Glutathione Dynamics in Subcellular Compartments and Implications for Drug Development

Abstract

Glutathione (GSH) is a pivotal tripeptide antioxidant essential for maintaining cellular redox homeostasis and regulating diverse cellular processes. Subcellular compartmentalization of GSH underscores its multifaceted roles across various organelles including the cytosol, mitochondria, endoplasmic reticulum, and nucleus, each exhibiting distinct regulatory mechanisms. Perturbations in GSH dynamics contribute to pathophysiological conditions, emphasizing the clinical significance of understanding its intricate regulation. This review consolidates current knowledge on subcellular GSH dynamics, highlighting its implications in drug development, particularly in covalent drug design and antitumor strategies targeting intracellular GSH levels. Challenges and future directions in deciphering subcellular GSH dynamics are discussed, advocating for innovative methodologies to advance our comprehension and facilitate the development of precise therapeutic interventions based on GSH modulation.

Introduction

Glutathione (GSH), a tripeptide composed of γ-glutamic acid, cysteine, and glycine, is the most abundant small-molecule thiol in cells and plays critical roles in various cellular processes. As the primary cellular antioxidant, GSH maintains redox homeostasis, protects cells from oxidative stress, and participates in detoxification processes [1]. Moreover, GSH is involved in protein modification through S-glutathionylation, a covalent post-translational modification impacting cell signaling [2–5]. Furthermore, GSH regulates gene expression and protein functions through non-covalent binding to proteins [6].

Only a very small percentage of GSH (< 5 %) exists in its oxidized form GSSG under normal conditions, when taken the overall cellular environment into consideration [7–9]. Certain subcellular organelles like ER might have higher local GSSG ratio compared to the overall cellular GSSG level [9–12]. The balance between GSH and GSSG is a critical determinant in the proper functioning of various cellular pathways [8,13,14]. Redox imbalances can lead to a cascade of cellular damage, inflammation, and cell death, contributing to pathological conditions observed in diseases such as cardiovascular disorders, neurodegenerative diseases, and diabetes [15–17]. For instance, one of the earliest biochemical abnormalities observed in patients with Parkinson’s disease is a reduction in overall GSH levels [18]. Therefore, understanding the regulation and dynamics of GSH in the cellular environment holds great clinical significance.

GSH is primarily synthesized in the cytosol but is not confined to this compartment [19,20]. It is distributed to various subcellular organelles, including mitochondria, the endoplasmic reticulum (ER), and the nucleus, each exhibiting distinctive regulation patterns. Some recent reviews provided good summary on the subcellular compartmentation of GSH distribution and the regulation on its transportation [21,22]. The concentration and redox state of GSH significantly impact organelle functions. The distinct patterns are usually characterized using redox potential (E_GSH) [23,24]. Cytosolic E_GSH is in the range of −280 to −320 mV depending on cell density [25]. Mitochondrial E_GSH generally ranges from −280 to −300 mV, while E_GSH in the ER is more oxidizing and has a wider range of values from −118 mV to −230 mV in mammalian cells [10,14,25–31]. This indicates a lower reducing potential in the ER lumen compared to other compartments, which facilitates protein folding and disulfide bond formation in nascent proteins. Disruptions in this equilibrium can lead to protein misfolding, potentially causing cellular dysfunction and cell death [32].

In this review, we summarize current knowledge on the subcellular GSH dynamics and its implication on drug targets regulating GSH abundance. Also, we provide discussion about the considerations of GSH under drug development, especially for covalent inhibitors.

1. Intracellular GSH dynamics

Intracellular GSH levels are maintained at a constant level, regulated by multiple processes [21,33–35]. GSH is primarily synthesized by glutamate-cysteine ligase (GCL) and glutathione synthetase (GS) in the cytosol (Figure 1), with GCL being the rate-limiting step [20]. Reactive oxygen species (ROS) can directly oxidize a portion of GSH to form GSSG, which can subsequently be reduced back to GSH by glutathione reductase (GR). Moreover, cytosolic GSH is consumed by glutathione peroxidases (GPx) in response to ROS like hydrogen peroxide [33]. Due to its inability to pass through membranes, GSH is transported into subcellular compartments (e.g., mitochondria, ER, nucleus, and peroxisomes) through distinct transporters [21,36,37]. Although subcellular GSH activities are less well-explored compared to overall cellular GSH, each organelle is believed to maintain an independent GSH pool [21].

Figure 1.

Overview of GSH distribution and transport pathways in cells. Glutathione (GSH) is predominantly synthesized within the cytosol from precursor amino acids including glutamate, cysteine, and glycine. Its conversion to oxidized glutathione (GSSG) can occur through various pathways. GSH is actively transported into different subcellular compartments. Within mitochondria, it is imported via carrier proteins belonging to the SLC25 family. Within this organelle, GSH plays a crucial role in mitigating reactive oxygen species (ROS) through redox reactions. In the ER, GSH is transported via facilitated diffusion catalyzed by Sec61, contributing significantly to protein folding processes. However, the regulatory mechanisms governing GSH levels within the nucleus remain poorly understood. To date, only Bcl2 has been identified as a potential carrier protein associated with GSH transport into the nucleus, indicating the complexity of intracellular GSH distribution and regulation.

1.1. GSH dynamics in mitochondria

Mitochondrial GSH, like cytosolic GSH, predominantly exists in its reduced state and serves as a critical buffer against excessive reactive oxygen species (ROS) [14]. Mitochondria is the primary organelle for ROS production. For example, H₂O₂, the main product of SOD2 from the detoxication process, can be generated at a rate of 50–70 pmol/min/mg in the mitochondria [38]. The peroxide detoxification process primarily relies on the GSH/Grx and Trx/Prx systems, with a collective scavenging rate over 100-fold higher than the production rate [39]. Under oxidative stress, either Grx or Trx can reduce disulfide bonds through GR or TR (thioredoxin reductase) and NADPH, preventing the accumulation of misfolded proteins [40,41]. The role of mitochondrial GSH in oxidative stress mediation has been well summarized in previous reviews [14,42,43].

Mitochondrial GSH comprises approximately 10–15% of total cellular GSH, with a similar concentration to cytosolic GSH, primarily maintained by GSH transport [42]. Cytosolic GSH crosses the mitochondrial outer membrane (MOM) through rapid porin-mediated exchange (Figure 1) and is then transported into the matrix primarily through inner mitochondria membrane (IMM) proteins of the SLC25 family: the dicarboxylate carrier (DIC) and the 2-oxoglutarate carrier (OGC) [36]. Interestingly, the authors observed that the distribution of GSH transport among DIC and OGC varies among cells from distinct organs. However, further exploration is needed to unmask the detailed contributions of these carriers in mitochondrial GSH transport from different cells.

Recently, another member of the SLC25 family, SLC25A39, was identified as a GSH carrier, offering a compensatory mechanism when mitochondrial GSH levels are low [44,45]. SLC25A39 is rapidly degraded under normal conditions by a mitochondrial protease (AFG3L2), but dissociates from the protease during GSH depletion to restore GSH levels. Atm1, identified as a GSH exporter in yeast, participates in delivering GSH-complexed iron-sulfur clusters into the cytosol [46]. These transportation systems are critical for maintaining mitochondrial GSH dynamics.

Disruption of GSH dynamics typically impairs redox buffering capacity, often manifested as reduced mitochondrial GSH (mtGSH) levels. mtGSH depletion is associated with ROS accumulation and dysregulated protein modifications, like S-glutathionylation [4,42]. One of the main causes of mtGSH depletion is impaired transport systems, as seen in alcoholic steatohepatitis (ASH) caused by excessive alcohol consumption. Studies in rat liver have shown that alcohol can reduce the GSH transport rate of OGC by altering the physical properties of the inner mitochondrial membrane due to increased cholesterol levels [47]. mtGSH depletion also sensitizes hepatocytes to tumor-necrosis factor (TNF), which can lead to rapid cell death [48]. Therefore, replenishing mtGSH levels is a common consideration for rescuing cells with activated TNF pathway. However, if mtGSH pool is strategically depleted during hypoxia, it can downregulate TNF-induced NF-κB activity, potentially activating apoptosis in cancer cells [49]. Further exploration of mtGSH regulation is essential for understanding both pathological mechanisms and potential therapeutic strategies.

1.2. GSH dynamics in endoplasmic reticulum

Similar to mitochondria, the ER relies on cytosolic GSH as it lacks GSH synthesis machinery [10,50]. While the ER shares a similar total GSH concentration with the cytosol and mitochondria, it possesses a higher GSSG ratio. GSH plays a critical role in nascent protein folding in the ER. Oxidized protein disulfide isomerase (PDI) facilitates disulfide bond formation in conjunction with endoplasmic reticulum oxidase 1 (Ero1), while GSH acts as a buffer to maintain the reduced form of PDI [50].

GSH is transported to the ER through Sec61 via facilitated diffusion, driven by the GSH concentration gradient [37]. Additionally, Ponsero and coworkers identified a negative feedback loop for GSH transportation regulated by Ero1 and Bip. Elevated GSH import leads to the Ero1-dependent oxidation of Bip, which impedes the transportation of GSH [37]. The regulation of GSH transport is also associated with the mobilization of Ca²⁺. Lizák and coworkers found that depleted Ca²⁺ in the ER lumen decreased PDI activities, leading to a reduced oxidation capacity locally. This can mediate the influx of GSH through a Ca²⁺-sensitive pathway [51]. This connection also contributes to the proximal cross-talk between mitochondria and ER [52–54]. Unlike other compartments, the ER lacks the reductase needed to convert GSSG back into GSH, making GSSG export vital for maintaining the GSH/GSSG balance. However, the mechanisms and players involved in GSSG export in the ER remain unclear.

Dysregulation of the GSH/GSSG ratio can lead to ER stress, which can be triggered by various pathological conditions [55]. For instance, during hepatitis C virus (HCV) infection, the expression of non-structural protein 5 (NS5) in the ER contributes to Ca²⁺ release and subsequent oxidative stress [56,57]. In response to ER stress, GSH levels are depleted due to the accumulation of misfolded proteins and increased ROS production [52]. Furthermore, the unfolded protein response (UPR) is stimulated during ER stress, leading to the activation of endoplasmic reticulum kinase (PERK) [58]. PERK, in turn, promotes increased GSH synthesis via the NRF2 pathway [59,60]. This enhanced GSH production serves as a cellular adaptive response to counteract elevated ER stress, restoring GSH equilibrium.

1.3. GSH dynamics in nucleus

The nucleus is also a main location where GSH highly resides [61]. GSH plays a vital role in essential functions including DNA synthesis, repair, and expression. During the cell cycle, GSH exhibits recruitment and sequestration within the nucleus. This recruitment is observed during the G1 and S phases in both animal and plant cells, while stimulation of GSH synthesis in the cytoplasm happens during G2 and M phases [62,63]. Meanwhile, the cell cycle could be blocked by the addition of GSSG, also indicating the participation of GSH in cell proliferation [64].

The precise mechanisms governing GSH transport to the nucleus remain incompletely understood, along with the question of the existence of an independent GSH regulation pool in the nucleus. One hypothesis is that GSH can freely diffuse among the nucleus and cytosol through nuclear pores. The Dick group developed Grx1-roGFP2 GSH redox sensor and did not observe differential GSH redox potentials between the nucleus and cytoplasm [25]. Our group developed a subcellular probe for the quantification of GSH concentrations in both the cytoplasm and nucleus, and the results suggested no significant difference under various conditions, supporting the hypothesis of the free distribution of GSH among the nucleus and cytoplasm [65].

Other studies also suggested a tightly regulated pool of nuclear GSH. Bcl-2, a protein with a GSH binding domain (BH-3), has been identified as a candidate involved in GSH transport [66,67]. It is hypothesized that Bcl-2 may facilitate GSH transport through Bcl-2-associated pores (BAP), maintaining a higher GSH concentration in the nucleus [68]. Another recent research from Emmert and coworkers has demonstrated that nuclear GSH is independently regulated during proliferation, with levels increasing during the early S phase and then stabilizing during the G2 phase, while whole-cell GSH levels remain relatively constant throughout the cell cycle [11]. These findings support the hypothesis of independent GSH regulation within the nucleus, also indicating that GSH transport might go through an active transport mechanism to achieve GSH enrichment in the nucleus, rather than free diffusion.

Currently, there is no consensus in terms of whether the nuclear GSH pool is independently regulated from the cytosol GSH. Future exploration needs to be done by employing different GSH or GSSG probes under the same cell culturing conditions.

2. Drugs downregulating intracellular GSH level as an antitumor strategy

Discovering small molecules that can modulate intracellular GSH levels is a compelling antitumor strategy, since GSH is upregulated in cancer cells to counter oxidative stress from elevated metabolic activities [7]. With a better understanding of the GSH dynamics and the relevant players, intervening these protein targets can alter the cellular GSH level and provides therapeutic benefits. The targets of current GSH depleting agents are mainly located in the cytosol or cell membrane, as the cytosol is the central hub for GSH biosynthesis (Figure 2).

Figure 2.

Pathways of GSH homeostasis and current available agents blocking the regeneration of GSH.

Here, we summarized current targets and emerging compounds that can lead to intracellular GSH depletion as an antitumor strategy.

Buthionine sulfoximine (BSO), a homocysteine derivative, is an irreversible GCL inhibitor that can achieve full inhibition at 10 μM in enzymatic assays and lower cytosolic and nuclear GSH levels [65,69]. Combination therapy with melphalan is under clinical trials to determine whether BSO can help sensitize resistant neuroblastoma [70]. Recently a novel covalent inhibitor EN25 and its derivatives targeting GCLM subunit were discovered through covalent library screening, with a reported IC₅₀ of 16 μM in 15 min reaction for EN25 and 6.8 μM for the most potent derivative EN25–8 [71].

γ-Glutamyl transferase (GGT) is a membrane-anchored ectoenzyme that salvages extracellular GSH derivatives [72]. The breakdown products glutamate and cys-gly serve as the major supply for intracellular GSH synthesis [72,73]. Therefore, inhibition of GGT leads to lower cellular GSH levels. Glutamate analogs, such as acivicin and DON, are representative GGT inhibitors but have notable glutaminase off-target and cytotoxic effects related to glutamine metabolism [74,75]. Two compounds, OU749 [76] and GGsTop [75,77], show better selectivity and are extensively used in related studies. OU749 is an uncompetitive inhibitor with a K_i of 17 uM [76], while GGsTop is a mechanism-based irreversible inhibitor, with an active ester of phosphonate mimicking the transition state of GSH in the GGT pocket, whose k_inact/K_I is 57 M⁻¹•s⁻¹ [77].

xCT, also known as SLC7A11, is a cystine/glutamate antiporter that exports intracellular glutamate in exchange of equimolar extracellular cystine [78]. Cytosolic cystine is further reduced by consuming NADPH to cysteine as a starting material for GSH synthesis [79]. Inhibiting xCT restricts cysteine supply for GSH replenishment and drives ferroptosis. Since the discovery of the first xCT inhibitor erastin [80], derivatives like imidazole ketone erastin (IKE) was developed to improve solubility meanwhile introducing lysine-reactive ketone as a covalent warhead, thereby achieving nanomolar IC₅₀ [81,82]. Interestingly, a xCT inhibitor, HG106 was identified that does not induce ferroptosis but apoptosis, indicating potential off-targets or cell-type specific response [83]. High extracellular glutamate level also leads to reduced transport efficiency. Inhibiting glutamine pathway, including downregulating glutamine transporter ASCT2 [84] or inhibit glutaminase by CB-839 [85] can deplete intracellular GSH, but such effect of CB-839 was only observed in cancer cell lines expressing xCT supplied with high cystine in the culture [86]. This indicates simultaneous inhibition of two different sources of glutamate – xCT and glutaminase – is necessary to bypass metabolic rewiring and effectively deplete GSH Restricting NADPH availability by inhibiting glucose transporter GLUT has a similar effect and can induce cell death only in xCT high-expression cancer cells [79,87]. Such cell death was recently described as disulfidptosis with the feature of cytoskeleton protein disulfide stress due to cystine accumulation in xCT high-expression cells [79].

NRF2 is a transcription factor regulating many redox regulating proteins, including aforementioned GCL, GGT and xCT [88]. KEAP1, as an E3 ligase, negatively regulates NRF2 level. Under oxidative stress, certain cysteine residues on KEAP1 undergo chemical modifications that decrease NRF2 binding affinity, thus facilitating the expression of protective redox-related genes [89]. The development on NRF2 inducers and KEAP1-NRF2 protein-protein interaction inhibitors is growing rapidly as a potential therapeutics to pathophenotypes including exacerbated oxidative stress, chronic inflammation, and metabolic alterations [90]. On the other hand, NRF2 inhibitors (ML385 (a direct Neh1 domain binder with an IC₅₀ of 1.9 μM) [91] and Nrf2-IN-1 [92]) induce apoptosis in cancer cells carrying mutations in the KEAP1-NRF2 axis by restricting the availability of oxidative stress protector proteins. On the contrary, NRF2 activators targeting KEAP1 thiols are recognized for their preventive potential for oxidative stress-related pathology such as Parkinson’s disease, but have not proven effective for chronic patients largely due to their electrophilic high off-target nature [93]. An indirect NRF2 activator through Bach1 derepression was developed to overcome these problems and relieved MPTP-induced neurodegeneration and associated oxidative stress [94].

3. Consideration of GSH in drug development

Understanding the dynamics of GSH in different organelles is not only important for studying cell biology but also crucial in drug development. As an important player in xenobiotics detoxification pathway, many existing drugs was known to alter GSH level through oxidative stress. Strategies has been developed to enhance the efficacy or mitigate the toxicity of these drugs, particularly those associated with oxidative stress and glutathione depletion. For example, cisplatin, anthracyclines and paracetamol (when overdosing) are all known for their capability of inducing oxidative stress, causing nephrotoxicity, cardiotoxicity, and hepatotoxicity respectively [95–97]. Concomitant administration of glutathione precursor N-acetylcysteine (NAC) has been shown to reduce cisplatin-induced nephrotoxicity and as a standard treatment for paracetamol overdose by replenishing depleted GSH. Cardiac mitochondria are a prominent site of injury by doxorubicin, an anthracycline, due to its high affinity for the di-anionic phospholipid cardiolipin on the inner mitochondrial membrane [98]. Thus, site-specific oxygen radical production by doxorubicin may be responsible for doxorubicin-induced mitochondrial damage [99]. Dexrazoxane, a chelating agent, has been approved for use in combination with anthracyclines to reduce the risk of cardiotoxicity. One of its proposed mechanisms of action is the preservation of glutathione levels in cardiomyocytes, thereby protecting against oxidative stress [99,100]. Interestingly, a recent research described a new mechanism by which selenomethione protects cardiomyocytes from doxorubicin-induced ferroptosis through activating GPX4 [101]. This represents a novel pharmacological strategy of unleashing the hidden GSH protection power by stimulating the cellular defense mechanisms against oxidative stress. Such insights into the role of glutathione in drug-induced toxicity and protective mechanisms open avenues for developing innovative therapeutic approaches to mitigate adverse drug effects, and have been reshaping the existing drug development paradigm.

The growing field of covalent drug discovery raises an important concern: are the electrophilic covalent warheads metabolically stable under high intracellular GSH concentrations? The pK_a of the thiol group on GSH is estimated to be around 8.6–8.7. At physiological pH 7.4, only 5% of GSH is in the active deprotonated form, making it not very reactive in the cellular environment [102]. But given the high GSH intracellular concentration (1–10 mM) in most cell types, this still accounts for ~50–500 μM thiolate concentration. The active site of glutathione S-transferase (GST) can further reduce the GSH thiol pK_a to 6, allowing for a more efficient reaction with electrophiles [103,104].

Since it is difficult to modify intrinsic GSH reactivity in the presence of cellular GST, warheads with reduced reactivity are preferred to minimize the non-specific covalent binding burden on proteins, which correlates with the in-vitro reaction rate of the drug and GSH [105,106]. (Figure 3A,B) Consider in vivo GSH concentration of 1–10 mM, second-order reaction rate k_GSH around 0.048 – 0.48 M⁻¹•min⁻¹ translates to a half-life of 24 h. The acrylamide warhead on ibrutinib and zanubrutinib has a GSH half-life of 33 h, while spebrutinib warhead acyl-acrylamide has a half-life of ~1.6 h [107]. Over-reactive warheads lead to significant non-specific covalent protein modifications - a major cause for immunotoxicity and idiosyncratic hypersensitivity reactions, as represented in β-lactam-induced allergic reactions [108,109]. In this scenario, with some sacrifice on warhead reactivity (k_inact), a high overall efficacy (k_eff) can only be achieved through optimizing for the non-covalent binding contribution (reflected by inactivation constant K_I) [107]. (Figure 3C). It is becoming routine for in vitro studies to monitor the reaction rates of commonly used warheads with GSH, offering guidance for warhead optimization [107,110–112]. An exemplary case of MRTX849, a covalent inhibitor targeting KRAS^G12C mutant, benefited from a lower reactivity warhead with an additional fluorine at the acrylamide α-carbon position, thus achieving better metabolic stability [113]. Similar optimization workflow by tuning acrylamide reactivity was also reported [114,115]. However, the GSH reactivity data predominantly focuses on acrylamide derivatives. In contrast, there is a notable scarcity of intrinsic reactivity data for other warhead classes. This under-representation renders the process of tuning warhead reactivity largely empirical. Further efforts are required to comprehensively investigate and understand the reactivity profiles across diverse warhead classes, thus enabling more informed and systematic optimization approaches. It is also noteworthy that drugs proposed as non-covalent mechanisms may still contain cryptic electrophilic groups capable of reacting with GSH and serum albumin in vivo [116]. On the other hand, traditional pharmacokinetics evaluation of covalent drug metabolism using hepatocytes or microsomes can potentially overlook the contribution from the non-oxidative metabolism pathway through extrahepatic GSH or albumin conjugation, accounting for up to 30% of total conversion in the case of ibrutinib [117].

Figure 3.

A. 25 most common cysteine-targeting covalent warhead groups and reaction types in irreversible small molecule inhibitors. Data retrieved from CovalentInDB [118]. B. GSH second-order reaction rate constant retrieved from literature, including acrylamides [107,112], conjugated carbonyls [119], aromatic halogen [110], nitrile [120], epoxide [110], quinone [121,122], disulfide [123,124], vinylsulfone [107], pyridylsulfone [125], lactone [126]. C. Two-step reaction scheme of acrylamide with thiol-containing molecules (GSH, cysteine, or Cys residue in the protein binding pocket). The first step involves the formation of non-covalent intermediate, which is more prominent in protein-ligand binding. The second step is the nucleophilic attack of thiolate ion towards the β-carbon on the acrylamide. k_eff denotes the second order rate constant for covalent product formation from the two reactants, assuming steady state approximation of the non-covalent intermediate. K_I is the inactivation constant for 2-step irreversible inhibitor, the value could be different from reversible inhibition equilibrium constant K_i when the forward reaction (k_inact) is faster than the disassociation event (k_off). The irreversible reaction rate (k_inact) is notably influenced by the substituent on the β-carbon position and whether it is an N-aryl or N-acyl-acrylamide. For detailed definition and measurement of k_inact and K_I, refer to [127].

In summary, the reactivity of glutathione (GSH) is a crucial consideration for covalent drug development, leading to a growing focus on designing lower-reactivity warheads to minimize non-specific covalent binding and reduce the risk of adverse reactions. High overall efficacy needs extensive optimization for non-covalent binding while delicately balancing the sacrifice on warhead reactivity.

Conclusions

The cellular redox environment is intricately linked to a multitude of essential cell functions [1]. GSH, the main components in redox system, plays a pivotal role in mediating and regulating critical cellular processes like proliferation, immune responses, and apoptosis [1,34,35,128]. Additionally, targeting intracellular GSH levels is a compelling antitumor strategy due to heightened GSH levels in cancer cells to counteract oxidative stress. Various targets (e.g., GCL, GGT, xCT, and NRF2) and compounds provide diverse avenues for modulating GSH levels, but challenges in achieving specificity and managing potential off-target effects underscore the complexity of this approach. As one of the most abundant redox molecules, unraveling the detailed dynamics of cellular GSH levels holds the key to demystifying redox biology. Comprehending the entirety of GSH-related biology requires more than just an understanding of the overall cellular GSH level.

A lot of progress has been made on GSH subcellular dynamics over the years, however numerous questions remain unanswered. For instance, the transportation of GSH in the nucleus remains enigmatic, hampering efforts to monitor and manipulate GSH levels within this compartment for further study. The mechanism regulating the balance of GSH levels in the ER is not fully elucidated [37]. Moreover, knowledge regarding the impact of drug treatments on subcellular GSH levels is limited. To address these questions, substantial investment in novel strategies and tools for studying subcellular GSH dynamics is essential. This knowledge, in turn, may lead to the development of more precise therapeutic strategies and approaches based on GSH modulation.

Highlights:

• GSH is crucial for cellular redox balance across various organelles. Dysregulation of subcellular GSH level could lead to various pathological conditions.

• Both mitochondria and the ER maintain separate pools of GSH, independently regulated apart from the cytosolic GSH pool.

• Current drug targets as intervening GSH levels includes GCL, GGT, xCT, GLUT, NRF2

• During drug and therapeutics development, extra attention should be paid to counterbalancing GSH depletion.

• For covalent drugs, routine monitoring of the warhead reactivity as well as in vivo modification burden should be carried out. Once the reaction rate has met the minimum requirement, efforts should be put on improving the non-covalent binding contribution.

Abbreviations

ER

endoplasmic reticulum

IMM

inner mitochondrial membrane

GCL

glutamate-cysteine ligase

GPx

glutathione peroxidases

GR

glutathione reductase

Grx

Glutaredoxin

GS

glutathione synthetase

GSH

reduced glutathione

GSSG

oxidized glutathione

GSH/GSSG

ratio between reduced and oxidized glutathione

GST

glutathione S-transferase

MOM

mitochondrial outer membrane

mtGSH

mitochondrial glutathione

OGC

2-oxoglutarate carrier

ROS

reactive oxygen species

SLC

soluble carrier protein

TR

thioredoxin reductase

Trx

Thioredoxin

xCT

cystine/glutamate antiporter SLC7A11