Ferroptosis-modulating small molecules for targeting drug-resistant cancer: Challenges and opportunities in manipulating redox signaling

Abstract

Ferroptosis is an iron-dependent cell death program that is characterized by excessive lipid peroxidation. Triggering ferroptosis has been proposed as a promising strategy to fight cancer and overcome drug resistance in antitumor therapy. Understanding the molecular interactions and structural features of ferroptosis-inducing compounds might therefore open the door to efficient pharmacological strategies against aggressive, metastatic, and therapy-resistant cancer. We here summarize the molecular mechanisms and structural requirements of ferroptosis-inducing small molecules that target central players in ferroptosis. Focus is placed on (i) glutathione peroxidase (GPX) 4, the only GPX isoenzyme that detoxifies complex membrane-bound lipid hydroperoxides, (ii) the cystine/glutamate antiporter system X_c ^- that is central for glutathione regeneration, (iii) the redox-protective transcription factor nuclear factor erythroid 2-related factor (NRF2), and (iv) GPX4 repression in combination with induced heme degradation via heme oxygenase-1. We deduce common features for efficient ferroptotic activity and highlight challenges in drug development. Moreover, we critically discuss the potential of natural products as ferroptosis-inducing lead structures and provide a comprehensive overview of structurally diverse biogenic and bioinspired small molecules that trigger ferroptosis via iron oxidation, inhibition of the thioredoxin/thioredoxin reductase system or less defined modes of action.

Keywords: drug-resistant cancer; ferroptosis; natural product; small molecule; structural requirement.

Figure 1

Major metabolic and regulatory pathways in ferroptosis targeted by small molecules. (A) Membrane peroxidation in ferroptosis depends on enzymatic and nonenzymatic mechanisms. LOX isoenzymes and oxidoreductases (POR) with Fe²⁺/Fe³⁺ in their active center introduce oxygen into polyunsaturated fatty acids (PUFAs), and free metal ions, in particular Fe²⁺, convert hydrogen peroxide into hydroxyl radicals via the Fenton reaction. GPX4 reduces lipid hydroperoxides, counteracts ferroptosis, and relies on the biosynthesis and regeneration of its substrate glutathione. (B) Additional protection against membrane peroxidation offers endogenous lipophilic radical traps such as ubiquinol and vitamin K (VitK), which are regenerated by FSP1 and DHODH in the cytosol and mitochondria, respectively. The mevalonate pathway is central for the biosynthesis of (i) cholesterol, (ii) the lipophilic radical trap CoQ10, and (iii) selenocysteine transfer RNA (sec‐tRNA), which inserts selenocysteine into GPX4. (C) System X_c ⁻ regulates cellular GSH levels by importing cystine in exchange for glutamate (Glu). Intracellular cystine is reduced to cysteine, which subsequently enters GSH biosynthesis and serves as a cofactor for GPX4. (D) Labile iron levels are kept within narrow thresholds by co‐ordinated regulation of iron uptake via the transferrin receptor and iron storage within ferritin. Other factors in the control of labile iron levels sequester iron into iron–sulfur clusters (NSF1) or liberate iron from heme oxygenase‐1 (HO‐1). The transmembrane glycoprotein CD44 mediates the endocytosis of iron‐bound hyaluronate. The scheme illustrates points of attack of selected small molecules that induce ferroptosis, with a focus on drug candidates, tool compounds, and natural products. The color of the boxes distinguishes between biogenic/bioinspired (gray) and synthesized small molecules (blue). (E) Central ferroptotic genes are under the control of the transcription factors NRF2 and p53. Mitochondrium in (B) was adapted from “Resting Metabolic Activity vs. Stimulated Metabolic Activity,” by BioRender.com (2020). Retrieved from https://app.biorender.com/biorender-templates. 2,2′‐BP, 2,2′‐bipyridine; 5‐ALA, 5‐aminolevulinic acid; ACO1, aconitase 1; ATPR, 4‐amino‐2‐trifluoromethyl‐phenyl retinate; BHT, butylhydroxy toluol; BSO, l ‐buthionine‐S,R‐sulfoximine; CLA conjugated linolenic acids; CPX, ciclopirox; DFO, DHODH, dihydroorotate dehydrogenase; deferoxamine; DMF, dimethyl fumarate; FPP, farnesyl pyrophosphate; FSP1, ferroptosis suppressor protein 1; FTH, ferritin heavy chain; GCL, glutamate cysteine ligase; GGT1, γ‐glutamyl transpeptidase 1; GLS2, glutaminase 2; GPX4, glutathione peroxidase 4; GSH, reduced glutathione; GSS, glutathione synthetase; GSSG, glutathione disulfide; HMG‐CoA, 3‐hydroxy‐3‐methylglutaryl‐coenzyme A; HMGR, 3‐hydroxy‐3‐methylglutaryl‐CoA reductase; IPP, isopentenyl pyrophosphate; IREB2, iron responsive element binding protein 2; KEAP1, kelch‐like ECH‐associated protein 1; LOX, lipoxygenase; NFS1, mitochondrial cysteine desulfurase; NRF2, nuclear factor erythroid 2‐related factor 2; PL, phospholipid; PL‐OOH, phospholipid hydroperoxide; PL‐OH, phospholipid alcohol; POR, cytochrome P450 oxidoreductase; ROS, reactive oxygen species; SQS squalene synthase; TFR, transferrin receptor; TRX, thioredoxin; TXNRD1, thioredoxin reductase 1; β‐ l ‐ODAP, β‐N‐oxalyl‐l‐α‐β‐diaminopropionic acid. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 2

Inhibitors of system X_c ⁻. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Inhibitors of glutathione peroxidase 4. MDM2, mouse double minute 2. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Human GPX4 (C66S) in complex with ML162 (S enantiomer). The surface of GPX4 with bound (S)‐ML162 (depicted with yellow carbons) (PDB entry 6HKQ) was superimposed to a structure that shows the peptide inhibitor GXpep3 (depicted with green carbons) bound to GPX4 (U46C) (PDB entry 5h5s). The surface of the catalytic tetrad is colored red (Sec46), blue (Gln81), dark blue (Trp136), and purple (Asn137). Two different orientations are shown. Reproduced with permission of the International Union of Crystallography. GPX4, glutathione peroxidase 4; PDB, Protein Data Bank. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Combined indirect glutathione peroxidase 4 inhibitors and heme oxygenase‐1 inducers. PEITC, phenylethyl isothiocyanate; TCBQ, tetrachloro‐1,4‐benzoquinone. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 5

Combined indirect glutathione peroxidase 4 inhibitors and heme oxygenase‐1 inducers. PEITC, phenylethyl isothiocyanate; TCBQ, tetrachloro‐1,4‐benzoquinone. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 6

Molecular characteristics related to cancer cell‐selective lethality shared by small molecules indirectly inhibiting GPX4 and inducing HO‐1. Genetic or pharmacological inhibition of HO‐1 decreases the viability of cancer cells under basal conditions. When cancer cells are, however, exposed to electrophilic small molecules that activate NRF2 and evoke a strong expression of HO‐1, the accumulation of lipid hydroperoxides and a drop in cell survival are diminished by selective HO‐1 inhibition. Whether the often associated increase of HO‐1 and decrease of GPX4 are independent events or functionally linked is poorly understood. In nontransformed, normal cells, the NRF2/HO‐1 axis instead rather protects from lipid peroxidation and cell death. This antiferroptotic activity seems at least partially to be mediated by HO‐1. GPX4, glutathione peroxidase 4; HO‐1, heme oxygenase‐1; NRF2, nuclear factor erythroid 2‐related factor 2; ROS, reactive oxygen species. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 7

Inducers of the nuclear factor erythroid 2‐related factor 2/heme oxygenase‐1 axis [Color figure can be viewed at wileyonlinelibrary.com]

Figure 8

NRF2‐regulatory pathways. The transcription factor NRF2 consists of multiple domains that are under tight regulatory control. In complexes with KEAP1, NRF2 is ubiquitinated and degraded by the proteasome. Electrophilic and oxidative modifications of cysteine residues in KEAP1 lower the affinity to NRF2, and factors like p21 or NF‐κB hamper the binding of KEAP1 to NRF2. Ubiquitin‐dependent NRF2 degradation is further determined through the availability of the substrate recognition component of the SKP1‐cullin 1‐F‐box protein E3 ligase complex β‐TrCP, which responds to metabolic changes and is regulated by GSK3‐β. Another NRF2‐regulatory factor is the E3 ubiquitin‐protein ligase HRD1 which participates in ER‐associated degradation during ER stress. A variety of signaling cascades regulate the expression, nuclear availability, DNA‐binding affinity, and transactivation activity of NRF2, and also posttranslational and epigenetic modifications essentially contribute to the regulation of NRF2 activity. These pathways and factors represent potential targets for NRF2‐inhibiting small molecules. AKT, protein kinase B; BACH1, BTB domain and CNC homolog 1; ERRβ, estrogen‐related receptor β; ERα, estrogen receptor α; GRα, glucocorticoid receptor α; GSK3‐β, glycogen synthase kinase‐3β; HRD1, HMG‐CoA reductase degradation protein 1; IGF‐1, insulin‐like growth factor 1; MAPK, mitogen‐activated protein kinase; NF‐κB, nuclear factor κ‐light‐chain‐enhancer of activated B cells; NRF2, nuclear factor erythroid 2‐related factor 2; PI3K, phosphatidylinositol 3‐kinase; PKC, protein kinase C, PPARγ, peroxisome proliferator‐activated receptor γ; RARα, retinoic acid receptor α; RXRα, retinoid X receptor α. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 9

Nuclear factor erythroid 2‐related factor 2 inhibitors

Figure 10

Inducers of (lipid) reactive oxygen species formation [Color figure can be viewed at wileyonlinelibrary.com]

Figure 11

Ferroptosis‐inducing ferroptocide covalently targets thioredoxin [Color figure can be viewed at wileyonlinelibrary.com]

Figure 12

Increasing the lysosomal iron load or inducing spontaneous lipid peroxidation [Color figure can be viewed at wileyonlinelibrary.com]

Figure 13

Inducers of (lipid) reactive oxygen species formation that trigger ferroptosis through less characterized mechanisms [Color figure can be viewed at wileyonlinelibrary.com]