Identification of Antagonistic Action of Pyrrolizidine Alkaloids in Muscarinic Acetylcholine Receptor M1 by Computational Target Prediction Analysis.

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.

Impact of plastic-related compounds on the gene expression signature of HepG2 cells transfected with CYP3A4.

The presence of plastic and microplastic within the oceans as well as in marine flora and fauna have caused a multitude of problems that have been the topic of numerous investigations for many years. However, their impact on human health remains largely unknown. Such plastic and microplastic particles have been detected in blood and placenta, underlining their ability to enter the human body. Plastics also contain other compounds, such as plasticizers, antioxidants, or dyes, whose impact on human health is currently being studied. Critical enzymes within the metabolism of endogenous molecules, especially of xenobiotics, are the cytochrome P450 monooxygenases (CYPs). Although their importance in maintaining cellular balance has been confirmed, their interactions with plastics and related products are poorly understood. In this study, the possible relationship between different plastic-related compounds and CYP3A4 as one of the most important CYPs was analyzed using hepatic cells overexpressing this enzyme. Beginning with virtual compound screening and molecular docking of more than 1000 plastic-related compounds, several candidates were identified to interact with CYP3A4. In a second step, RNA-sequencing was used to study in detail the transcriptome-wide gene expression levels affected by the selected compounds. Three candidate molecules ((2,2'-methylenebis(6-tert-butyl-4-methylphenol), 1,1-bis(3,5-di-tert-butyl-2-hydroxyphenyl)ethane, and 2,2'-methylenebis(6-cyclohexyl-4-methylphenol)) had an excellent binding affinity to CYP3A4 in-silico as well as cytotoxic effects and interactions with several metabolic pathways in-vitro. We identified common pathways influenced by all three selected plastic-related compounds. In particular, the suppression of pathways related to mitosis and 'DNA-templated DNA replication' which were confirmed by cell cycle analysis and single-cell gel electrophoresis. Furthermore, several mis-regulated metabolic and inflammation-related pathways were identified, suggesting the induction of hepatotoxicity at different levels. These findings imply that these compounds may cause liver problems subsequently affecting the entire organism.

Investigating the Cytotoxicity of Ru(II) Polypyridyl Complexes by Changing the Electronic Structure of Salicylaldehyde Ligands.

A novel class of Ru(II)-based polypyridyl complexes with an auxiliary salicylaldehyde ligand [Ru(phen)2(X-Sal)]BF4 {X: H (1), 5-Cl (2), 5-Br (3), 3,5-Cl2 (4), 3,5-Br2 (5), 3-Br,5-Cl (6), 3,5-I2 (7), 5-NO2 (8), 5-Me (9), 4-Me (10), 4-OMe (11), and 4-DEA (12), has been synthesized and characterized by elemental analysis, FT-IR, and 1H/13C NMR spectroscopy. The molecular structure of 4, 6, 9, 10, and 11 was determined by single-crystal X-ray diffraction analysis which revealed structural similarities. DFT and TD-DFT calculations showed that they also possess similar electronic structures. Absorption/emission spectra were recorded for 2, 3, 10, and 11. All Ru-complexes, unlike the pure ligands and the complex lacking the salicylaldehyde component, displayed outstanding antiproliferative activity in the screening test (10 µM) against CCRF-CEM leukemia cells underlining the crucial role of the presence of the auxiliary ligand for the biological activity. The two most active derivatives, namely 7 and 10, were selected for continuous assays showing IC50 values in the submicromolar and micromolar range against drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells, respectively. These two compounds were investigated in silico for their potential binding to duplex DNA well-matched and mismatched base pairs, since they showed remarkable selectivity indexes (2.2 and 19.5 respectively) on PBMC cells.

Two palladium (II) complexes derived from halogen-substituted Schiff bases and 2-picolylamine induce parthanatos-type cell death in sensitive and multi-drug resistant CCRF-CEM leukemia cells.

The use of cisplatin and its derivatives in cancer treatment triggered the interest in metal-containing complexes as potential novel anticancer agents. Palladium (II)-based complexes have been synthesized in recent years with promising antitumor activity. Previously, we described the synthesis and cytotoxicity of palladium (II) complexes containing halogen-substituted Schiff bases and 2-picolylamine. Here, we selected two palladium (II) complexes with double chlorine-substitution or double iodine-substitution that displayed the best cytotoxicity in drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells for further biological investigation. Surprisingly, these compounds did not significantly induce apoptotic cell death. This study aims to reveal the major mode of cell death of these two palladium (II) complexes. We performed annexin V-FITC/PI staining and flow cytometric mitochondrial membrane potential measurement followed by western blotting, immunofluorescence microscopy, and alkaline single cell electrophoresis (comet assay). J4 and J6 still induced neither apoptosis nor necrosis in both leukemia cell lines. They also insufficiently induced autophagy as evidenced by Beclin and p62 detection in western blotting. Interestingly, J4 and J6 induced a novel mode of cell death (parthanatos) as mainly demonstrated in CCRF-CEM cells by hyper-activation of poly(ADP-ribose) polymerase 1 (PARP) and poly(ADP-ribose) (PAR) using western blotting, flow cytometric measurement of mitochondrial membrane potential collapse, nuclear translocation of apoptosis-inducing factor (AIF) by immunofluorescence microscopy, and DNA damage by alkaline single cell electrophoresis (comet assay). AIF translocation was also observed in CEM/ADR5000 cells. Thus, parthanatos was the predominant mode of cell death induced by J4 and J6, which explains the high cytotoxicity in CCRF-CEM and CEM/ADR5000 cells. J4 and J6 may be interesting drug candidates and deserve further investigations to overcome resistance of tumors against apoptosis. This study will promote the design of further novel palladium (II)-based complexes as chemotherapeutic agents.

Modes of Action of a Novel c-MYC Inhibiting 1,2,4-Oxadiazole Derivative in Leukemia and Breast Cancer Cells.

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.

The Effect of Plastic-Related Compounds on Transcriptome-Wide Gene Expression on CYP2C19-Overexpressing HepG2 Cells.

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.

In Silico and In Vitro Identification of P-Glycoprotein Inhibitors from a Library of 375 Phytochemicals.

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.

Cynaropicrin disrupts tubulin and c-Myc-related signaling and induces parthanatos-type cell death in multiple myeloma.

The majority of blood malignancies is incurable and has unforeseeable remitting-relapsing paths in response to different treatments. Cynaropicrin, a natural sesquiterpene lactone from the edible parts of the artichoke plant, has gained increased attention as a chemotherapeutic agent. In this study, we investigated the effects of cynaropicrin against multiple myeloma (MM) cells in vitro and assessed its in vivo effectiveness in a xenograft tumor zebrafish model. We showed that cynaropicrin exerted potent cytotoxicity against a panel of nine MM cell lines and two leukemia cell lines with AMO1 being the most sensitive cell line (IC50 = 1.8 ± 0.3 µM). Cynaropicrin (0.8, 1.9, 3.6 µM) dose-dependently reduced c-Myc expression and transcriptional activity in AMO1 cells that was associated with significant downregulation of STAT3, AKT, and ERK1/2. Cell cycle analysis showed that cynaropicrin treatment arrested AMO1 cells in the G2M phase along with an increase in the sub-G0G1 phase after 24 h. With prolonged treatment times, cells accumulated more in the sub-G0G1 phase, implying cell death. Using confocal microscopy, we revealed that cynaropicrin disrupted the microtubule network in U2OS cells stably expressing α-tubulin-GFP. Furthermore, we revealed that cynaropicrin promoted DNA damage in AMO1 cells leading to PAR polymer production by PARP1 hyperactivation, resulting in AIF translocation from the mitochondria to the nucleus and subsequently to a novel form of cell death, parthanatos. Finally, we demonstrated that cynaropicrin (5, 10 µM) significantly reduced tumor growth in a T-cell acute lymphoblastic leukemia (T-ALL) xenograft zebrafish model. Taken together, these results demonstrate that cynaropicrin causes potent inhibition of hematopoietic tumor cells in vitro and in vivo.

Synthesis and Bioactivity of Isoflavones from Ficus carica and Some Non-Natural Analogues.

Ficucaricone D (1) and its 4'-demethyl congener 2 are isoflavones isolated from fruits of Ficus carica that share a 5,7-dimethoxy-6-prenyl-substituted A-ring. Both natural products were, for the first time, obtained by chemical synthesis in six steps, starting from 2,4,6-trihydroxyacetophenone. Key steps are a microwave-promoted tandem sequence of Claisen- and Cope-rearrangements to install the 6-prenyl substituent and a Suzuki-Miyaura cross coupling for installing the B-ring. By using various boronic acids, non-natural analogues become conveniently available. All compounds were tested for cytotoxicity against drug-sensitive and drug-resistant human leukemia cell lines, but were found to be inactive. The compounds were also tested for antimicrobial activities against a panel of eight Gram-negative and two Gram-positive bacterial strains. Addition of the efflux pump inhibitor phenylalanine-arginine-ß-naphthylamide (PAßN) significantly improved the antibiotic activity in most cases, with MIC values as low as 2.5 µM and activity improvement factors as high as 128-fold.

Coronavirus Inhibitors Targeting nsp16.

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.

Antileukemia Activity and Mechanism of Platinum(II)-Based Metal Complexes.

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.