Discovery of spirooxadiazoline oxindoles with dual-stage antimalarial activity

© 2022 Published by Elsevier Masson SAS.Malaria remains a prevalent infectious disease in developing countries. The first-line therapeutic options are based on combinations of fast-acting artemisinin derivatives and longer-acting synthetic drugs. However, the emergence of resistance to these first-line treatments represents a serious risk, and the discovery of new effective drugs is urgently required. For this reason, new antimalarial chemotypes with new mechanisms of action, and ideally with activity against multiple parasite stages, are needed. We report a new scaffold with dual-stage (blood and liver) antiplasmodial activity. Twenty-six spirooxadiazoline oxindoles were synthesized and screened against the erythrocytic stage of the human malaria parasite P. falciparum. The most active compounds were also tested against the liver-stage of the murine parasite P. berghei. Seven compounds emerged as dual-stage antimalarials, with IC50 values in the low micromolar range. Due to structural similarity with cipargamin, which is thought to inhibit blood-stage P. falciparum growth via inhibition of the Na + efflux pump PfATP4, we tested one of the most active compounds for anti-PfATP4 activity. Our results suggest that this target is not the primary target of spirooxadiazoline oxindoles and further studies are ongoing to identify the main mechanism of action of this scaffold.This work was supported by FCT (Fundação para a Ciência e a Tecnologia, I.P.) through iMed.ULisboa (UID/DTP/04138/2019), project PTDC/QUI-QOR/29664/2017, and PhD fellowship SFRH/BD/137544/2018 (E. Lopes). The NMR spectrometers are part of the National NMR Network (PTNMR) and are partially supported by Infrastructure Project Nº 022161 (co-financed by FEDER through COMPETE 2020, POCI and PORL and FCT through PIDDAC). Financial support from FCT and Portugal 2020 to the Portuguese Mass Spectrometry Network (Rede Nacional de Espectrometria de Massa – RNEM; LISBOA-01-0145-FEDER-402-022125) is also acknowledged.info:eu-repo/semantics/publishedVersio

A G358S mutation in the Plasmodium falciparum Na⁺ pump PfATP4 confers clinically-relevant resistance to cipargamin

Diverse compounds target the Plasmodium falciparum Na(+) pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4(G358S) parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na(+) regulation. The G358S mutation reduces the affinity of PfATP4 for Na(+) and is associated with an increase in the parasite’s resting cytosolic [Na(+)]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4(G358S) parasites, and that their combination with unrelated antimalarials may mitigate against resistance development

Identification of Loop D Domain Amino Acids in the Human Aquaporin-1 Channel Involved in Activation of the Ionic Conductance and Inhibition by AqB011

Aquaporins are integral proteins that facilitate the transmembrane transport of water and small solutes. In addition to enabling water flux, mammalian Aquaporin-1 (AQP1) channels activated by cyclic GMP can carry non-selective monovalent cation currents, selectively blocked by arylsulfonamide compounds AqB007 (IC50 170 μM) and AqB011 (IC50 14 μM). In silico models suggested that ligand docking might involve the cytoplasmic loop D (between AQP1 transmembrane domains 4 and 5), but the predicted site of interaction remained to be tested. Work here shows that mutagenesis of two conserved arginine residues in loop D slowed the activation of the AQP1 ion conductance and impaired the sensitivity of the channel to block by AqB011. Substitution of residues in loop D with proline showed effects on ion conductance amplitude that varied with position, suggesting that the structural conformation of loop D is important for AQP1 channel gating. Human AQP1 wild type, AQP1 mutant channels with alanines substituted for two arginines (R159A+R160A), and mutants with proline substituted for single residues threonine (T157P), aspartate (D158P), arginine (R159P, R160P), or glycine (G165P) were expressed in Xenopus laevis oocytes. Conductance responses were analyzed by two-electrode voltage clamp. Optical osmotic swelling assays and confocal microscopy were used to confirm mutant and wild type AQP1-expressing oocytes were expressed in the plasma membrane. After application of membrane-permeable cGMP, R159A+R160A channels had a significantly slower rate of activation as compared with wild type, consistent with impaired gating. AQP1 R159A+R160A channels showed no significant block by AqB011 at 50 μM, in contrast to the wild type channel which was blocked effectively. T157P, D158P, and R160P mutations had impaired activation compared to wild type; R159P showed no significant effect; and G165P appeared to augment the conductance amplitude. These findings provide evidence for the role of the loop D as a gating domain for AQP1 ion channels, and identify the likely site of interaction of AqB011 in the proximal loop D sequence

Divalent Cations Regulate the Ion Conductance Properties of Diverse Classes of Aquaporins

Aquaporins (AQPs) are known to facilitate water and solute fluxes across barrier membranes. An increasing number of AQPs are being found to serve as ion channels. Ion and water permeability of selected plant and animal AQPs (plant Arabidopsis thaliana AtPIP2;1, AtPIP2;2, AtPIP2;7, human Homo sapiens HsAQP1, rat Rattus norvegicus RnAQP4, RnAQP5, and fly Drosophila melanogaster DmBIB) were expressed in Xenopus oocytes and examined in chelator-buffered salines to evaluate the effects of divalent cations (Ca2+, Mg2+, Ba2+ and Cd2+) on ionic conductances. AtPIP2;1, AtPIP2;2, HsAQP1 and DmBIB expressing oocytes had ionic conductances, and showed differential sensitivity to block by external Ca2+. The order of potency of inhibition by Ca2+ was AtPIP2;2 > AtPIP2;1 > DmBIB > HsAQP1. Blockage of the AQP cation channels by Ba2+ and Cd2+ caused voltage-sensitive outward rectification. The channels with the highest sensitivity to Ca2+ (AtPIP2;1 and AtPIP2;2) showed a distinctive relief of the Ca2+ block by co-application of excess Ba2+, suggesting that divalent ions act at the same site. Recognizing the regulatory role of divalent cations may enable the discovery of other classes of AQP ion channels, and facilitate the development of tools for modulating AQP ion channels. Modulators of AQPs have potential value for diverse applications including improving salinity tolerance in plants, controlling vector-borne diseases, and intervening in serious clinical conditions involving AQPs, such as cancer metastasis, cardiovascular or renal dysfunction

The Purified Extract from the Medicinal Plant Bacopa monnieri, Bacopaside II, Inhibits Growth of Colon Cancer Cells In Vitro by Inducing Cell Cycle Arrest and Apoptosis

Aquaporin-1 (AQP1), a transmembrane pore-forming molecule, facilitates the rapid movement of water and small solutes across cell membranes. We have previously shown that bacopaside II, an extract from the medicinal herb Bacopa monnieri, blocks the AQP1 water channel and impairs migration of cells that express AQP1. The aim of this study was to further elucidate the anti-tumour potential of bacopaside II in colon cancer cells. Expression of AQP1 in HT-29, SW480, SW620 and HCT116 was determined by quantitative PCR and western immunoblot. Cells were treated with bacopaside II, and morphology, growth, autophagy, cell cycle and apoptosis assessed by time-lapse microscopy, crystal violet, acridine orange, propidium iodide (PI) and annexin V/PI staining respectively. AQP1 expression was significantly higher in HT-29 than SW480, SW620 and HCT116. Bacopaside II significantly reduced growth at ≥20 µM for HT-29 and ≥15 µM for SW480, SW620 and HCT116. Inhibition of HT-29 at 20 µM was primarily mediated by G0/G1 cell cycle arrest, and at 30 µM by G2/M arrest and apoptosis. Inhibition of SW480, SW620 and HCT116 at ≥15 µM was mediated by G2/M arrest and apoptosis. These results are the first to show that bacopaside II inhibits colon cancer cell growth by inducing cell cycle arrest and apoptosis

The Aquaporin 1 Inhibitor Bacopaside II Reduces Endothelial Cell Migration and Tubulogenesis and Induces Apoptosis

Expression of aquaporin-1 (AQP1) in endothelial cells is critical for their migration and angiogenesis in cancer. We tested the AQP1 inhibitor, bacopaside II, derived from medicinal plant Bacopa monnieri, on endothelial cell migration and tube-formation in vitro using mouse endothelial cell lines (2H11 and 3B11) and human umbilical vein endothelial cells (HUVEC). The effect of bacopaside II on viability, apoptosis, migration and tubulogenesis was assessed by a proliferation assay, annexin-V/propidium iodide flow cytometry, the scratch wound assay and endothelial tube-formation, respectively. Cell viability was reduced significantly for 2H11 at 15 μM (p = 0.037), 3B11 at 12.5 μM (p = 0.017) and HUVEC at 10 μM (p < 0.0001). At 15 μM, the reduced viability was accompanied by an increase in apoptosis of 38%, 50% and 32% for 2H11, 3B11 and HUVEC, respectively. Bacopaside II at ≥10 μM significantly reduced migration of 2H11 (p = 0.0002) and 3B11 (p = 0.034). HUVECs were most sensitive with a significant reduction at ≥7.5 μM (p = 0.037). Tube-formation was reduced with a 15 μM dose for all cell lines and 10 μM for 3B11 (p < 0.0001). These results suggest that bacopaside II is a potential anti-angiogenic agent