Biology subjects

Yool, A. J.

Publications and source records attributed to Yool, A. J..

4 recordsLinked to original sources

bioRxiv2024

AER-270 and TGN-020 are not aquaporin-4 water channel blockers

Aquaporin-4 (AQP4) is the most abundant water channel protein in the brain. It controls water homeostasis, facilitates glymphatic function and is a drug target for brain edema following injury or stroke. Dysregulation of brain water homeostasis affects millions of people every year leading to death, disability and cognitive decline, for which no medicines are available. Two compounds, AER-270 and TGN-020, are sold as AQP4 inhibitors and a prodrug of AER-270 is currently in a phase I human trial. However, the direct effect of these compounds on AQP4 function has not been unequivocally demonstrated. Our data across multiple cellular and molecular assay systems demonstrate, unexpectedly, that AER-270 and TGN-020 do not inhibit AQP4. Although we observed an apparent inhibitory effect of AER-270 and TGN-020 in the Xenopus laevis oocyte assay, there was no effect in assays using reconstituted recombinant AQP4 or mammalian cells expressing exogenous or endogenous AQP4. We identify alternative mechanisms of action for both molecules that may explain previously reported in vivo results that were interpreted in the context of AQP4 inhibition. Overall, we conclude that AER-270 and TGN-020 should not be used to investigate the AQP4-dependence of biological processes in the brain.

biophysics↗

bioRxiv2024

Validating the Target Functions and Synergistic Multi-Target, Multi-Pathway Action Mode of Compound Kushen Injection Using CRISPR/CAS

Background and PurposeDue to the complexity of traditional Chinese medicine based on complex mixtures of natural products and their multi-target mechanism of action, the discovery and validation of relevant targets have always been challenging. In previous studies, using transcriptomic methods and Compound Kushen Injection (CKI) as a model drug, we identified multiple pathways and target genes through which CKI exerts its pharmacological effects. Therefore, we wished to verify these targets by perturbing those genetic pathways. Experimental ApproachIn this study, we selected eight key genes from four candidate pathways and used CRISPR/CAS technology to knock out these genes in four cell lines, validating their role in CKI activity. Key ResultsFirstly, we found that although the sensitivity of different cell lines to gene knockout varied, overall, it led to a reduction in various cellular activities. After the addition of CKI, we observed that, except for the minor impact of CDKN1A gene knockout on the effect of CKI, knocking out the other genes significantly affected the pharmacological efficacy of CKI in different assays. Among them, knockout of MYD88 and NFkB genes enhanced the efficacy of CKI. At the same time, we found that the genes IL24 and CYP1B1 play a crucial role in CKI inhibition of tumour cell migration, and the CYP1A1 gene is critical for the cell cycle arrest induced by CKI. Conclusion and ImplicationsThese findings not only validate the results of our previous transcriptomic analysis but also further demonstrate the complexity of pharmacological mechanisms of multi-target synergistic action of natural product mixtures. What is already knownCKI demonstrates antitumor effects in both clinical and pharmacological research. Transcriptomic analysis shows CKI can perturb the expression of numerous genes in pathways related to cancer. What does this study addThe knockout of most selected genes whose expression is altered by CKI can significantly affect the pharmacological effects of CKI. IL24 and CYP1B1genes are essential to CKIs inhibition of cancer cell migration and CYP1A1 is important for CKIs G2M cell cycle arrest effect. What is the clinical significanceThe efficacy of CKI is demonstrated to arise from the synergistic action of multiple pathways and targets.

pharmacology and toxicology↗

bioRxiv2018

Effect of Compound Kushen Injection, a natural compound mixture, and its identified chemical components on migration and invasion of colon, brain and breast cancer cell lines

Cancer metastasis is a major cause of death. Traditional Chinese medicines (TCM) are promising sources of new anti-metastatic agents. Compound Kushen Injection (CKI), extracted from medicinal plants, Kushen (Sophora flavescens) and Baituling (Heterosmilax chinensis), contains a mixture of alkaloids and flavonoids known to disrupt cell cycle and induce apoptosis in breast cancer (MCF7). However, effects on cancer cell migration and invasion have remained unknown. CKI, fractionated mixtures, and single identified components were tested in migration assays with colon (HT-29, SW-480, DLD-1), brain (U-87 MG, U-251 MG), and breast (MDA-MB-231) cancer cell lines. Human embryonic kidney (HEK-293) and human foreskin fibroblast (HFF) served as non-cancerous controls. Wound closure, transwell invasion, and live cell imaging assays showed that CKI reduced motility in all eight cell lines. The greatest inhibition of migration occurred in HT-29 and MDA-MB-231, and the least in HEK-293. Fractionation and reconstitution of CKI showed that combinations of compounds were required for activity. Live cell imaging confirmed CKI strongly reduced migration of HT-29 and MDA-MB-231 cells, moderately slowed brain cancer cells, and had no effect on HEK-293. CKI uniformly blocked invasiveness through extracellular matrix. Apoptosis was increased by CKI in MDA-MB-231 cells but not in non-cancerous cells. Cell viability in CKI was unaffected in all cell lines. Transcriptomic analyses of MDA-MB-231 with and without CKI indicated down-regulated expression of actin cytoskeletal and focal adhesion genes, consistent with the observed impairment of cell migration. The pharmacological complexity of CKI is important for its effective block of cancer cell migration and invasion.

cancer biology↗

bioRxiv2018

FRACTIONAL DELETION OF COMPOUND KUSHEN INJECTION, A NATURAL COMPOUND MIXTURE, INDICATES CYTOKINE SIGNALING PATHWAYS ARE CRITICAL FOR ITS PERTURBATION OF THE CELL CYCLE.

We have used computational and experimental biology approaches to identify candidate mechanisms of action of a traditional Chinese medicine. Compound Kushen Injection (CKI), in a breast cancer cell line in which CKI causes apoptosis. Because CKI is a complex mixture of plant secondary metabolites, we used a high-performance liquid chromatography (HPLC) fractionation and reconstitution approach to define chemical fractions required for CKI to induce apoptosis in MDA-MB-231 cells. Our initial fractionation separated major from minor compounds, and showed that the major compounds accounted for little of the activity of CKI. By systematically perturbing the major compounds in CKI we found that removal of no single major compound could alter the effect of CKI on cell viability and apoptosis. However, simultaneous removal of two major compounds identified oxymatrine and oxysophocarpine as critical compounds with respect to CKI activity. We then used RNA sequencing and transcriptome analysis to correlate compound removal with gene expression and phenotype data. We determined that many compounds in CKI are required for its effectiveness in triggering apoptosis but that significant modulation of its activity is conferred by a small number of compounds. In conclusion, CKI may be typical of many plant based extracts that contain many compounds in that no single compound is responsible for all of the bioactivity of the mixture and that many compounds interact in a complex fashion to influence a network containing many targets.

pharmacology and toxicology↗