key: cord-0287732-72zrkaan
authors: Delehouzé, Claire; Comte, Arnaud; Hauteville, Marcelle; Goekjian, Peter; Dimanche-Boitrel, Marie-Thérèse; Rousselot, Morgane; Bach, Stéphane
title: Nigratine as first-in-class dual inhibitor of necroptosis and ferroptosis regulated cell death
date: 2020-12-15
journal: bioRxiv
DOI: 10.1101/2020.12.15.422885
sha: 2c9acd2e037c6f0642bdf872cf319336abd5f877
doc_id: 287732
cord_uid: 72zrkaan

Nigratine (also known as 6E11), a natural flavanone derivative, was characterized as highly specific non-ATP competitive inhibitor of RIPK1 kinase, one of the key component of necroptotic cell death signaling. We show here that nigratine inhibited both necroptosis (induced by Tumor Necrosis Factor-α) and ferroptosis (induced by glutamate, erastin or RSL3 small chemical compounds) with EC50 in the µM range. Altogether, the data obtained showed that nigratine is the first-in-class dual inhibitor of necroptosis and ferroptosis cell death routes and opened new therapeutic avenues for treating complex necrosis-related diseases.

Advances in systems biology have revealed that single-target compounds are less efficient in preventing or cure complex diseases such as neurodegenerative diseases 1, 2 . Drug discovery failures in complex cases, including Alzheimer's disease (AD), may suggest that the definition of a "magic bullet" (drug selective for a single molecular target), the scientific concept developed by Paul Ehrlich more than one century ago 3 , should be tempered. The development of multi-target-directed ligands (MTDLs) for AD treatment is a notable example of promising therapeutic strategies 2 . Moreover, the most clinically effective central nervous system (CNS) drugs -such as clozapine for treatment of schizophrenia-act as "magic shotguns": a nonselective drugs with pleiotypic actions 4 . These compounds are effective in treating complex human disorders because they are able to modulate the multiple targets involved in the pathophysiological processes, a strategy called polypharmacology. Synergistic effects are also among the main advantages of this combined therapeutic approach 5 . This strategy is now widely used in drug development as, from 2015 to 2017, 21% of the new molecular entities (NMEs) approved by the food and drug administration (FDA) are multi-target drugs (34% were single-target small molecules) 6 .

The systemic breakdown of physiological networks is not only restricted to the brain tissue and was described in numerous others including kidneys and liver. Functionally redundancy is typical features of such diseased networks and is well described for regulated cell death (RCD) signaling 7 . Indeed, this redundancy may reflect a particular evolutionary history for cell suicide and autophagic, apoptotic or necrotic elements might have been added to an ancestral death mechanism. Ancestral cell death routes probably include ferroptosis, a non-apoptotic cell death that is catalyzed by iron 8 , shown to be functional in cyanobacteria, components of phytoplankton communities evolving over 2.7 billions years ago 9 . As mentioned by Golstein and Kroemer in 2005, "the resulting redundancy of cell death mechanisms has pathophysiological implications" 10 . Cell death is inevitable and can be either physiological (e.g.

for removing unwanted cells, such as cancer cells) or pathological (e.g. in neurodegenerative disorders). Nowadays, researchers noticed that there are at least 12 distinct types of RCD including various forms of regulated necrosis 7 . The term "regulated" indicates that RCD relies on fine-tuned molecular machinery involving signaling cascades and effectors. Thus, it is possible to find a way to characterize small chemical compounds that can modulate these pathways (such as with necrostatins for necroptosis 11 ). These results led to the hypothesis that RCDs are « druggable », an emerging breakthrough that carries the potential to find new therapeutic approaches for unmet therapeutic needs including inflammatory and neurodegenerative diseases 12 . Necroptosis, is a regulated cell necrosis route that can be activated under apoptosis-deficient conditions. Necroptosis depends notably on the serine/threonine kinase activity of RIPK1 (Receptor-Interacting Protein Kinase 1) and RIPK3 and on the trafficking and accumulation at the plasma membrane of the pseudokinase MLKL (Mixed lineage kinase domain-like) 12 We contributed to this intensive hunt for potent RIPK1 inhibitors with the characterization of synthetic and natural-derivatives compounds including the 7-azaindole derivative Sibiriline 15 , the marine-derivative 2-aminobenzothiazole MBM105 16 and flavanone nigratine (also known as 6E11) 17 . Nigratine (2-(4-(benzyloxy)phenyl)-2,5-dihydroxy-7-methoxychroman-4-one) is a synthetic derivative of the naturally occurring 2,5-dihydroxy-2-phenylchroman-4-ones isolated from Populus nigra buds. We previously showed that nigratine is a non-ATP competitive inhibitor with a remarkable selectivity toward RIPK1 and able to protect human aortic endothelial cells (HAEC) from cold hypoxia/reoxygenation-induced cell death 17 .

We now reported here the characterization of nigratine as new inhibitor of ferroptosis cell-death (with EC50 in the µM range). Nigratine is thus considered as the first-in-class dual inhibitor (or "magic shotgun") of both necroptosis and ferroptosis regulated cell death that can be used in polypharmacological approaches for treatments of regulated-necrosis related disorders.

Nigratine was characterized by Delehouzé et al. as new necroptosis inhibitor using a TNFinduced FADD-deficient human Jurkat necroptosis assay 17 . TNF-a can induce necroptosis in Jurkat cells when FADD is deleted. MTS assay was used to measure the viability of lymphocytes and the effect on the RIPK1-kinase activity was evaluated to study the mechanism of action of nigratine. In this study, we compared the bioactivity of nigratine with necrostatin-1 (nec-1) as control inhibitor of necroptosis 11 . These compounds were tested at the concentration of 10 and 50µM. The results obtained and the chemical structures of these compounds are depicted in Figure 1 . In this assay, nigratine inhibited both necroptosis cell death and RIPK1 kinase activity with a similar activity than nec-1 (with both EC50 and IC50 in the μM range). The value of the protection index (dubbed ProtecD) for nigratine at 10µM (45.0) compared to the value at 50µM (26.0) suggested a lesser effect of the molecule on Jurkat FADD-deficient cells at high concentration.

In Delehouzé et al., we previously showed that treatment of human aortic endothelial cells (HAEC) with nigratine during cold hypoxia or during cold hypoxia and reoxygenation brought measurable benefits on cell survival. Compared to nec-1s, as specific RIPK1 inhibitor of necroptosis, the effect of nigratine was significantly better 17 . We thus suggested that this observed effect of nigratine was not fully related to the inhibition of RIPK1 kinase. Indeed, when compared to nigratine, nec-1s is a more potent inhibitor of RIPK1 17 .

We next analyzed the effect of nigratine treatment on ferroptotic cell-based models. The dose-dependent effect of nigratine on cell viability was analyzed using MTS reduction assay. In order to validate the effect of nigratine on neuroprotection and necrosis, the extracellular lactate dehydrogenase (LDH) detection assay was also used as an independent cell death assay 19 . Indeed, the LDH is released into extracellular space when the plasma membrane is damaged. The necrotic cell death is essentially associated with the membrane permeabilization resulting to the rapid release of the cellular contents (including LDH and various damage-associated molecular patterns) 20 . As shown in Figure 3b , the treatment of SH-SY5Y with increasing doses of nigratine protected cells against cell-death in a dose dependent manner that correlated with the similar dose-dependent increase in cell survival. The results obtained also showed that nigratine has an acceptable cytotoxic effect on SH-SY5Y neuroblastoma cell line (Figure 3b) . Interestingly, nigratine is active against ferroptosis on SH-SY5Y with EC50 around 6.5µM (EC50(LDH)=6.0µM and EC50(MTS)=6.9µM) in the same range than the activity against necroptosis (EC50(MTS)=4.6µM). Ferrostatin-1 was used as model inhibitor of ferroptosis and showed a better cellular effect compared to nigratine (Figure 2 and 3) . (Figure 4b-c) . This dye is a wellknown sensitive indicator of free radical processes that have the potential to oxidize lipids in membranes. Taken together, these results suggested that nigratine has an effect on lipid peroxydation, one major hallmark of ferroptosis.

Ferroptosis is characterized as a consequence of defects in antioxidant defenses. Indeed, ferroptosis is catalyzed by iron and is due to a loss of activity of the lipid repair enzyme glutathione peroxidase 4 (GPX4) 21 

Nigratine was previously described as a putative non-ATP competitive type III RIPK1 kinase inhibitor that can block the necroptosis cell death on various cellular models including Jurkat lymphocytes with EC50 in the µM range 17 . Necroptosis, a programmed cell death route, is clearly distinct from apoptosis as it does not involve key apoptosis regulators, such as caspases, Bcl-2 family members or cytochrome c release from mitochondria. In the present study, we showed that nigratine is also an inhibitor of ferroptosis: nigratine suppressed cell death induced by class I and II ferroptosis inducers: excess of glutamate, erastin and RSL3, respectively. We also showed that nigratine inhibits phospholipid peroxidation induced by RSL3 in epithelial LLC-PK1 porcine renal cells, an inhibition that was also observed for ferrostatin-1 as control molecule.

As mentioned here before, polypharmacological approaches -using drugs acting on multiple targets-appear suited to improve the outcome of complex diseases. Thus, nigratine appears to be very attractive in therapy for preventing and/or treating disorders associated with the induction of both necroptosis and ferroptosis. As example, the therapeutic benefit of nigratine can be evaluated in animal models of cisplatin-induced nephrotoxic acute kidney injury (AKI) where these two regulated necrosis were shown to be involved in proximal tubular cell death [23] [24] [25] . Ischemia/reperfusion injuries (IRI), that have been attributed to cell necrosis for decades 26 , should also be explored with nigratine.

Canonical ferroptosis inhibitors like the lipophilic antioxidant ferrostatin-1, prevent the accumulation of toxic lipid and cytosolic ROS, inhibiting ferroptotic cell death. As observed here using the DPPH assay, nigratine is comparatively less potent antioxidant than the two aromatic amines ferrostatin-1 and a-tocopherol. We consequently hypothesized that nigratine does not use a mechanism linked to the peroxyl radical scavenging for the inhibition of ferroptosis -or may inhibit another cellular target contributing to the overall cell-death protection. We now have to identify the molecular target of nigratine in ferroptosis using various approaches and notably by target fishing (see 27 for review). This identification is prerequisite for improving the efficacy of dual-target inhibitors on ferroptosis-related phenotypes without affecting the activity of its primary target, the receptor-interacting protein kinase 1 (RIPK1).

Numerous approaches are available to increase the efficacy of this class of drugs on rational basis. They include notably the combination of medicinal chemistry and computational strategies as it was already described for dual-target kinase inhibitors 28 .

Taken together these results obtained on nigratine indicate that pharmaceutical agents acting on both necroptosis and ferroptosis cell death routes can be designed and used to treat complex diseases involving the activation of multiple regulated necrosis. This study shed light on the emergence of polypharmacological approaches for treating multiple disorders where necrosis is of central pathophysiological relevance, such as: ischemia-reperfusion injury in brain, heart and kidney, inflammatory diseases, sepsis, retinal disorders or neurodegenerative diseases. Cell death was determined by measurement of lactate dehydrogenase (LDH) leakage using the LDH Cytotoxicity assay kit (Invitrogen, Carlsbad, CA, USA) following manufacturer's recommendations. LDH is a cytosolic enzyme that is rapidly released into the supernatant after cell damage. After 24h of treatment 50µl of supernatants were transferred into a clean 96-well plates, reagents were added and LDH activity was measured using a microplate reader.

Percentage of cytotoxicity was calculated by dividing the LDH activity of the compounds with RSL3 by that of the RSL3 and DMSO. Results are plotted in % of LDH release measured when cells are treated with RSL3 (left axis, colored blue). Cell viability was measured by MTS reduction assay. The results obtained (colored red) were plotted in % of maximal viability (detected in DMSO-treated cells, right axis).

Data are shown as the mean ± SD of three replicates. 

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LLC-PK1 cell line was treated with 2µM of RSL3 and increasing concentration of Nigratine or ferrostatin-1 (Fer-1). (a) Cell viability was estimated by MTS assay. Data are shown as the mean ± SD of three replicates. (b) Lipid peroxidation was detected by cellular BODIPY 581/591 C11 staining. Fluorescence was recorded with the IncuCyte S3 live cell imaging apparatus. Data are shown as the mean ± SD of three replicates of nine replicates. (c) Representative phase-contrast and fluorescence images of cells stained with BODIPY 581/591

Claire Delehouzé, Marie-Thérèse Dimanche-Boitrel, Morgane Rousselot and Stéphane Bach are the founders and members of the scientific advisory board of SeaBeLife Biotech, which is developing novel therapies for treating liver and kidney acute disorders. Peter Goekjian is member of the scientific advisory board of SeaBeLife Biotech.