GSTO1 Human

What is GSTO1 and how does it differ from other GST family members?

GSTO1 (Glutathione S-transferase omega 1) belongs to an atypical class of cytosolic glutathione transferases with distinct structural and functional attributes that differentiate it from other GST classes. Unlike conventional GSTs, GSTO1 possesses an active site cysteine residue that enables it to catalyze unique reactions . GSTO1 can perform glutaredoxin (thioltransferase) reactions, reduce methylated arsenic species, dehydroascorbic acid, and S-phenacylglutathiones, and catalyze protein deglutathionylation . This diverse catalytic profile suggests that GSTO1 plays roles beyond the typical xenobiotic detoxification functions associated with other GST family members. Structurally, the omega class GSTs have specific conformational features that contribute to their specialized functions in redox homeostasis and cellular signaling pathways .

What are the primary cellular functions of GSTO1?

GSTO1 has been implicated in several critical cellular processes beyond typical GST detoxification roles. Research indicates that GSTO1 plays important roles in:

• Redox homeostasis through its ability to catalyze various reduction reactions and protein deglutathionylation

• Inflammatory responses, particularly in TLR4-mediated signaling pathways

• Cellular metabolism regulation, including impacts on glycolysis and oxidative phosphorylation in response to inflammatory stimuli

• Cancer cell proliferation, migration, and invasion, suggesting oncogenic potential

• Protection against oxidative stress by contributing to cellular antioxidant defense mechanisms

Functionally, GSTO1 has been shown to influence the dephosphorylation of adenosine monophosphate kinase (AMPK), a key metabolic stress regulator, during inflammatory responses . Additionally, GSTO1 appears critical for proper innate immune function, as deficiency affects monocyte counts and alters inflammatory responses to various stimuli .

What experimental models are available for studying GSTO1 function?

Several experimental models have been developed to investigate GSTO1 function:

• Genetic knockout models: GSTO1-deficient (Gsto1^-/-) mice have been characterized and show normal development but exhibit altered inflammatory responses . These mice show resistance to LPS-induced inflammatory shock, reduced susceptibility to high-fat diet-induced obesity, and differential responses in colitis models .

• Cell-based knockdown/overexpression systems: Researchers have utilized shRNA to silence GSTO1 in macrophage cell lines (e.g., J774.1A) and cancer cell lines . Similarly, overexpression models using cDNA constructs in cell lines like H2122 have been employed to study gain-of-function effects .

• Biochemical assays: Enzymatic activity can be measured using specific substrates such as 4-NPG that allow for quantitative assessment of GSTO1 function .

• Insect models: Studies have examined GSTO1 in species such as Apis cerana cerana to investigate evolutionary conservation of function, particularly in oxidative stress responses .

When selecting experimental models, researchers should consider that GSTO1 may have species-specific and tissue-specific functions, with particular importance in macrophages, liver, and certain cancer types.

How can GSTO1 activity be accurately measured in experimental settings?

Measuring GSTO1 activity requires specialized approaches due to its unique catalytic properties. Recommended methodologies include:

• Substrate-specific enzymatic assays: GSTO1 activity can be quantified using the specific substrate 4-NPG (4-nitrophenacyl glutathione) . This assay differentiates GSTO1 activity from other GST isoforms.

• Thioltransferase activity measurement: Since GSTO1 catalyzes glutaredoxin reactions, measuring thioltransferase activity provides insight into its functional capacity .

• Protein deglutathionylation assays: Monitoring the deglutathionylation of specific protein substrates can assess GSTO1 activity in a physiologically relevant context .

• Redox state analysis: Examining the reduction of substrates like dehydroascorbic acid or methylated arsenic species allows for functional assessment of GSTO1's reductive capacity .

For accurate results, activity measurements should be performed under standardized conditions with appropriate positive and negative controls. In the case of genetic models, complete absence of activity should be confirmed in knockout systems, while heterozygotes typically show approximately half the wildtype activity levels . Researchers should also be aware that environmental stressors and cellular redox state can influence GSTO1 activity.

What approaches are effective for studying GSTO1's role in inflammatory pathways?

GSTO1 plays a significant role in inflammatory responses, particularly through TLR4-mediated signaling. Effective approaches for investigating these functions include:

• In vivo inflammatory challenge models: Lipopolysaccharide (LPS) administration in GSTO1-deficient vs. wildtype animals allows for assessment of systemic inflammatory responses . Parameters to monitor include body temperature changes, survival rates, cytokine production, and metabolic alterations.

• Disease-specific inflammation models:

  • Dextran sodium sulfate (DSS)-induced colitis for studying inflammatory bowel disease mechanisms

  • High-fat diet (HFD) models for investigating metabolic inflammation

  • Bacterial infection models to assess pathogen-associated molecular pattern (PAMP) responses

• Macrophage activation studies: Since GSTO1 influences macrophage responses to LPS, examining parameters such as ROS production, NADPH oxidase expression, lactate production, and AMPK phosphorylation status in macrophages provides insight into mechanistic pathways .

• Inhibitor studies: Small molecule inhibitors of GSTO1 can be employed to examine acute inhibition effects on inflammatory responses and validate GSTO1 as a therapeutic target .

• Transcriptional profiling: Techniques such as Bru-seq can identify novel GSTO1-regulated pathways in inflammation .

When designing these studies, researchers should consider the potentially divergent roles of GSTO1 in different inflammatory contexts, as demonstrated by its contrasting effects in LPS-induced shock versus DSS-induced colitis .

What strategies can be employed to investigate GSTO1's role in cancer biology?

Given GSTO1's emerging significance in cancer, several specialized approaches can be utilized:

• Expression correlation analyses: Examining GSTO1 expression across cancer types and correlating with clinical outcomes to identify relevant cancer models .

• Functional assessment in cancer cell lines:

  • Proliferation assays (e.g., Cell Counting Kit-8) following GSTO1 manipulation

  • Migration and invasion assays using Transwell systems

  • Apoptosis quantification via flow cytometry with appropriate markers

  • Combination studies with chemotherapeutic agents like cisplatin to assess drug resistance modulation

• Xenograft tumor models: Utilizing GSTO1-manipulated cancer cells in animal models to assess in vivo tumor growth and response to therapies .

• Mechanistic pathway analysis: Examining GSTO1's impact on:

  • Cholesterol metabolism pathways

  • Oxidative and endoplasmic stress responses

  • Cytoskeleton organization and cell migration machinery

  • Apoptotic signaling through markers like Bax and caspases

• Inhibitor screening and development: Testing small molecule compounds for GSTO1 inhibitory activity and assessing their effects on cancer cell phenotypes .

Comprehensive investigation requires integration of molecular, cellular, and in vivo approaches. Researchers should also consider potential isoform-specific effects and the contextual dependency of GSTO1's roles across different cancer types.

How does the structure of GSTO1 contribute to its unique functions?

GSTO1's distinct functional properties derive from unique structural features:

• Active site cysteine: Unlike most GSTs that utilize a tyrosine or serine residue, GSTO1 contains a critical cysteine residue (Cys-32 in humans) at its active site that enables thiol-transferase activity and other reductive functions .

• Cysteine network: Beyond the active site, additional cysteine residues (such as Cys-28, Cys-70, and Cys-124 identified in some species) contribute to GSTO1's function . Mutational analysis demonstrates that alterations in these residues affect catalytic activity and antioxidant function.

• G-site and H-site coordination: The glutathione binding site (G-site) and hydrophobic substrate binding site (H-site) in GSTO1 have unique structural arrangements that accommodate distinct substrates compared to classical GSTs .

• Structural plasticity: Co-crystal structures of GSTO1 with inhibitors reveal binding modes that may induce conformational changes relevant to function . This structural flexibility likely contributes to GSTO1's diverse substrate profile.

• Structural changes upon mutation: Three-dimensional structural models indicate that cysteine mutations influence protein conformation, potentially altering substrate accessibility and catalytic efficiency .

Advanced techniques including X-ray crystallography, site-directed mutagenesis, and molecular dynamics simulations have been valuable in elucidating these structural features. Understanding structural determinants provides critical insights for rational drug design targeting GSTO1.

What are the methodological challenges in developing specific GSTO1 inhibitors?

Developing selective GSTO1 inhibitors presents several methodological challenges:

• Target selectivity: Creating compounds that specifically target GSTO1 without affecting other GST isoforms requires detailed understanding of unique binding pockets. Structural studies using co-crystallization with chloroacetamide-containing compounds have provided insights into covalent binding mechanisms at the active site cysteine .

• Mechanistic validation: Confirmation of the inhibitory mechanism is essential. For example, researchers have developed inhibitors that form covalent bonds with the active site cysteine, ensuring specific targeting . This requires comprehensive biochemical characterization.

• Functional consequences: Effective inhibitors must not only bind GSTO1 but also functionally impair its biological activities. Assessing effects on multiple GSTO1 functions (deglutathionylation, thioltransferase activity, etc.) provides more complete inhibitor profiling .

• In vivo efficacy and safety: Small molecule inhibitors must demonstrate efficacy in relevant in vivo models while maintaining acceptable toxicity profiles. Studies have shown that GSTO1 inhibitors can ameliorate LPS-induced inflammatory responses in mice, validating the approach .

• Target engagement metrics: Developing reliable methods to confirm GSTO1 inhibition in complex biological systems remains challenging. Activity-based assays and target engagement biomarkers need further development.

Researchers have made progress with chloroacetamide-containing compounds that covalently bind to the active site cysteine, demonstrating both anti-cancer and anti-inflammatory properties in preclinical models . These serve as valuable chemical tools and potential therapeutic leads.

How does GSTO1 contribute to inflammation-associated diseases?

GSTO1 plays multifaceted roles in inflammatory conditions through several mechanisms:

• TLR4 signaling modulation: GSTO1 appears critical for proper TLR4-mediated inflammatory responses, as evidenced by GSTO1-deficient mice showing resistance to LPS-induced inflammatory shock . This implicates GSTO1 in sepsis and acute inflammatory disorders.

• Metabolic inflammation: GSTO1-deficient mice are protected from high-fat diet-induced obesity and associated metabolic changes, suggesting GSTO1 promotes inflammation-driven metabolic dysfunction . This parallels observations in TLR4 and MyD88 deficient mice, further connecting GSTO1 to these inflammatory pathways.

• Divergent roles in gut inflammation: Interestingly, GSTO1-deficient mice show more severe inflammatory responses in dextran sodium sulfate (DSS)-induced colitis models, with increased bacterial translocation from the colon to lymphatics . This suggests context-dependent functions in different inflammatory settings.

• Mechanistic pathways: GSTO1 influences:

  • ROS production through regulation of NADPH oxidase expression

  • Metabolic reprogramming during inflammation, affecting lactate production and AMPK phosphorylation

  • Potentially protein glutathionylation status, which can regulate multiple inflammatory signaling pathways

The seemingly contradictory roles of GSTO1 in different inflammatory contexts (protective in colitis but pathogenic in endotoxic shock and metabolic inflammation) highlight the complexity of its functions and suggest disease-specific targeting strategies may be required for therapeutic development.

What evidence supports GSTO1 as a therapeutic target in cancer?

Multiple lines of evidence support GSTO1 as a potential cancer therapeutic target:

• Altered expression in cancer: GSTO1 is overexpressed in several cancer types, suggesting pathophysiological relevance . In non-small cell lung cancer and colorectal cancer, higher expression appears to correlate with more aggressive disease characteristics.

• Functional impact on cancer hallmarks: Experimental evidence demonstrates that GSTO1:

  • Promotes cancer cell proliferation when overexpressed

  • Enhances migration and invasion capabilities of cancer cells

  • Inhibits apoptosis, potentially contributing to treatment resistance

  • Modulates cellular responses to oxidative and endoplasmic reticulum stress

• Pharmacological validation: Small molecule GSTO1 inhibitors have demonstrated promising anti-cancer effects:

  • Suppression of cancer cell growth in vitro

  • Enhancement of cisplatin cytotoxicity, suggesting combination therapy potential

  • Inhibition of tumor growth in colorectal cancer models as single agents

• Mechanistic insights: Transcriptional profiling has revealed GSTO1 involvement in cancer-relevant pathways including:

  • Cholesterol metabolism, which is frequently dysregulated in cancer

  • Cytoskeletal organization and cell migration mechanisms

  • Stress response pathways that influence cancer cell survival

• Genetic validation: siRNA-mediated silencing of GSTO1 significantly impairs cancer cell viability, validating its functional importance .

These findings collectively suggest that GSTO1 inhibition may offer therapeutic benefits across multiple cancer types, potentially through both direct anti-cancer effects and chemosensitization mechanisms.

What methods can assess GSTO1 polymorphisms and their impact on disease susceptibility?

GSTO1 polymorphisms have been implicated in disease susceptibility, particularly in conditions like Alzheimer's disease and various cancers. Methodological approaches to investigate these associations include:

• Genotyping techniques:

  • PCR-RFLP (Restriction Fragment Length Polymorphism) analysis for common GSTO1 variants

  • TaqMan SNP genotyping assays for high-throughput screening

  • Next-generation sequencing approaches for comprehensive polymorphism identification

• Case-control association studies: Comparing polymorphism frequencies between affected individuals and matched controls, with appropriate statistical analysis accounting for multiple testing and population stratification .

• Functional characterization of variants:

  • Recombinant expression of variant proteins to assess catalytic activity changes

  • Cellular models expressing different variants to evaluate functional consequences

  • Structural modeling to predict impacts of amino acid substitutions on protein function

• Clinical correlation analyses: Assessing associations between specific polymorphisms and:

  • Age of disease onset (as reported for Alzheimer's disease)

  • Treatment response variations

  • Disease progression rates

  • Comorbidity patterns

• Mechanistic studies: Investigating how specific polymorphisms affect:

  • Protein stability and expression levels

  • Substrate specificity and catalytic efficiency

  • Protein-protein interactions relevant to disease pathways

  • Stress response capabilities

When designing such studies, researchers should consider potential gene-environment interactions, as GSTO1's roles in detoxification and stress response suggest that environmental factors may modify genetic associations. Sample size considerations are also critical for achieving adequate statistical power.

What emerging technologies could advance our understanding of GSTO1 biology?

Several cutting-edge technologies offer promising avenues for deeper investigation of GSTO1 biology:

• CRISPR-Cas9 genome editing: Precise modification of GSTO1 and interacting genes can create more sophisticated cellular and animal models beyond traditional knockouts, including:

  • Specific polymorphism knock-in models to study variant effects

  • Domain-specific mutations to dissect structure-function relationships

  • Conditional knockout systems for tissue-specific and temporal control

• Single-cell omics approaches: These technologies can reveal cell-type specific functions of GSTO1:

  • Single-cell RNA sequencing to identify GSTO1-dependent transcriptional programs across diverse cell populations

  • Single-cell proteomics to examine GSTO1's impact on protein expression and modification states

  • Spatial transcriptomics to map GSTO1 expression and activity in complex tissues

• Advanced protein analysis techniques:

  • Redox proteomics to identify GSTO1-dependent protein glutathionylation targets

  • Hydrogen-deuterium exchange mass spectrometry to detect conformational changes upon substrate/inhibitor binding

  • Proximity labeling approaches to identify novel GSTO1 interaction partners

• Systems biology integration:

  • Multi-omics data integration to position GSTO1 within broader cellular networks

  • Machine learning approaches to predict GSTO1 functions and disease associations

  • Pathway analysis tools to identify previously unrecognized GSTO1-regulated processes

• Advanced imaging techniques:

  • Live cell imaging with fluorescent GSTO1 reporters to track subcellular localization and dynamics

  • Super-resolution microscopy to examine GSTO1 interactions at nanoscale resolution

These technologies will likely provide unprecedented insights into GSTO1's cellular functions and disease relevance beyond what conventional approaches have revealed.

How might GSTO1 research inform personalized medicine approaches?

GSTO1 research has several potential applications in personalized medicine:

• Pharmacogenomics: GSTO1 polymorphisms may influence drug metabolism and efficacy, particularly for:

  • Chemotherapeutic agents, where GSTO1 has been implicated in resistance

  • Anti-inflammatory medications, given GSTO1's role in inflammatory pathways

  • Drugs targeting metabolic disorders, considering GSTO1's influence on metabolic inflammation

• Biomarker development:

  • GSTO1 expression levels could serve as prognostic or predictive biomarkers in certain cancers

  • GSTO1 activity measurements might indicate inflammatory status in chronic conditions

  • GSTO1-dependent metabolite profiles could reflect disease activity or treatment response

• Therapeutic targeting strategies:

  • Patient stratification based on GSTO1 expression or polymorphism status for clinical trials

  • Development of companion diagnostics for GSTO1 inhibitors to identify likely responders

  • Combination therapy approaches based on GSTO1 status (e.g., combining GSTO1 inhibitors with chemotherapy for enhanced efficacy)

• Disease risk assessment:

  • GSTO1 polymorphism testing could contribute to risk profiling for conditions where associations have been established

  • Integration of GSTO1 status with other genetic and environmental risk factors for improved prediction models

• Therapeutic monitoring:

  • Development of assays to measure GSTO1 inhibition as pharmacodynamic biomarkers during treatment

  • Assessment of GSTO1-dependent pathway activation to monitor disease activity

As our understanding of GSTO1 biology deepens, these personalized medicine applications are likely to expand, potentially providing more precise approaches to patient care in inflammatory conditions and cancer.

What are the key considerations for interpreting contradictory findings in GSTO1 studies?

Researchers investigating GSTO1 may encounter seemingly contradictory results across studies. Key considerations for reconciling these findings include:

• Context-dependent functions: GSTO1 demonstrates opposing effects in different disease models, as evidenced by its pro-inflammatory role in LPS response versus a seemingly protective role in DSS-induced colitis . This suggests that GSTO1's functions may be highly context-dependent.

• Model system variations: Different experimental systems may yield disparate results:

  • Cell line-specific effects due to varying genetic backgrounds and signaling pathway configurations

  • Differences between in vitro cellular models and in vivo physiological conditions

  • Species differences in GSTO1 structure, regulation, and function

  • Acute versus chronic models of disease or stress exposure

• Technical methodology differences:

  • Varying methods of GSTO1 manipulation (siRNA, shRNA, CRISPR, chemical inhibition)

  • Differences in assay sensitivity and specificity

  • Timing of interventions relative to disease progression

  • Dosing considerations for inhibitors or stimuli

• Genetic compensation mechanisms: Complete GSTO1 knockout may trigger compensatory changes not observed with transient knockdown or inhibition. For instance, GSTO1-deficient mice show altered expression of other GSTs and NQO1 .

• Experimental endpoint selection: Different studies may focus on varied aspects of GSTO1 function:

  • Enzymatic activity versus signaling pathway effects

  • Cellular versus systemic responses

  • Acute versus chronic outcomes

When evaluating seemingly contradictory findings, researchers should carefully consider these factors and design experiments that directly address discrepancies by systematically varying conditions while maintaining consistent core methodologies.

What are best practices for quantifying GSTO1 expression in clinical samples?

Accurate quantification of GSTO1 in clinical samples requires careful methodological considerations:

Adherence to these practices enhances reliability and reproducibility of GSTO1 quantification in clinical research, enabling more meaningful comparisons across studies and potential translation to clinical applications.

How can researchers integrate findings from different disciplines to advance GSTO1 understanding?

Advancing GSTO1 research requires integrating insights from multiple scientific disciplines:

• Structural biology and biochemistry integration:

  • Combining crystallographic data with functional enzymatic studies to understand structure-function relationships

  • Using structural insights to guide inhibitor design and optimization

  • Applying biochemical activity profiles to validate structural predictions

• Molecular and cell biology coordination:

  • Connecting molecular mechanisms to cellular phenotypes through targeted experiments

  • Validating in vitro findings in physiologically relevant cell types

  • Examining cell type-specific effects of GSTO1 modulation

• Translational research approaches:

  • Correlating basic science discoveries with clinical observations

  • Testing hypotheses generated from patient data in appropriate experimental models

  • Developing biomarkers based on mechanistic understanding for clinical application

• Computational and experimental synergy:

  • Using computational predictions to guide experimental design

  • Validating in silico models with experimental data

  • Employing systems biology approaches to position GSTO1 within broader networks

• Collaborative infrastructure development:

  • Establishing shared resources such as validated reagents, cell lines, and animal models

  • Creating common data repositories with standardized formats

  • Developing consistent methodological protocols across research groups

Successful integration requires regular communication between researchers across disciplines, shared conceptual frameworks, and willingness to adopt methodologies from different fields. Collaborative research networks, consortia, and interdisciplinary grant programs can facilitate these connections.

What are the recommended experimental designs for testing GSTO1 inhibitors as potential therapeutics?

Development of GSTO1 inhibitors as therapeutics requires systematic experimental approaches:

• Target validation phase:

  • Confirm GSTO1 dependence in disease models using genetic approaches (knockout, knockdown)

  • Validate expression and activity of GSTO1 in relevant disease tissues

  • Establish clear phenotypic readouts associated with GSTO1 modulation

• Compound screening and optimization:

  • Structure-based design informed by GSTO1 crystal structures

  • Biochemical assays using recombinant GSTO1 to assess direct binding and inhibition

  • Counter-screening against other GST family members to establish selectivity

  • Medicinal chemistry optimization for potency, selectivity, and drug-like properties

• Cellular activity assessment:

  • Target engagement confirmation in intact cells

  • Phenotypic evaluation in disease-relevant cell types

  • Dose-response studies to establish potency in cellular context

  • Combination studies with standard-of-care agents

• Preclinical in vivo evaluation:

  • Pharmacokinetic/pharmacodynamic relationship studies

  • Efficacy testing in disease-relevant animal models:

    • LPS-induced inflammatory shock models for anti-inflammatory applications

    • Xenograft models for cancer applications

    • Metabolic disease models for obesity/diabetes applications

  • Safety assessment including toxicology studies

• Translational biomarker development:

  • Identification of pharmacodynamic markers of GSTO1 inhibition

  • Development of patient stratification strategies based on GSTO1 expression or activity

  • Correlation of biomarker changes with clinical outcomes in animal models

These experimental designs should be tailored to the specific therapeutic indication being pursued, with particular attention to the context-dependent functions of GSTO1 across different disease settings.

What specialized reagents and tools are available for GSTO1 research?

Researchers investigating GSTO1 can access various specialized resources:

• Genetic tools:

  • GSTO1 knockout mouse models: Characterized Gsto1^-/- mice are available and show normal development but altered inflammatory responses

  • Expression vectors: cDNA constructs for wildtype and mutant GSTO1 overexpression

  • siRNA/shRNA: Validated sequences for GSTO1 knockdown in various cell types

  • CRISPR-Cas9 constructs: Guide RNA sequences targeting GSTO1 for genetic editing

• Biochemical reagents:

  • Recombinant GSTO1 proteins: Purified proteins for enzymatic and structural studies

  • Specific antibodies: Validated for Western blotting, immunohistochemistry, and immunoprecipitation

  • Enzyme activity substrates: 4-NPG (4-nitrophenacyl glutathione) for specific activity measurement

• Chemical tools:

  • Small molecule inhibitors: Characterized chloroacetamide-containing GSTO1 inhibitors with known binding modes

  • Activity-based probes: Tools for monitoring GSTO1 activity in complex biological samples

• Structural resources:

  • Crystal structures: Published co-crystal structures with inhibitors providing structural insights

  • Homology models: Computational models for species where crystal structures are unavailable

  • Molecular dynamics simulations: For understanding dynamic aspects of GSTO1 function

• Cellular models:

  • Characterized cell lines: Systems with confirmed GSTO1 dependence for specific phenotypes

  • Reporter systems: Constructs for monitoring GSTO1 expression or activity

Researchers should validate these tools in their specific experimental systems, as the performance of reagents may vary across different contexts. Documentation of validation results in publications enhances resource sharing within the research community.

How can researchers effectively design experiments to control for GSTO1 isoform specificity?

Ensuring isoform specificity in GSTO1 research requires careful experimental design:

• Isoform identification and characterization:

  • Comprehensive sequence analysis to identify all relevant GSTO isoforms in the study system

  • Expression profiling of different isoforms across tissues or cell types of interest

  • Functional characterization of enzymatic activities of individual isoforms

• Specific genetic manipulation approaches:

  • Design of siRNA/shRNA sequences that target unique regions of GSTO1 not present in other isoforms

  • CRISPR-Cas9 guide RNA selection targeting GSTO1-specific exons

  • Site-directed mutagenesis targeting residues unique to GSTO1 function

• Antibody validation for specificity:

  • Testing antibodies against recombinant GSTO1 and related isoforms

  • Validation in knockout/knockdown systems to confirm specificity

  • Epitope mapping to ensure recognition of GSTO1-specific regions

• Enzymatic assay specificity:

  • Selection of substrates with preferential reactivity toward GSTO1

  • Inclusion of appropriate controls with other GST isoforms

  • Kinetic analysis to differentiate GSTO1-mediated reactions

• Complementation experiments:

  • Rescue experiments with isoform-specific expression in knockout/knockdown systems

  • Comparison of phenotypic rescue efficiency across different isoforms

  • Structure-function analysis using chimeric proteins or domain swaps between isoforms

• Small molecule selectivity profiling:

  • Comprehensive testing of inhibitors against all relevant GST family members

  • Structure-activity relationship studies focused on enhancing selectivity

  • Target engagement confirmation in complex biological systems