (nsp16) as a key protein of SARS-CoV-2, SARS-CoV-1 and MERS-CoV to develop broad-spectrum inhibitors of nsp16. Computational methods were used to filter candidates from a natural product-based library of 224,205 compounds obtained from the ZINC database. The binding of the candidates to nsp16 was assessed using virtual screening with VINA LC, and molecular docking with AutoDock 4.2.6. The top 9 compounds were bound to the nsp16 protein of SARS-CoV-2, SARS-CoV-1, and MERS-CoV with the lowest binding energies (LBEs) in the range of −9.0 to −13.0 kcal with VINA LC. The AutoDock-based LBEs for nsp16 of SARS-CoV-2 ranged from −11.42 to −16.11 kcal/mol with predicted inhibition constants (pKi) from 0.002 to 4.51 nM, the natural substrate S-adenosyl methionine (SAM) was used as control. In silico results were verified by microscale thermophoresis as in vitro assay. The candidates were investigated further for their cytotoxicity in normal MRC-5 lung fibroblasts to determine their therapeutic indices. Here, the IC₅₀ values of all three compounds were >10 µM. In summary, we identified three novel SARS-CoV-2 inhibitors, two of which showed broad-spectrum activity to nsp16 in SARS-CoV-2, SARS-CoV-1, and MERS-CoV. All three compounds are coumarin derivatives that contain chromen-2-one in their scaffolds.

The c-MYC oncogene regulates multiple cellular activities and is a potent driver of many highly aggressive human cancers, such as leukemia and triple-negative breast cancer. The oxadiazole class of compounds has gained increasing interest for its anticancer activities. The aim of this study was to investigate the molecular modes of action of a 1,2,4-oxadiazole derivative (ZINC15675948) as a c-MYC inhibitor. ZINC15675948 displayed profound cytotoxicity at the nanomolar range in CCRF-CEM leukemia and MDA-MB-231-pcDNA3 breast cancer cells. Multidrug-resistant sublines thereof (i.e., CEM/ADR5000 and MDA-MB-231-BCRP) were moderately cross-resistant to this compound (<10-fold). Molecular docking and microscale thermophoresis revealed a strong binding of ZINC15675948 to c-MYC by interacting close to the c-MYC/MAX interface. A c-MYC reporter assay demonstrated that ZINC15675948 inhibited c-MYC activity. Western blotting and qRT-PCR showed that c-MYC expression was downregulated by ZINC15675948. Applying microarray hybridization and signaling pathway analyses, ZINC15675948 affected signaling routes downstream of c-MYC in both leukemia and breast cancer cells as demonstrated by the induction of DNA damage using single cell gel electrophoresis (alkaline comet assay) and induction of apoptosis using flow cytometry. ZINC15675948 also caused G2/M phase and S phase arrest in CCRF-CEM cells and MDA-MB-231-pcDNA3 cells, respectively, accompanied by the downregulation of CDK1 and p-CDK2 expression using western blotting. Autophagy induction was observed in CCRF-CEM cells but not MDA-MB-231-pcDNA3 cells. Furthermore, microarray-based mRNA expression profiling indicated that ZINC15675948 may target c-MYC-regulated ubiquitination, since the novel ubiquitin ligase (ELL2) was upregulated in the absence of c-MYC expression. We propose that ZINC15675948 is a promising natural product-derived compound targeting c-MYC in c-MYC-driven cancers through DNA damage, cell cycle arrest, and apoptosis.

Cancer therapy with clinically established anticancer drugs is frequently hampered by the development of drug resistance of tumors and severe side effects in normal organs and tissues. The demand for powerful, but less toxic, drugs is high. Phytochemicals represent an important reservoir for drug development and frequently exert less toxicity than synthetic drugs. Bioinformatics can accelerate and simplify the highly complex, time-consuming, and expensive drug development process. Here, we analyzed 375 phytochemicals using virtual screenings, molecular docking, and in silico toxicity predictions. Based on these in silico studies, six candidate compounds were further investigated in vitro. Resazurin assays were performed to determine the growth-inhibitory effects towards wild-type CCRF-CEM leukemia cells and their multidrug-resistant, P-glycoprotein (P-gp)-overexpressing subline, CEM/ADR5000. Flow cytometry was used to measure the potential to measure P-gp-mediated doxorubicin transport. Bidwillon A, neobavaisoflavone, coptisine, and z-guggulsterone all showed growth-inhibitory effects and moderate P-gp inhibition, whereas miltirone and chamazulene strongly inhibited tumor cell growth and strongly increased intracellular doxorubicin uptake. Bidwillon A and miltirone were selected for molecular docking to wildtype and mutated P-gp forms in closed and open conformations. The P-gp homology models harbored clinically relevant mutations, i.e., six single missense mutations (F336Y, A718C, Q725A, F728A, M949C, Y953C), three double mutations (Y310A-F728A; F343C-V982C; Y953A-F978A), or one quadruple mutation (Y307C-F728A-Y953A-F978A). The mutants did not show major differences in binding energies compared to wildtypes. Closed P-gp forms generally showed higher binding affinities than open ones. Closed conformations might stabilize the binding, thereby leading to higher binding affinities, while open conformations may favor the release of compounds into the extracellular space. In conclusion, this study described the capability of selected phytochemicals to overcome multidrug resistance.

Pyrrolizidine alkaloids (PAs) are one of the largest distributed classes of toxins in nature. They have a wide range of toxicity, such as hepatotoxicity, pulmonary toxicity, neuronal toxicity, and carcinogenesis. Yet, biological targets responsible for these effects are not well addressed. Using methods of computational biology for target identification, we tested more than 200 PAs. We used a machine-learning approach that applies structural similarity for target identification, ChemMapper, and SwissTargetPrediction. The predicted targets with high probabilities were muscarinic acetylcholine receptor M1. The predicted interactions between these two targets and PAs were further studied by molecular docking-based binding energies using AutoDock and VinaLC, which revealed good binding affinities. The PAs are bound to the same binding pocket as pirenzepine, a known M1 antagonist. These results were confirmed by in vitro assays showing that PAs increased the levels of intracellular calcium. We conclude that PAs are potential acetylcholine receptor M1 antagonists. This elucidates for the first time the serious neuro-oncological toxicities exerted by PA consumption.

Transition metal complexes have continued to constitute an appealing class of medicinal compounds since the exceptional discovery of cisplatin in the late 1960s. Pt(II)-based complexes are endowed with a broad range of biological properties, which are mainly exerted by targeting DNA. In this study, we report a significant biological investigation into and computation analyses of four Pt(II)-complexes, namely, LDP-1–4, synthesized and characterized according to previously reported procedures. Molecular-modelling studies highlighted that the top two LDP compounds (i.e., LDP-1 and LDP-4) might bind to both matched and mismatched base pair sites of the oligonucleotide 5′-(dCGGAAATTACCG)₂-3′, supporting their anticancer potential. These two complexes displayed noteworthy cytotoxicity in vitro (sub-micromolar–micromolar range) against two leukaemia cell lines, i.e., CCRF-CEM and its multi-drug-resistant counterpart CEM/ADR5000, and remarkable anti-angiogenic properties (in the sub-micromolar range) evaluated in an in vivo model, i.e., a chick embryo chorioallantoic membrane (CAM) assay.

In recent years, plastic and especially microplastic in the oceans have caused huge problems to marine flora and fauna. Recently, such particles have also been detected in blood, breast milk, and placenta, underlining their ability to enter the human body, presumably via the food chain and other yet-unknown mechanisms. In addition, plastic contains plasticizers, antioxidants, or lubricants, whose impact on human health is also under investigation. At the cellular level, the most important enzymes involved in the metabolism of xenobiotic compounds are the cytochrome P450 monooxygenases (CYPs). Despite their extensive characterization in the maintenance of cellular balance, their interactions with plastic and related products are unexplored. In this study, the possible interactions between several plastic-related compounds and one of the most important cytochromes, CYP2C19, were analyzed. By applying virtual compound screening and molecular docking to more than 1000 commercially available plastic-related compounds, we identified candidates that are likely to interact with this protein. A growth inhibition assay confirmed their cytotoxic activity on a CYP2C19-transfected hepatic cell line. Subsequently, we studied the effect of the selected compounds on the transcriptome-wide gene expression level by conducting RNA sequencing. Three candidate molecules were identified, i.e., 2,2′-methylene bis(6-tert-butyl-4-methylphenol), 1,1-bis(3,5-di-tert-butyl-2-hydroxyphenyl) ethane, and 2,2′-methylene bis(6-cyclohexyl-4-methylphenol)), which bound with a high affinity to CYP2C19 in silico. They exerted a profound cytotoxicity in vitro and interacted with several metabolic pathways, of which the ‘cholesterol biosynthesis process’ was the most affected. In addition, other affected pathways involved mitosis, DNA replication, and inflammation, suggesting an increase in hepatotoxicity. These results indicate that plastic-related compounds could damage the liver by affecting several molecular pathways.