The potential for OGG1 inhibition to be a therapeutic strategy for pulmonary diseases

Abstract

Introduction:

Pulmonary diseases impose a daunting burden on healthcare systems and societies. Current treatment approaches primarily address symptoms, underscoring the urgency for the development of innovative pharmaceutical solutions. A noteworthy focus lies in targeting enzymes recognizing oxidatively modified DNA bases within gene regulatory elements, given their pivotal role in governing gene expression.

Areas Covered:

This review delves into the intricate interplay between the substrate-specific binding of 8-oxoguanine DNA glycosylase 1 (OGG1) and epigenetic regulation, with a focal point on elucidating the molecular underpinnings and their biological implications. The absence of OGG1 distinctly attenuates the binding of transcription factors to cis elements, thereby modulating pro-inflammatory or pro-fibrotic transcriptional activity. Through a synergy of experimental insights gained from cell culture studies and murine models, utilizing prototype OGG1 inhibitors (O8, TH5487, and SU0268), a promising panorama emerges. These investigations underscore the absence of cytotoxicity and the establishment of a favorable tolerance profile for these OGG1 inhibitors.

Expert opinion:

Thus, the strategic targeting of the active site pocket of OGG1 through the application of small molecules introduces an innovative trajectory for advancing redox medicine. This approach holds particular significance in the context of pulmonary diseases, offering a refined avenue for their management.

1. Introduction

Inflammation-associated pulmonary diseases encompass a diverse group of respiratory disorders, including asthma, chronic obstructive pulmonary disease (COPD), and pulmonary fibrosis. These conditions are characterized by chronic inflammation in the lungs, leading to tissue damage and impaired lung function. One common factor in this complex process is the involvement of reactive oxygen species (ROS) as signaling entities. ROS are highly reactive molecules produced during cellular metabolism and in response to environmental stimuli. While traditionally associated with tissue damage and oxidative stress, emerging research highlights their critical role in regulating tissue homeostasis.

ROS function as signaling molecules that influence cellular responses, including proliferation, differentiation, and immune regulation. This dual role of ROS, both as mediators of tissue damage and regulators of tissue homeostasis, adds complexity to the pathogenesis of inflammation-initiated pulmonary diseases. Understanding the multifaceted role of ROS in these conditions may pave the way for novel therapeutic strategies aimed at preserving tissue integrity and promoting lung repair. With an increasing appreciation for homeostasis, it becomes better understood that regulation or response is key in physiological processes. ROS can modify several important targets, including genomic DNA, where 7, 8-dihydro-8-oxoguanine (8-oxoGua) is considered a marker of oxidative stress, but in gene regulatory sequences function as an epigenetic-like mark. This review explores the association of repairing oxidatively modified nucleobase with pulmonary inflammation, the use of OGG1 inhibitors in experimental models, and provides recommendations for the potential translation of these studies to clinical applications.

1.1. The canonical function of OGG1

To counteract ROS-induced DNA damage, cells have a particular need for a functional repair system. Certain enzymes seek out oxidatively modified base, representing the predominant BER pathway for repairing lesions that do not cause steric hindrance or DNA strand breakage. The substrate specificity depends on DNA glycosylases, which initiate BER by hydrolyzing the N-glycosidic bond attached to the lesion, generating an apurinic/apyrimidinic (AP) site referred to as monofunctional glycosylases. Bifunctional glycosylases possess AP-lyase activity that processes the substrate into different ends through a β- or β-δ-elimination mechanism. DNA glycosylase reactions are followed by AP endonucleases and polymerases that incorporate a new deoxynucleotide at the 3’-hydroxyl to fill the gap, finally sealed by ligases [1].

The protein responsible for removing 8-oxoGua is 8-oxoguanine DNA glycosylase 1 (OGG1), owing to its robust activity on 8-oxoGua embedded in double-stranded oligodeoxynucleotides when paired with cytosine (Cyt). OGG1 also excises ring-fragmented purine, 2-6-diamino-4-hydroxy 5-formamidopyrimidine (FapyGua), and 7,8-dihydro-8-oxoadenine (8-oxoAde) when the lesion is paired with Cyt or 5-methylcytosine [2,3]. The X-ray structure of OGG1 bound to 8-oxoGua: Cyt-containing DNA provides insight into the mechanistic basis for damage recognition and repair [4]. In the catalytic core of OGG1 bound to DNA, substrate is flipped out and the extrahelical base inserted into active-site pocket on the enzyme, causing a sharp kink in the DNA. This extrahelical repair mechanism is conversed in most DNA glycosylases [5]. The strong bias of OGG1 in contacting the lesion-containing backbone hints at the scanning process used to locate damaged bases in the genome [6]. OGG1 was originally described as a DNA glycosylase-AP endo-deoxyribonuclease (lyase); however, by using site-directed mutants revealed that OGG1’ glycosylase and lyase activity is independent [7]. In fact, studies support the view that in cellulo OGG1 is functioning as a monofunctional DNA glycosylase without lyase activity [8,9]. Additionally, this BER activity, which involves the replacement of a single nucleotide, is also implicated in mismatch repair pathways (MMR) characterized by short excision repair tract (≤ 10 nucleotides) [10]. The correction of oxidative DNA lesions can also be carried out by nucleotide excision repair (NER), a more complex process involving the removal of a lesion with helical distortion. This interplay of multiply DNA repair pathways recognizing oxidative lesions suggests that proteins work in unison to faithfully process DNA lesions.

Mutations in the OGG1 gene are a pivotal tool in studying the enzyme's role in DNA repair mechanisms [4,11]. For example, a mutation from lysine to glutamine at position 249 has been observed to eliminate catalytic activity while preserving the enzyme's ability to recognize its substrate. Similarly, substituting cysteine with alanine at position 253 results in diminished substrate recognition capabilities. These specific OGG1 mutations have been linked to the modulation of pro-inflammatory gene expression in response to oxidative stress [12]. Additionally, a notable genetic polymorphism involving the substitution of serine with cysteine at position 326 of the OGG1 protein varies significantly across populations, occurring in 23% to 41% of Caucasians and 40% to 60% of Asians [13]. Experimental studies have shed light on the reduced activity of the OGG1-Cys326 variant, which is attributed to the oxidation of Cys326, leading to the formation of a disulfide bond [14]. This nuanced understanding of OGG1's genetic variations and their functional implications plays a critical role in our comprehension of DNA repair processes and their impact on cellular and organismal health.

2. Transcriptional activation requires scheduled 8-oxoGua generation.

Both DNA repair and transcription involve intimate interactions with DNA, which are associated with the action of repair enzymes or transcription factors (TFs) that efficiently recognize specific sites of DNA damage for transcriptional regulation. Especially under certain circumstances, the cell is obliged to replicate or transcribe DNA containing persistent damage, giving the impression of inefficiency for DNA repair machinery to repair lesions. In a defined location and time frame, oxidative DNA bases can also play an epigenetic role [15], blurring the fine line between DNA damage and epigenetic marks.

2.1. 8-oxoGua as an epigenetic mark

Early observations indicated that 8-oxoGua is compatible with the transcription process [16-18], suggesting that its effect is not merely mutational. Another key parameter is the genomic structure, as only 1.5% of the human genome consists of protein-coding gene [19]. The distribution of 8-oxoGua is mostly in non-coding regions [20-22], which are representative of both transcriptionally active and silenced genes. Advancements in modern technology have revealed that most guanine-rich sequences, including potential quadruplex-forming sequences (PQS), are located within the telomeres [23], ribosomal DNA [24], immunoglobulin heavy chain switch regions [25], minisatellites [26], and promoters of inflammatory cytokines [27].

Data from antibody based OxiDIP-Seq has shown that after oxidative stress, the distribution of 8-oxoGua accumulates at DNA replication origins within the body of transcribed long genes [20], enhancer [22] and promoter [21] regions that are physically associated with RNA pol II. This has sparked considerable interest in the possibility that oxidatively modified guanine bases have specific functions in the regulation of genetic stability or gene expression. 8-oxoGua itself overcomes the intrinsic stiffness of the DNA strand, thus affecting both initial recognition complex with the repair enzyme [28] and TFs binding [29-33]. The Gillespie laboratory reported that hypoxia-induced ROS actively oxidized the promoter of the endothelial cell growth factor (VEGF) gene to modulate gene expression [34], pointing to a common biological role of signaling-related oxidative base damage [35]. ROS generation is ubiquitous; thus, it is challenging to speculate on the site-specific formation of 8-oxoGua in the genome. However, it is interesting to note that the ROS released from the demethylation of histone lysine by the lysine-specific demethylase 1 (LSD1) [36] was shown to induce 8-oxoGua in the promoter [37]. This writer function of LSD1 provides a strategy to relax the promoter at the site of histone modification (Figure 1A). The associated reports suggest that 8-oxoGua present within a restricted time frame of stress response and specific DNA sequences may be widely used for transcriptional regulation.

Figure 1: OGG1 as an epigenetic reader in gene expression.

A). Targeted 8-oxoGua generation and OGG1 recruitment for modulation of gene expression. Histone demethylation by LSD1-produced ROS oxidize Gua to 8-oxoGua, which is then excised by OGG1. The AP-site is processed by OGG1 or APE1, and the resulting single-strand gap is recognized by Topo II. Topo II-unwinds DNA double helix to facilitate the occupancy of transcription factors on promoters.

B) OGG1 modulates transcription through Gua quadruplexes. OGG1 excises 8-oxoGua from potential quadruplex forming sequences (PQS), resulting in an AP-site that is subsequently occupied by APE1/Ref1. This occupancy leads to the folding of the PQS into G-quadruplex structure. APE1/Ref1 then recruits transcription factors for gene expression.

C) The role of repurposed OGG1 in transcriptional activation. ROS produced by inflammatory agents oxidize both Gua to 8-oxoGua and OGG1 at cysteines (OGG1S-OH). This enzymatically inactive form of OGG1 (OGG1S-OH) binds to 8-oxoGua at the promoter region and changes the local sequence topography, inducing DNA bending at the site itself as well as in adjacent DNA sequences. These modifications facilitate the binding of transcription factors. LSD1: Lysine-specific demethylase 1; Topo II, topoisomerase II; AP-site, apurinic/apyrimidinic-site, APE1/Ref1, AP-endonuclease1 or redox factor1; PQS, potential guanine quadruplex forming sequences. (Created with BioRender.com)

Another consideration for the epigenetic role of 8-oxoGua comes from its repair intermediate, an AP site (Figure 1B). After 8-oxoGua is removed by OGG1, the resulting AP site is acted upon by an AP endonuclease, such as apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) [38]. Similar to the property of 8-oxoGua on DNA structure, the AP site also introduces considerable tortional flexibility [39]. The AP site enables melting of the duplex to unmask the PQS, driving the spatiotemporal formation of G4-fold by APE1 binding and recruiting multiple transacting factors [40]. There is also a discrepancy in the role of G4 in transcription, as it may strongly inhibit of KRAS transcription without OGG1 function [41,42]. An in-depth study from the Burrows laboratory have reported that under oxidative stress conditions, the AP site in the promoter bound by APE1 is inefficiently cleaved, thus coupling DNA repair with transcription [43]. They also propose that the G4 occupancy in the strand (coding versus non-coding) is an on-off switch for transcription [44,45]. The Bhakat laboratory further showed that APE1 is a G4 DNA binding protein independent of AP site and stabilizes G4 folding structures in the promoter/enhancer regions to facilitate G4-mediated gene expression [46,47].

2.2. OGG1 doubles as an epigenetic reader

The substrate-specific binding functions of OGG1 directly modify DNA structure and interact with transcription components, bringing attention to the epigenetic role of 8-oxoGua as cellular repair attempts. The impact of 8-oxoGua on gene expression remained controversial until several laboratories, including ours, documented in vivo that 8-oxoGua initiates transcription in the model of hypoxia-inducible genes [48], estrogen-responsive genes [37], Myc targeted genes [49], nuclear factor (NF)-κB targeted genes [50], and epithelial-to-mesenchymal transition (EMT) genes [51].

NF-κB activation is central to protect against lethal challenges upon exposure to environmental stimuli [52]. Not only is the consensus motif recognized by NF-κB sensitive to 8-oxoGua generation, but the cysteines within OGG1 polypeptide chain are also prone to be oxidized by ROS [53]. This leads to the suppression of OGG1 glycosylase activity, but not its DNA scanning or interaction with 8-oxoGua. During oxidative stress conditions, 8-oxoGua binding by OGG1 at regulatory regions modifies local sequence topography [4], resulting in a sharp (~70°) bending of the DNA duplex both at the site of action and in adjacent DNA sequences (Figure 1C). The active site of OGG1 possesses flexibility [54], which lowers the energy required for TFs to twist DNA and create a stereo-specific interface suitable for recognizing the consensus motif by TFs. In this regard, the efficient surveillance mechanisms of OGG1 for detecting 8-oxoGua are transiently utilized as a reader function to facilitate NF-κB binding [50,55]. Once redox homeostasis is reached, OGG1 excises 8-oxoGua, converting to an eraser function. For genes that have the potential to be redox switches for transcriptional regulation, the substrate-specific reading function of OGG1 at functionally κB sites may be a part of the elaborate operations allowing innate immunity. It involves a redox switch, during which OGG1 serves as an epigenetic “reader” and “eraser”.

On the other hand, the coordinated function of 8-oxoGua and OGG1 may affect transcriptional elongation [56]. Phosphorylated NF-κB/RelA translocated to the nucleus where it binds to cis-elements facilitated by OGG1 and forms an enhanceosome containing CBP/p300 coactivator, the cyclin-dependent kinase 9/cyclin T1 complex, the positive transcription elongation factor b (P-TEFb) and bromodomain-containing protein 4 (BRD4) [57]. BRD4 actively links chromatin decompaction [58], allowing OGG1-nucleated NF-κB/RelA complexes to take part in activating transcription at target genes.

2.3. Characterization of OGG1 in gene expression

Notably, chromatin immunoprecipitation-coupled next generation sequencing (ChIP-seq) results further suggest that OGG1 facilitates gene expression by regulating NF-κB/RelA enrichment on DNA [59]. This study revealed that nearly fifty percentage of the thirteen thousand OGG1-enriched peaks were found on Gua-rich regulatory sequences in proximity to the transcription start site (TSS). Enrichment peaks for NF-κB/RelA were strikingly similar but found only on 8800 genes. For both OGG1 and NF-κB/RelA, fewer enrichment peaks were observed on exons, introns, untranslated or intragenic regions. Hierarchical structure and relationship analysis identified inflammation and immune processes as the primary responses regulated by OGG1. Specifically, these processes are associated with the positive regulation of cytokine production, such as TNFα, interleukin (IL)1, 6, 17 and 23, C-C and C-X-C motif chemokine ligands, and interferons. Recently, a novel function of OGG1 was documented in the expression of MYC (encoded by the human homolog of avian myelocytomatosis viral oncogene)-dependent genes, such as those involved in DNA damage responses, cell cycling, hematopoiesis, and apoptotic processes [60]. Specifically, the protein-protein interaction of MYC with OGG1 led to the inhibition of its glycosylase activity. Enzymatically disabled OGG1 at its DNA substrate increases the binding of MYC to its E-box promoter sequence, leading to the expression of the MYC-controlled transcriptional network.

In another comprehensive studies, murine lungs underwent challenges with single or multiple doses of free 8-oxoGua base [61]. The specificity for OGG1 was ensured by employing this pro-inflammatory agent, which triggers ROS generation [62]. The RNA-seq data analysis showed a rapid increase in the expression of C-C, C-X-C motif chemokines, cytokines and interleukins, accompanied by the rapid recruitment of neutrophil to the airways after a single challenge, indicating an acute inflammatory response. However, after multiple 8-oxoGua challenges, upregulation of transforming growth factor β (TGF-β), fibroblast growth factors (FGF), epidermal growth factor (EGF) signaling, as well as the expression of collagens, alpha-smooth muscle actin (α-Sma), cadherins, along with pro-inflammatory cytokines and chemokines were observed. Moreover, the recruitment of neutrophils, inflammatory macrophages, and eosinophils was significantly more pronounced after multiple challenges and were present for a prolonged period of time. These data collectively suggest that OGG1-facilitated gene expression plays a role in both acute and chronic inflammation, cell cycle progression which is tightly associated with progress to pulmonary diseases.

3. Active site inhibitors of OGG1 decrease inflammatory responses

Targeting DNA glycosylases has long been recognized as a potential pharmaceutical intervention in cancer therapies [63]. Attempts to target OGG1 in tumorigenesis through knockout approaches have yielded different and sometimes contradictory findings [64,65], highlighting the complex nature of 8-oxoGua tolerance. Mice lacking OGG1 are viable [66] and show normal growth [67], indicating that inhibiting OGG1 activity may have minimal on-target toxicity in rodent models and may not always induce synthetic lethality. Interestingly, the loss of OGG1 has been shown to suppress the inflammatory phenotype in BER-defective murine models of endotoxic shock, diabetes, contact hypersensitivity [68] and allergic airway inflammation [69,70], suggesting a major role of OGG1 in regulating the inflammatory response. As dysfunctional redox status can be exploited for anti-inflammatory treatment, researchers hypothesized that the BER protein OGG1 might facilitate pro-inflammatory gene expression under oxidative stress conditions.

Prototypic OGG1 inhibitors have recently been developed to target different stages of OGG1 activity. Lloyd laboratory identified O8, a compound that inhibits Schiff base formation during OGG1-mediated catalysis but not substrate-binding, using 8-oxoGua: Cyt as the OGG1 substrate in a duplex oligonucleotide [71]. On the other hand, the Helleday laboratory developed TH5487, a selective inhibitor that inhibits the substrate-specific binding by employing a similar approach but using 8-oxoAde: Cyt as the OGG1 substrate and involving APE1 in the reaction system [72]. These inhibitors interfere with different BER activities of OGG1, with O8 affecting β-lyase activity. Additionally, the Dr Kool laboratory reported a fluorogenic probe that directly reflects 8-oxoGua: Cyt excision activity [73] and identified another noncovalent small-molecule, SU0268 [72]. While the O8 can be utilized in many other applications, including specific inflammatory processes. As these data are preliminary, O8 potential clinical utility is not discuss further. Both TH5487 and SU0268 bind to the catalytic pocket of OGG1, preventing its fixation to the 8-oxoGua: Cyt-containing DNA.

Due to the well-desired and favorable chemical properties of these inhibitors, TH5487 and SU0268 exhibit good membrane permeability and show no cytotoxicity. In experimental murine models, TH5487 has demonstrated decreased lung inflammation after TNFα and lipopolysaccharide (LPS) challenges by reducing DNA occupancy of transcription factors [72]. Similarly, SU0268 inhibited proinflammatory responses during Pseudomonas aeruginosa infection and promoted type I interferon (IFN) responses [74]. Furthermore, TH5487 showed potential in treating human respiratory syncytial virus infection by reducing exuberant innate immune responses and increasing type III (IFN-λ) expression, resulting in decreased lung pathology and viral progeny [75,76]. Both TH5487 and SU0268 were found to enhance IFN-β production during African swine fever virus infection and decrease viral yield [77]. These findings highlight the promising therapeutic applications of TH5487 and SU0268 in various inflammatory and viral infections.

3.1. Fate of 8-oxoGua in the absence of OGG1 repair

TH5487 and SU0268 treatment in cells or experimental animals prevents the binding of OGG1 to intrahelical 8-oxoGua, leading to its accumulation in the genome. Originally considered a prototype for exploring mutagenesis of damaged bases, 8-oxoGua is known for its miscoding property and pairs with adenine. While the syn- and anti-form of 8-oxoGua are in rapid equilibrium, the syn conformation is energetically favored, forming a stable Hoogsteen base-pair with 8-oxoGua (syn)·A (anti), leading to G:C → T:A transversions after replication [78]. However, studies on OGG1 knockout (Ogg1^−/−) mice showed no correlation between 8-oxoGua accumulation and malignancies. Although spontaneous mutation frequencies were significantly increased in the liver, the Ogg1^−/− mice remained physically normal without developing malignancies or marked pathological changes [66,67]. The spontaneous consequences of 8-oxoGua accumulation appear to be tissue-specific and associated with the aging process [79].

In cellular DNA, 8-oxoGua can undergo further oxidation to more stable products. Compared to the four normal bases or their oxidatively modified forms, 8-oxoGua acts as the ultimate sink for charges in DNA due to its low oxidation potential [80]. Notably, 8-oxoGua can be further oxidized to several products, with spiroiminodihydantoin (Sp) and 5-guanidinohydantoin (Gh) being the most studied [81]. The bifunctional glycosylases Endonuclease VIII-like 1–3 (NEIL1–3) have been observed to remove Sp and Gh from double-stranded oligodeoxynucleotides [82-84], indicating the wide substrate specificity of BER enzymes that can mitigate the mutagenic potential of 8-oxoGua. Moreover, the interplay between BER and other repair pathway such as NER or MMR provides another mechanism [85], ensuring that even in the absence of OGG1 function, 8-oxoGua can be repaired by backup systems.

To support the backup hypothesis, a comprehensive proteomic analysis was conducted using liquid chromatography tandem mass spectrometry (LC-MS/MS) on lung lysates from bleomycin-induced fibrotic tissues and tissues treated with TH5487 [86]. In the TH5487-treated lungs, there were significant increases in the expression of proteins functioning in NER and MMR pathways, such as cyclin-H, CDK-activating kinase assembly factor MAT1/Tfb3, xeroderma pigmentosum complementation group C (XPC), X-ray repair cross-complementing protein 1, DNA polymerase delta subunit 2/3, DNA ligase 1, and G/T mismatch-specific thymine DNA glycosylase (TDG), likely due to the expansion of fibrotic tissues and bleomycin-induced DNA damage signaling.

4. OGG1 function in pulmonary diseases

The complex mechanisms underlying major respiratory diseases, such as asthma, COPD, acute respiratory distress syndrome (ARDS), idiopathic pulmonary fibrosis (IPF) and lung cancer, are not well understood. However, they share a common theme, which is airway inflammation [87]. For instance, individuals with allergic asthma often exhibit chronic inflammation, mucus secretion and hyperresponsiveness [88]. Rather than being solely a consequence of sustained airway inflammation, structural change in response to the destruction of the epithelial barrier and the loss of homoeostatic control can provide positive feedback to the inflammatory response [89]. In the wound healing response, the formation of connective tissue primarily composed of extracellular matrix (ECM) components plays a crucial role in maintaining organ functionality [90]. However, senescence accompanying normal lung regeneration can influence the outcome of wound healing response, determining whether it leads to efficient repair or progressive fibrosis [91]. Intriguingly, senescent cells secrete a plethora of modulators [92], including pro-inflammatory cytokines and chemokines, growth factors, angiogenic factors, and matrix metalloproteinases that can trigger fibrosis [93]. The similarity in inflammation among these various pathologies allows us to develop new therapeutics targeting OGG1 in inflammatory lung diseases. By understanding the function of OGG1, we can potentially address the common inflammatory processes in diseases like asthma, COPD, ARDS, IPF, and lung cancer, paving the way for innovative treatment strategies.

4.1. Targeting OGG1 in allergic lung diseases

One of the most discussed respiratory chronic lung diseases is asthma. It is a highly heterogeneous, displaying diverse pathogenetic mechanisms with overlapping inflammatory and anatomical features. Due to an individual’s genetic makeup, the inflammatory diversity in asthma extends from T helper 2 (Th2) eosinophilic phenotypes to Th17 (Th1) neutrophilic ones, often with overlapping immune traits [94]. The complexity of chronic lung inflammation is significantly enhanced by reactive oxygen species (ROS) and redox reactions. Therefore, disease intervention is complicated, primarily symptomatic, requiring a step-by-step treatment approach, regular monitoring, and dose adjustment to minimize airflow limitations. Among these treatments, redox modulators, short/long-acting bronchodilators, and inhaled corticosteroids are commonly used—the latter have considerable side effects, underlining importance for development of novel inflammatory inhibitors.

The specific role of OGG1 in repairing ROS-induced 8-oxoGua has led to increasing research on its regulatory function in allergic airway inflammation. Initial evidence stemmed from observations that Ogg1^−/− mice subjected to LPS-induced inflammation displayed reduced neutrophil infiltration and diminished Th2 cytokine levels compared to wildtype mice [68]. Subsequent studies consistently highlighted OGG1's impact on airway allergic inflammation following sensitization and challenge with ovalbumin (OVA) or house dust mite, notably suppressing IL-4 and IL-17 production [69]. In an asthma model induced by ragweed pollen grain extract inhalation after intraperitoneal sensitization [70], OGG1 depletion in the airway epithelium led to attenuated inflammatory responses. This manifested as reduced expression of Th2 cytokines, decreased eosinophilia, epithelial metaplasia, and alleviated airway hyperresponsiveness (AHR).

Moreover, pharmacological inhibition of OGG1 using TH5487 demonstrated a reduction in plasma IgE levels, goblet cell hyperplasia, mucus production and AHR [95]. Molecular analysis by Tanner and colleagues revealed that TH5487 reduced the expression of soluble inflammatory mediators including Tnfrsf4, Arg1, Ccl12, and Ccl11 (eotaxin). Intriguingly, an unexpected outcome was the heightened expression of B-cell lymphoma 6 protein (Bcl6), a negative regulator of type 2 inflammation that acts as a transcriptional repressor. BCL6 restrains the function of CD4⁺ T cells, Th2 cells, B-cells, and T follicular helper cells, subsequently downregulating IL-4, IL-5, IL-13, and monocyte chemoattractant protein-1 (also known as CCL2) expression. Thus, the inhibition of OGG1 decreased this cascade, resulting in a significant reduction in eosinophils and inflammatory macrophages in the lungs. Furthermore, TH5487 treatment mitigated plasma IgE levels, alleviated goblet cell hyperplasia, mucus accumulation and notably decreased AHR, suggesting its potential for alleviating asthma-related symptoms in humans (Figure 2).

Figure 2: Proposed mechanism of OGG1 inhibitors in ameliorating allergic inflammation.

Upper panel, TH5487 or SU0268 inhibits OGG1 binding to 8-oxoGua in the promoters, thereby preventing its nucleation function and DNA occupancy of transcription factors. This results in decreased expression of pro-inflammatory chemokines, cytokines, recruitment of inflammatory cells. Studies also provided evidence for decreased levels of IgE antibodies after OGG1 inhibition.

Lower panel, Schematic depiction of pathophysiology of allergic asthma. Exposure to allergen leads to prompt release of the mediators (e.g., histamine) from mast cells and further activation of Th2 cells with recruitment of inflammatory cells (eosinophil, neutrophils), inflammation, increased mucus secretion and airway hyperresponsiveness. IL, interleukin, TSLP, thymic stromal lymphopoietin, C-C, and C-X-C, chemokines (Created with BioRender.com).

While asthma research traditionally emphasizes environmental factors like aeroallergens, pathogens exposure, and environmental oxidants, epidemiological studies highlight a connection between the metabolic syndrome and an elevated risk of asthma development [96,97]. Data documented by Dr. Lloyd and colleagues showed novel roles for OGG1 in obesity and metabolic syndrome [9,98]. Ogg1^−/− mice when compared to wildtype mice, showed increased weight gain, adiposity, and fatty livers on high-fat diet. This was associated with hyper-insulinemia, and altered glucose tolerance, consistent with insulin resistance. In line, obese Ogg1^−/− mice had significantly decreased respiratory ratio (expired volume of CO₂ being divided by inspired O₂) consistent with decrease in pulmonary function characteristic of obstructive lung disease [98]. Specific gene expression profiles, supporting the observed lower fat oxidation in the liver, provide further evidence for these observations. Although there are no studies using the Ogg1^−/− mouse model of allergic inflammation in the context of metabolic disorders, population-based investigations underline the role of insulin resistance in obesity and asthma/allergy development [88, 89]. Remarkably, overexpression of OGG1 in the mitochondrial compartment mediated a favorable metabolic phenotype (resistant to diet-induced obesity, fat tissue inflammation, and hyperphagia) involving enhanced mitochondrial respiration in the white adipose tissue of OGG1 transgenic mice [99]. These collective findings suggest a contributive role of OGG1 in allergic pulmonary diseases through its regulation of metabolic homeostasis and provide support for targeting OGG1 as a viable approach in combating pulmonary diseases.

4.2. Targeting OGG1 in TGFβ-induced pulmonary fibrosis

Pulmonary fibrosis arises from dysregulated tissue repair responses following tissue injury, particularly as a consequence of chronic inflammatory processes in small and terminal airways. The role of OGG1 in inflammatory airway remodeling was first documented by Luo et. al. [100] and Aguilera-Aguirre et. al., [101], utilizing cultured airway epithelial cells and murine models. In these models, chronic inflammatory injury to epithelium increased expression of TGF-β, inducing airway epithelial cells to differentiate towards a mesenchymal phenotype, fostering OGG1-dependent wound healing. Comprehensive transcriptome analysis, complemented by biomolecular and tissue histological characterization, pinpointed the involvement of OGG1 in EMT and fibroblast-to-myofibroblast transition (FMT).

The recent work by Pan et. al. [51] delved into the role of OGG1 in the essential molecular reprogramming required for TGF-β-driven EMT and the conversion to fibroblast-like cells. These studies utilized human diploid small airway epithelial cells (hSAECs), revealing that TGF-β exposure induced histone modifications at regulatory sequences, leading to chromatin opening essential for phosphorylated SMAD occupancy in cis-regulatory regions of target genes. The consensus SMAD binding elements are GC-rich [GGC(GC)|(CG)], yet recognition is not solely determined by this consensus binding site due to chromatin architecture constraints. Pan et. al. demonstrated that TGF-β exposure triggers ROS generation via NADPH oxidases, concurrently activating LSD1 at specific sites. This oxidative environment oxidizes guanine to 8-oxoGua within regulatory regions, including those proximal to SMAD binding elements. Furthermore, cysteine is converted to sulfenic acid in the OGG1 molecule, thus establishing OGG1 as an epigenetic reader of 8-oxoGua. Experimental data illuminated that the initial steps of the OGG1-BER process induce structural DNA changes, fostering the nucleation of SMAD binding and facilitating their assembly into the transcription machinery. The study also documented the necessity of OGG1 in the transcriptional network governing EMT. Depletion of OGG1 through siRNA or inhibition using TH5487 impeded this network, underscoring OGG1's pivotal role in EMT regulation.

To investigate the role of OGG1 in TGF-β-induced fibrotic development in the lungs, murine airways were subjected to repeated challenges with TGF-β alone or TGF-β followed by the addition of TH5487. Similar to findings in cultured hSAECs, TGF-β exposure led to a noticeable increase in the levels of 8-oxoGua, particularly in the lower airway epithelial cells. This rise in 8-oxoGua levels was closely associated with an enhanced enrichment of OGG1 on promoters of fibrotic genes associated with fibrosis. However, the addition of TH5487 effectively counteracted this effect by inhibiting OGG1's enrichment on these fibrotic gene promoters. Notably, the expression patterns of genes involved in EMT/FMT closely mirrored the changes in OGG1 enrichment, and treatment with TH5487 resulted in a significant reduction in gene expression related to these transitions. Further investigation through chromatin immunoprecipitation (ChIP) assays revealed that the inhibition of OGG1 led to a decrease in the DNA occupancy of specific transcription factors such as SMAD2/3, erythroblast transformation specific variant transcription factor 4, and NF-κB at the promoter regions of key fibrotic genes including collagen1a2 (Col1a2), fibronectin 1 (FN1), and vimentin (Vim). In line with these molecular findings, lung exposure to TGF-β resulted in increased expression of keratin 14, a marker associated with fibrotic lungs. However, inhibition of OGG1 by TH5487 led to a reduction in keratin 14 expression. Immunoblotting analysis further demonstrated that TH5487-induced functional inactivation of OGG1 significantly decreased the protein levels of molecules linked to the mesenchymal phenotype, such as COL1a2, FN1, and VIM. Global gene ontology (GO) analysis provided insights into the pathways regulated by OGG1, which were found to be intricately involved in the networks of EMT and FMT. Moreover, histological examination of lung tissue sections revealed characteristic fibrotic changes, including extensive collagen accumulation, which were shown to be dependent on the presence of functional OGG1.

Importantly, specificity of TH5487 treatment was reinforced through the demonstration that pharmacological inhibition of OGG1 had no impact on TGF-β signaling pathway, which is essential for the post-translational modifications of SMAD2/3. These findings strongly suggest that for the existence of an OGG1-dependent network that drives distinct cellular behaviors within the lungs. Remarkably, the results also unveiled a noteworthy phenomenon: the shift of acute inflammatory signals during transitions in cellular states towards an extracellular matrix (ECM)-activated program. Consequently, epithelial-immune reactions contribute to the establishment of a localized microenvironment that drives EMT and fibroblast-to-myofibroblast transition (FMT), processes effectively counteracted by TH5487 (Figure 3). Furthermore, numerous research laboratories have independently elucidated OGG1's role in epigenetic reprogramming [21], which involves facilitating the occupancy of SMAD proteins on DNA to regulate gene expression during the EMT process.

Figure 3: Schematic depiction of OGG1 targeting in fibrotic gene expression.

A, Inhibition of OGG1 at 8-oxoGua prevents the assembly of transcriptional machinery for activation of fibrotic and inflammatory genes.

B, Pathophysiological and structurer changes in lower airways attenuated by OGG1 inhibition. EMT, epithelial to mesenchymal transition. FMT, fibroblast to myofibroblast transition. FGF, fibroblast growth factors. EGF, epidermal growth factor. IL, interleukins. C-C and C-X-C, chemokines. (Created with BioRender.com).

4.3. Targeting OGG1 in a model of IPF

Bleomycin is utilized in chemotherapy for various conditions including lymphomas, cervical and uterine cancer, and head and neck cancers. However, due to the deficiency of the bleomycin hydrolase enzyme activity in the lungs, the drug accumulates and leads to injury and fibrosis. Consequently, investigating the molecular mechanisms and therapeutic effectiveness of drugs in experimental animal models with bleomycin-induced fibrosis holds great relevance for future applications in humans.

Recent comprehensive pre-clinical research has revealed that depleting OGG1 through siRNA or functionally inhibiting it with TH5487 significantly alleviated bleomycin-induced clinical symptoms, lung tissue damage, density of fibrotic lesions, and inflammatory responses [86]. Notably, inhibition of OGG1 notably decreased levels of soluble inflammatory mediators (e.g., IL4, IL5, IL6, IL9, eotaxin, GCSF, MIP-1α, MIP-1β, MCP-1) in bronchoalveolar lavage fluid (BALF) and lung lysates. Additionally, TGF-β, a pivotal driver of fibrosis, exhibited significantly reduced levels in animals treated with TH5487 compared to those treated with bleomycin alone. The efficacy of TH5487 was compared to that of the steroid dexamethasone treatment, showing similar decreases in cellular immune responses. Moreover, TH5487's efficacy was comparable to approved fibrosis drugs, nintedanib and pirfenidone, but latter drugs exhibited insignificant decreases in chemokine, cytokine, and immune cell accumulation in BALF and lungs. Collagen deposition and histological changes were significantly diminished by TH5487 treatment, in contrast to those treated with bleomycin alone. These findings emphasize the potential of TH5487 as a promising therapeutic candidate for fibrosis treatment.

The same cutting-edge study employed state-of-the-art proteomic LC-MS/MS analysis on BALF and lung lysates, with a specific focus on pathways associated with fibrosis. In contrast to treatment with bleomycin alone, administration of TH5487 led to significant downregulation of various biosynthetic processes linked to fibrosis. These encompassed collagen biosynthesis, wound healing implicated in inflammatory response, endothelial cell proliferation, collagen metabolic processes, response to fibroblast growth factor, regulation of cytokine production, wound healing, and response to wounding. Crucial proteins participating in these processes exhibited logarithmic two-fold reductions following TH5487 treatment compared to bleomycin treatment alone. Among these proteins were hyaluronidase 1, secreted frizzled-related protein 1, metalloproteinase with thrombospondin motifs 15, cathepsin S, C-type lectin domain family 10 member A, disintegrin A, heme oxygenase 2, arginase 1, and collagen 1A1. Significantly, LC-MS/MS analysis demonstrated that TH5487 increased levels of SMAD 2 and 5, while decreasing levels of SMAD 2/3 and 4 in animal groups treated with bleomycin. The findings unveiled heightened OGG1 levels correlated with SMAD in IPF patients compared to healthy individuals, pointing towards their potential role as etiologically relevant regulators in human fibrotic pathologies.

Moreover, it was demonstrated that mice treated with bleomycin-TH5487 exhibited significantly lower levels of albumin and lactate dehydrogenase activity in BALF compared to those treated with bleomycin alone. This observation reflects the protective effects of OGG1 inhibition on endothelial function and cellular toxicity induced by bleomycin, respectively. While LC-MS/MS analysis of protein changes in lungs treated solely with TH5487 did not reach statistical significance using strict multiple testing correction. This and similar steps are crucial to guarantee the safety and efficacy of TH5487 as a pharmaceutical intervention for fibrotic diseases. While these studies overpoweringly support OGG1's engagement in fibrotic progressions and the effectiveness of the OGG1 inhibitor, further studies in primates are required to reinforce TH5487's clinical utility in humans.

4.4. Targeting OGG1 in chronic obstructive pulmonary disease (COPD)

COPD, a progressive disease, ranks as the third leading cause of death globally [102]. This condition presents with multiple pathological features including emphysema, chronic bronchiolitis, excessive goblet cell metaplasia, and small airway remodeling, all of which severely impact lung functions. COPD diversity is further increased by redox-driven cell activation pathways that lead to exacerbation of chronic inflammation and impaired tissue repair. Current treatment primarily addresses symptoms and encompasses the use of steroids, bronchodilators (β2-adrenergic receptor and muscarinic antagonists), and phosphodiesterase type 4 inhibitors. Given the prominence of redox signaling in COPD, thioredoxin therapy is being considered as a potential approach [103].

Using ozone(O₃) in murine models has long been established as a reliable method for simulating human COPD, and the effectiveness of TH5487 was evaluated within this context [104]. Repeated exposure of the lungs to O₃ led to epithelial injury, resulting in both functional and structural alterations, accompanied by increased expression of inflammatory cytokines in the airways. The initial acute inflammatory response was succeeded by a chronic phase, characterized mainly by the recruitment of neutrophils. This study clearly demonstrated that the inhibition of OGG1 using TH5487 provided a protective effect on epithelial integrity, leading to significant reductions in the expression of inflammatory mediators, lung inflammation, collagen accumulation, and histological changes. It is noteworthy that these beneficial outcomes were predominantly observed in the lower airways, including a noticeable decrease in alveolar enlargement in the experimental animals.

At the molecular level, investigations have unveiled that oxidative stress induced by O₃ triggers the generation of 8-oxoGua in gene regulatory regions located upstream and downstream of the TSS. In the specific context of this study, researchers directed their attention towards the regulation of the tissue inhibitor of metalloproteinase-1 (TIMP1) gene. This particular protein functions as a naturally inhibitor of matrix metalloproteinases, pivotal players in ECM turnover and the orchestration of inflammatory responses. Through meticulous molecular analysis, it was elucidated that OGG1 binds to the 8-oxoGua present in intron 1 of the TIMP-1 gene via a DNA: RNA hybrid interaction. This intricate interplay halts the elongation and maturation process of nascent RNA into mRNA, consequently affecting the synthesis of TIMP1. As a result, the beneficial effects observed following TH5487 treatment could potentially stem from its regulation of ECM homeostasis. These studies not only provide insights into OGG1's role in the development of COPD but also introduce a novel mechanism by which OGG1 is implicated in gene expression downstream of TSS through DNA: RNA hybrid interactions, contributing to the progression of the disease.

5. Potential risks associated with OGG1 inhibitors treatment

When evaluating the potential risks associated with OGG1 inhibitors treatment, it is crucial to consider the diverse regenerative capabilities of tissues in adult vertebrates. Some tissues contain stem cell lineages, while others consist of fully differentiated and functional cells. However, in response to damage (e.g., under disease conditions), these cells can gain activities similar to progenitor cells and their activities are essential for both homeostatic maintenance and tissue regeneration [105]. In this context, it is noteworthy to consider the roles of OGG1 and 8-oxoGua in gene expression, as well as the mutagenicity of 8-oxoGua. However, the lack of phenotype in Ogg1^−/− mice may suggest that transient inhibition of OGG1 does not significantly impair cellular function, viability, mutation rate, or exacerbate disease processes in human. Supporting this, under oxidative stress, the liver of Ogg1^−/− mice regenerated to the same extent as the non-treated heterozygous or wild-type control mice [106]. The balance between DNA damage accumulation and the tissue's innate repair mechanisms will be critical in determining the overall impact."

Another risk associated with the use of OGG1 inhibitors is their potential impact on the body's inflammatory processes [107]. OGG1 has been implicated in the regulation of inflammation, and inhibiting this enzyme could disrupt the delicate balance of the immune response. While reducing acute and chronic inflammation can be beneficial in certain conditions, an overly suppressed inflammatory response raises serious concerns about immunosuppression. Such weakened immunity could lead to increased susceptibility to life-threatening infections, as the body's natural defense mechanisms against pathogens are compromised. Therefore, the risks of immunosuppression must be carefully weighed against the potential benefits of OGG1 inhibitors, especially in patients with existing immune system vulnerabilities or those at high risk of infectious diseases. To this end, OGG1 inhibitors demonstrated excellent clinical utility in animal models of viral and bacterial pneumonia, suggesting no immunosuppression by these drugs [74].

Inhibiting OGG1 may also come with certain side effects. While most studies found that functional inactivation (inhibition) of OGG1 resulted in no toxicity, however, a recent study reported off-target effects of TH5487 and SU0268. Both OGG1 inhibitors inhibit ABC B1 (multidrug resistance 1: MDR1) and ABC G2 (breast cancer resistance protein; BCRP) transporters [108]. Additionally, SU0268 has an anti-mitotic activity resulting in increased cellular toxicity when combined with cytotoxic agents. We note that these studies have utilized established cell lines (U-2OS, A549, HMEC, and SH-SY5Y), while these side effects were not tested in primary cells (or animal models) where MDR1 or BCRP is not or poorly expressed. On the other hand, the inhibition of MDR1 or BCRP by OGG1 inhibitors can be advantageous to increase the cytotoxic effect of anti-cancer drugs and support their utility in cancer chemotherapy [109,110]. The clinical application of OGG1 inhibitors in human would necessitate rigorous monitoring for signs of impaired immune function and tailored strategies to mitigate such risks.

6. Conclusion

Chronic airway diseases are highly complex, with clinical characteristics and fundamental molecular mechanisms that can vary significantly among patients, even though they may share common regulatory pathways. Due to these reasons, standardized treatments for these disease conditions are not appealing to both physicians and patients. However, the recently developed OGG1 inhibitors, modulating expression of genes of which regulatory sequences bearing the epigenetic mark 8-oxoGua is common to chronic airway inflammatory diseases. Targeting the interaction between OGG1 and 8-oxoGua in regulatory sequences of genes involved in inflammation and those associated with airway remodeling in conditions such as asthma, COPD, or fibrosis, using compounds like TH5487 or SU0286, could yield unexpected benefits. The primary role of OGG1 is to maintain genome integrity; so inhibiting OGG1 function raises mutagenic potential of 8-oxoGua. However, mice lacking OGG1 exhibit no apparent consequences of 8-oxoGua accumulation, maintaining fertility and full viability without displaying significant phenotypical pathologies.

OGG1 remains a critical redox switch in response to oxidative stress. Given OGG1’ pivotal role in expression of redox regulated genes, OGG1 represents a potential drug target for treating diseases associated with oxidative stress, particularly those involving airway inflammation. Encouragingly, experimental results, along with the molecular level information presented, strongly suggest that compounds like TH5487 and SU0268, specifically targeting the 8-oxoGua binding site of OGG1, hold promise for future trials in primates and humans, for inflammatory processes. By focusing on OGG1's specific interactions and its involvement in redox signaling, these small molecules pave the way for a more refined approach to precision medicine. Embracing this targeted strategy represents an exciting novel direction for advancing the treatment of respiratory diseases and beyond. As research progresses, targeting OGG1 and related pathways may become central to more effective and personalized therapies in the battle against oxidative stress-related disorders.

Finally, targeting OGG1 with specific inhibitors presents a promising and distinct approach to addressing acute and chronic inflammatory airway diseases. While preliminary data suggests favorable outcomes, thorough research and clinical trials are needed to fully grasp the efficacy, safety, and long-term effects of OGG1 inhibitors. If proven effective, OGG1 inhibitors could provide a targeted therapeutic option for patients with respiratory diseases and potentially pave the way for advancements in precision medicine.

7. Expert opinion

The data from the experimental animal studies described above give rise to the possibility that antagonizing OGG1 could potentially lead to novel therapies for both acute and chronic airway inflammatory diseases. While the results obtained from knock-out mice are promising, they do not inherently translate to therapeutic outcomes in humans. Small molecule targeting and siRNA depletion in mice primarily result in a reduction, rather than a complete elimination, of the influence of genes like OGG1. Such decreases in seem to be highly advantageous in animal models, suggesting that attenuating OGG1 function to a certain extent can effectively inhibit key pathways responsible for pathophysiological changes. To enhance clinical applicability, additional experiments using species that close to human, including possibly primates, is valuable to gain insights with chronic airway inflammation. These models should encompass relevant risk factors and comorbidities. Equally important are the establishment of dosing protocols, the determination of drug metabolites, and the identification of appropriate biomarkers relevant to medical contexts. The subsequent step would involve the implementation of human clinical trials, tailored to the most suitable disease conditions. In animal models, the daily administration of OGG1 inhibitors has proven effective. However, the therapeutic window in human should be defined through dosing studies that also assess potential undesired effects. A more comprehensive analysis is imperative, encompassing insights into asthma, COPD, or IPF subtypes and the clinical characteristics of variants. Moreover, genetic variations within human populations should be considered, as certain gene combination combinations of OGG1 variants elevate the risk of malignancies and other disorders.

In clinical practice, corticosteroids are widely used for decreasing symptoms in asthma [88], COPD [102] and IPF [111]. Similarly, nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly employed to address a wide range of inflammatory diseases. Although these pharmaceuticals have shown considerable efficacy in improving lung function, relieving symptoms, and reducing exacerbations, they are associated with certain limitations and potential side effects. Given the heterogeneity of these diseases, characterized by various endotypes and phenotypes reflecting diverse pathophysiological mechanisms, there is a pressing need for new targeted therapies and a more precision-based approach.

The concept of targeting OGG1 for anti-inflammation and developing OGG1 inhibitors represents a departure from the mechanism of action of steroids or NSAIDs. OGG1 inhibitors are specifically designed to target the active site pocket of OGG1, inhibiting its nucleation function and reducing the DNA occupancy of TFs in gene regulatory sequences. This impairment of TFs-driven pro-inflammatory gene expression is a unique mechanism not shared by corticosteroids or NSAIDs. Corticosteroids, on the other hand, primarily exert their anti-inflammatory effects by interacting with the glucocorticoid receptor, limiting target gene transcription at the glucocorticoid response element [112]. NSAIDs, by inhibiting the activity of cyclooxygenase (COX-1 and COX-2), suppress the biosynthesis of prostaglandins [113], which contributes to their anti-inflammatory properties.

Unlike corticosteroids and NSAIDs, inactivating OGG1 function specifically affects gene expression from promoters that acquired 8-oxoGua, ensuring selectivity. This is highly relevant to the initiation and exacerbation of inflammatory processes leading to chronic inflammatory diseases. However, the impact of systemic administration of OGG1 inhibitors on the immune system remains a question that needs further investigation. While current experimental data suggests that OGG1 inhibitors do not compromise the immune system, long-term effects and potential immune-related adverse events, such as lymphocyte activation or proliferation, require careful evaluation. Previous studies on knockout mice have shown ablation of OGG1 to be well-tolerated but understanding the potential side effects and physiological changes associated with long-term application is crucial.

To assess the potential of OGG1 inhibitors in comparison to available drugs used in the therapy of chronic airway inflammation steroids and NSAIDs, more comprehensive studies are necessary. Direct comparisons of OGG1 inhibitors with traditional treatments in various disease conditions would provide valuable insights. Additionally, investigating the metabolism of OGG1 inhibitors through diverse data sources to analyze absorption, excretion, intoxication outcomes, basic physiological changes, and toxico-kinetics would be beneficial. Applying OGG1 inhibitors clinically in the context of disease heterogeneity would further contribute to understanding their therapeutic potential and safety profile.

Article highlights.

• Preclinical studies indicate the potential clinical relevance of highly selective small molecule inhibitors targeting OGG1 in both acute and chronic airway inflammatory diseases.

• These OGG1 inhibitors decrease the expression of inflammatory and remodeling genes that harbor the epigenetic modification, 8-oxoGua, within their regulatory sequences.

• The interaction between OGG1 and 8-oxoGua induces alterations in the local DNA structure, thereby facilitating the binding of transcriptional effectors to DNA.

• Ogg1^−/− mice, which accumulate 8-oxoGua in DNA, show no symptoms, age normally, and become resistant to acute and systemic inflammation, indicating the potential dispensability of the mutagenic effects of 8-oxoGua.

• The targeted approach of using small molecules to inhibit OGG1's active site shows promise in experimental models of asthma, COPD, pulmonary fibrosis, and acute airway infections.