DHODH Human

What is DHODH and what is its primary function in human cells?

Dihydroorotate dehydrogenase (DHODH) is a mitochondrial enzyme that plays a crucial role in the de novo pyrimidine biosynthesis pathway. It catalyzes the rate-limiting step in this pathway, specifically the conversion of dihydroorotate to orotate. This reaction is essential for the subsequent production of pyrimidines, which are fundamental building blocks of DNA and RNA .

Methodologically, researchers studying DHODH function typically employ enzymatic assays measuring the conversion of dihydroorotate to orotate, often using spectrophotometric methods that track changes in absorbance associated with the reduction of electron acceptors.

Where is DHODH localized within human cells and how does this affect its function?

DHODH is primarily localized in the mitochondria of human cells. Specifically, it is found in the inner mitochondrial membrane where it participates in the fourth step of pyrimidine biosynthesis . This localization is significant because it enables the enzyme to connect pyrimidine synthesis to the mitochondrial electron transport chain.

To study DHODH localization, researchers commonly use subcellular fractionation techniques followed by Western blotting, or fluorescence microscopy with antibodies specific to DHODH. The enzyme's association with the mitochondrial membrane means that proper solubilization techniques are essential when isolating it for functional studies.

What is the relationship between DHODH gene variants and Miller syndrome?

Several variants in the DHODH gene have been identified as causative for Miller syndrome, a rare condition affecting facial, arm, and leg development. These variants typically result in amino acid substitutions in the dihydroorotate dehydrogenase enzyme .

Miller syndrome appears to disrupt the development of the pharyngeal arches, which are paired structures that form during embryonic development and ultimately develop into the bones, nerves, and muscles of the head and neck. Although DHODH is active in these structures during development, the exact mechanism by which DHODH variants lead to the specific facial features of Miller syndrome remains unclear .

Additionally, the bones of the arms and legs often develop abnormally in people with Miller syndrome. While DHODH appears to be active in the limb buds during early development, the precise relationship between DHODH variants and the specific bone abnormalities observed is still being investigated .

How does DHODH expression correlate with neuroblastoma prognosis and progression?

DHODH expression has been identified as an independent unfavorable prognostic marker in neuroblastoma. High DHODH expression significantly correlates with poor survival across all stages of the disease. Particularly, the highest levels of DHODH expression are observed in high-risk MYCN-amplified tumors, and expression increases with disease stage .

Methodologically, researchers can assess DHODH expression using RNA sequencing or quantitative PCR for mRNA levels, or immunohistochemistry for protein expression. Kaplan-Meier survival analyses with stratification based on DHODH expression levels can help establish its prognostic value in neuroblastoma patient cohorts.

What are the major classes of DHODH inhibitors currently used in research and clinical settings?

Several classes of DHODH inhibitors have been developed and are being researched:

• Established clinical inhibitors:

  • Teriflunomide: An approved drug that has been established in the market with known DHODH inhibitory activity

  • Brequinar: One of the strongest known DHODH inhibitors, which has been explored in various cancer types

• Novel synthetic inhibitors:

  • Compounds with a 6-isopropyl-1,5,6,7-tetrahydro-4H-benzo[d] triazol-4-one scaffold (e.g., compounds 1289 and 1291) have shown high in vitro inhibitory activity against human DHODH with nanomolar IC50 values

• Natural product inhibitors:

  • Piperine has been identified as a novel DHODH inhibitor with potential applications in treating multiple sclerosis and other autoimmune conditions

For researchers, selection of an appropriate inhibitor depends on the specific research question, desired potency, and pharmacokinetic/pharmacodynamic requirements.

How do the binding modes of different DHODH inhibitors compare, and how can this inform new inhibitor design?

High-resolution crystal structures have revealed detailed interactions between DHODH inhibitors and the enzyme. Human DHODH has an α/β-barrel fold, and inhibitors bind at a tunnel within the enzyme structure together with FMN .

Compounds like 1289 and 1291 are stabilized by numerous hydrophobic interactions involving specific amino acid residues including M43, L46, L58, F62, F98, M111, and L359 . The DHODH inhibitor binding site demonstrates intrinsic plasticity, allowing it to accommodate diverse inhibitor scaffolds, which explains why structurally different molecules can effectively inhibit the enzyme.

The crystal structures of human DHODH complexed with various inhibitors provide valuable insights for structure-based drug design. Key methodological approaches include X-ray crystallography (as shown in the table below), molecular docking studies, and structure-activity relationship analysis.

Table 1: Crystallographic data for DHODH-inhibitor complexes

What methodologies are used to evaluate DHODH inhibitors in experimental models of autoimmune disease?

For evaluating DHODH inhibitors in autoimmune disease models, researchers employ several methodological approaches:

• In vitro assessment:

  • Enzymatic kinetic analysis to determine inhibitory potency

  • Thermofluor assays to assess binding affinity through thermal stabilization

  • Isothermal titration calorimetry for quantitative binding measurements

  • Concanavalin A-triggered T-cell assays to assess immunomodulatory effects

  • Mixed lymphocyte reaction assays to evaluate impact on immune cell proliferation

• In vivo assessment in experimental autoimmune models:

  • MOG-induced experimental allergic encephalomyelitis (EAE) in mice as a model for multiple sclerosis

  • In vivo imaging to evaluate myelin destruction using specific dyes (e.g., DBT dye administered intravenously)

  • Blood-brain barrier integrity assessment

  • Histochemical analysis of tissue sections using hematoxylin and eosin (H&E) staining and luxol fast blue (LFB) staining

The natural product piperine has been evaluated using these methodologies and identified as a DHODH inhibitor with potential therapeutic effects in experimental models of multiple sclerosis .

How can multi-omic approaches be applied to study DHODH's role in cancer metabolism?

Multi-omic approaches have proven valuable for identifying DHODH as a metabolic vulnerability in cancers like neuroblastoma. These approaches integrate:

• Metabolomics: Analysis of metabolite profiles revealed that neuroblastoma cell lines accumulate specific metabolites compared to other solid tumor cell lines, including pyrimidines (2-deoxycytidine and cytidine), the purine derivative hypoxanthine, and glutamine, which provides nitrogen for nucleotide synthesis .

• Transcriptomics: Gene expression analysis across tumor types showed that DHODH is expressed at high levels in neuroblastoma and rhabdoid tumors compared to other solid tumors and pediatric samples .

• Functional genomics: CRISPR screening data from the Cancer Cell Line Encyclopedia (CCLE) identified a correlation between DHODH dependency and MYCN dependency in neuroblastoma cell lines .

• Partial least-squares discriminant analysis: This statistical approach can be used to identify metabolites that specifically accumulate in certain cancer types, such as neuroblastoma .

For researchers, integration of these multi-omic datasets requires sophisticated computational approaches and can provide comprehensive insights into the metabolic dependencies of cancer cells that would not be apparent from any single analysis method.

What techniques are used to investigate the relationship between DHODH and cellular differentiation states in cancer?

Research has revealed an important relationship between DHODH dependency and cellular differentiation states, particularly in neuroblastoma. Key methodological approaches include:

• Transcriptional profiling to classify cell lines based on differentiation status:

  • Adrenergic (ADRN) and mesenchymal (MES) scoring systems have been developed to quantify the differentiation state of neuroblastoma cell lines

  • Cell lines can be plotted on a spectrum from high ADRN/low MES to low ADRN/high MES

• Correlation analysis between differentiation status and DHODH dependency:

  • DHODH CERES scores (a measure of gene dependency from CRISPR screens) can be compared across cell lines with different differentiation states

  • Neuroblastoma cell lines with mesenchymal features (high MES scores) typically show lower DHODH dependency

  • Conversely, cell lines most dependent on DHODH generally have low MES scores

• Sensitivity testing of differentiated cells to DHODH inhibitors:

  • Comparing drug sensitivity (e.g., to brequinar) between cell lines with different differentiation states

  • Assessing whether cells with mesenchymal features (like SK-N-AS and SH-EP) show resistance similar to non-malignant stromal cells

These findings suggest that cellular differentiation state is an important factor to consider when evaluating DHODH as a therapeutic target in cancer.

How do researchers evaluate the combination of DHODH inhibitors with standard chemotherapeutics?

Combination approaches involving DHODH inhibitors and standard chemotherapeutics have shown promising results in preclinical models. Key methodological considerations include:

• In vitro combination studies:

  • Dose-response matrices testing various concentrations of DHODH inhibitors and chemotherapeutics

  • Cell viability/cytotoxicity assays to quantify therapeutic effects

  • Calculation of combination indices to determine synergy, additivity, or antagonism

• In vivo combination studies:

  • Animal models, such as transgenic neuroblastoma mouse models

  • Evaluation of tumor response, survival, and toxicity profiles

  • The combination of DHODH inhibition and the standard chemotherapeutic temozolomide has shown curative potential in a transgenic neuroblastoma mouse model

• Mechanistic investigations of combination effects:

  • Analysis of cell cycle effects

  • Assessment of DNA damage and repair mechanisms

  • Evaluation of changes in pyrimidine metabolism that might sensitize cells to chemotherapy

• Biomarker identification for combination therapy response:

  • MYCN status in neuroblastoma may predict sensitivity to DHODH inhibitor combinations

  • DHODH expression levels could serve as predictive biomarkers

These methodological approaches are critical for translating promising preclinical findings with DHODH inhibitor combinations into clinical applications.

What are the current challenges in translating DHODH inhibitor research into clinical applications?

Translating DHODH inhibitor research into clinical applications faces several challenges that researchers must address:

• Target specificity and off-target effects:

  • Developing highly selective DHODH inhibitors to minimize off-target effects

  • Differentiating between effects due to DHODH inhibition versus other mechanisms

  • Employing rescue experiments with pyrimidine supplementation to confirm on-target activity

• Biomarker development:

  • Identifying and validating predictive biomarkers of response

  • In neuroblastoma, high DHODH expression identifies a "highest risk group" that might benefit most from DHODH inhibition

  • Determining whether DHODH expression alone or in combination with other markers (like MYCN status) best predicts response

• Resistance mechanisms:

  • Understanding potential mechanisms of resistance to DHODH inhibition

  • Investigating whether cellular differentiation state (e.g., mesenchymal features) contributes to inherent resistance

  • Developing strategies to overcome or prevent resistance

• Optimal dosing and scheduling:

  • Determining optimal dose and schedule for DHODH inhibitors, particularly in combination regimens

  • Minimizing toxicity while maintaining efficacy

  • Considering pharmacokinetic and pharmacodynamic relationships

These challenges require rigorous preclinical investigation using appropriate disease models and careful clinical study design to advance DHODH inhibitors as therapeutic agents.

How might artificial intelligence and computational approaches advance DHODH inhibitor discovery?

Artificial intelligence (AI) and computational methods offer promising approaches to accelerate DHODH inhibitor discovery:

• Structure-based drug design:

  • Using the high-resolution crystal structures of human DHODH-inhibitor complexes

  • Employing molecular docking and virtual screening to identify new chemical scaffolds

  • Applying deep learning models trained on known DHODH inhibitor structures to predict novel inhibitors

• Quantitative structure-activity relationship (QSAR) modeling:

  • Developing predictive models based on known DHODH inhibitors and their potencies

  • Identifying key molecular features that contribute to DHODH inhibitory activity

  • Utilizing these models to optimize lead compounds

• Molecular dynamics simulations:

  • Investigating the dynamic behavior of DHODH and its interactions with inhibitors

  • Exploiting the intrinsic plasticity of the DHODH binding site to design adaptable inhibitors

  • Predicting binding free energies to prioritize compounds for synthesis and testing

• Systems biology approaches:

  • Modeling the impact of DHODH inhibition on pyrimidine metabolism in different cellular contexts

  • Predicting combination strategies that might synergize with DHODH inhibition

  • Identifying potential biomarkers of response to DHODH inhibition

These computational approaches can significantly reduce the time and resources required for traditional drug discovery methods while increasing the likelihood of identifying effective DHODH inhibitors.

What novel experimental models might advance our understanding of DHODH in development and disease?

Advancing our understanding of DHODH in development and disease will benefit from several novel experimental models:

• Patient-derived organoids:

  • Three-dimensional cultures derived from patient tumors that better recapitulate tumor heterogeneity

  • Evaluation of DHODH inhibitor response across organoids derived from different patients

  • Correlation of organoid response with patient characteristics and outcomes

• Developmental models for Miller syndrome:

  • CRISPR-engineered animal models harboring specific DHODH variants identified in Miller syndrome

  • Studies of pharyngeal arch and limb bud development in these models

  • Investigation of how DHODH function impacts specific developmental processes

• Single-cell approaches:

  • Single-cell RNA sequencing to assess heterogeneity in DHODH expression within tumors

  • Spatial transcriptomics to understand DHODH expression in the context of tissue architecture

  • Single-cell metabolomics to evaluate cell-to-cell variation in pyrimidine metabolism

• Humanized mouse models:

  • Mice with human immune systems for studying DHODH inhibition in autoimmune conditions

  • Evaluation of both therapeutic efficacy and potential immune-related adverse effects

These experimental models will provide more nuanced insights into DHODH biology and facilitate the translation of basic research findings into clinical applications.