Peroxide antimalarial drugs target redox homeostasis in Plasmodium falciparum infected red blood cells

Plasmodium falciparum causes the most lethal form of malaria. Peroxide antimalarials based on artemisinin underpin the frontline treatments for malaria, but artemisinin resistance is rapidly spreading. Synthetic peroxide antimalarials, known as ozonides, are in clinical development and offer a potential alternative. Here, we used chemoproteomics to investigate the protein alkylation targets of artemisinin and ozonide probes, including an analogue of the ozonide clinical candidate, artefenomel. We greatly expanded the list of protein targets for peroxide antimalarials and identified significant enrichment of redox-related proteins for both artemisinins and ozonides. Disrupted redox homeostasis was confirmed by dynamic live imaging of the glutathione redox potential using a genetically encoded redox-sensitive fluorescence-based biosensor. Targeted LC-MS-based thiol metabolomics also confirmed changes in cellular thiol levels. This work shows that peroxide antimalarials disproportionately alkylate proteins involved in redox homeostasis and that disrupted redox processes are involved in the mechanism of action of these important antimalarials.

Acidity and nucleophilic reactivity of glutathione persulfide

Persulfides (RSSH/RSS−) participate in sulfur trafficking and metabolic processes, and are proposed to mediate the signaling effects of hydrogen sulfide (H2S). Despite their growing relevance, their chemical properties are poorly understood. Herein, we studied experimentally and computationally the formation, acidity, and nucleophilicity of glutathione persulfide (GSSH/GSS−), the derivative of the abundant cellular thiol glutathione (GSH). We characterized the kinetics and equilibrium of GSSH formation from glutathione disulfide and H2S. A pKa of 5.45 for GSSH was determined, which is 3.49 units below that of GSH. The reactions of GSSH with the physiologically relevant electrophiles peroxynitrite and hydrogen peroxide, and with the probe monobromobimane, were studied and compared with those of thiols. These reactions occurred through SN2 mechanisms. At neutral pH, GSSH reacted faster than GSH because of increased availability of the anion and, depending on the electrophile, increased reactivity. In addition, GSS− presented higher nucleophilicity with respect to a thiolate with similar basicity. This can be interpreted in terms of the so-called α effect, i.e. the increased reactivity of a nucleophile when the atom adjacent to the nucleophilic atom has high electron density. The magnitude of the α effect correlated with the Brønsted nucleophilic factor, βnuc, for the reactions with thiolates and with the ability of the leaving group. Our study constitutes the first determination of the pKa of a biological persulfide and the first examination of the α effect in sulfur nucleophiles, and sheds light on the chemical basis of the biological properties of persulfides.

Medicinal Thiols: Current Status and New Perspectives

The thiol (-SH) functional group is found in a number of drug compounds and confers a unique combination of useful properties. Thiol-containing drugs can reduce radicals and other toxic electrophiles, restore cellular thiol pools, and form stable complexes with heavy metals such as lead, arsenic, and copper. Thus, thiols can treat a variety of conditions by serving as radical scavengers, GSH prodrugs, or metal chelators. Many of the compounds discussed here have been in use for decades, yet continued exploration of their properties has yielded new understanding in recent years, which can be used to optimize their clinical application and provide insights into the development of new treatments. The purpose of this narrative review is to highlight the biochemistry of currently used thiol drugs within the context of developments reported in the last five years. More specifically, this review focuses on thiol drugs that represent the standard of care for their associated conditions, including N-acetylcysteine, 2,3-meso-dimercaptosuccinic acid, British anti-Lewisite, D-penicillamine, amifostine, and others. Reports of novel dosing regimens, delivery strategies, and clinical applications for these compounds were examined with an eye toward emerging approaches to address a wide range of medical conditions in the future.

Intracellular Thiol Oxidation Is Linked with Loss of Δψm and Disease Progression in Acute Promyelocytic Leukaemia

Acute promyelocytic leukaemia (APL) is a type of myeloid malignancy defined by the chromosomal translocation t(15;17) and subsequent expression of the PML-RARα fusion protein. Long term remission in APL is achieved through a combination of high dose all-trans retinoic acid (ATRA) and arsenic trioxide (ATO), the latter of which has potential to induce mitochondrial derived oxidative stress and initiate the intrinsic apoptosis pathway. ATO is particularly effective in the treatment of APL when compared with acute myeloid leukaemia (AML), suggesting that the mitochondrial membrane potential (Δψm) of APL cells is compromised. Here we show that loss of Δψm is associated with intracellular thiol oxidation and disease progression in APL. The mitochondrial probe 3,3'-diethyloxacrbocyanine iodide (DiOC2(3)) was used to evaluate MRP8-PML/RARA transgenic mice Δψm in bone marrow mononuclear cells (BMMC) at three disease stages: 1) pre-APL, 2) APL and 3) ATRA treated APL. Our data show that MRP8-PML/RARA BMMC cells during the APL stage accumulate significantly less DiOC2(3) compared with pre-APL cells (0.05±0.02 vs 0.24±0.05, p<0.05; data are normalised FL1/FL3 MFI ratio). The DiOC2(3) accumulation in BMMC from ATRA treated APL mice was equivalent to that of pre-APL cells (p>0.05). This finding indicates a loss of Δψm in APL. This may result in free radical leakage from the mitochondria, which in turn could oxidise intracellular thiols. To evaluate intracellular thiol oxidation, a previously optimised flow cytometric assay that incorporates the thiol reactive probe fluorescein-5 maleimide (F5M) was used. Our data show a significant decrease in F5M MFI for MRP8-PML/RARA BMMC cells during the APL stage, compared with pre-APL cells (219.01±47.67 vs 672.66±131.04, p<0.05; data are F5M MFI relative to unstained controls). This finding signifies that there is more thiol oxidation at the APL stage. ATRA treated APL mice showed an equivalent F5M MFI to that of pre-APL cells. Confocal microscopy confirmed that the F5M signal was intracellular. This observation shows that there is an increase in intracellular thiol oxidation in APL, which may be tracked during disease progression and treatment course. This finding may explain why APL cells are more sensitive to pro-oxidant treatments i.e. ATO. To understand these observations further, the APL cell line (NB4) was subjected to glucose oxidase (GOX) mediated oxidative stress in cell culture over a 4 hour time-course. F5M MFI signal was compared with the THP1 and Kasumi-1 AML cell lines subjected to the same treatment. All cell lines showed similar significant increases in F5M MFI after 1 hour GOX treatment relative to control (p<0.05), indicating an increase in 'reduced' cellular thiol i.e. reductive stress. However, after 4 hours NB4 cells showed a significant decrease in F5M MFI relative to control (p<0.05), indicating an increase in 'oxidised' cellular thiol i.e. oxidative stress. In contrast, THP1 cells showed no difference in MFI after 4 hours (p>0.05), whereas F5M MFI Kasumi-1 remained significantly increased after 4 hours (p<0.05) relative to control. This finding indicates differences in the sensitivity to oxidative stress between APL and AML cells. Taken together, this study shows that APL cells are more sensitive to oxidative stress compared with AML cells. Our F5M flow cytometric assay illustrates that an increase in cellular thiol oxidation is linked to disease progression in APL, with loss of Δψm associated with this process. Our F5M flow cytometric assay may therefore have clinical utility in monitoring treatment efficacy in APL. Disclosures No relevant conflicts of interest to declare.

Fluorescence “off” and “on” signalling of esculetin in the presence of copper and thiol: a possible implication in cellular thiol sensing

The fluorescence intensity of esculetin was drastically reduced in the presence of a copper ion, which was regenerated in the presence of GSH. The copper–esculetin system was able to detect GSH in a cellular model.