NUDT15 Antibody

Basic Research Questions

• What is NUDT15 and why is it important in biomedical research?

NUDT15 (Nudix Nucleoside Diphosphate Linked Moiety X-Type Motif 15) is a nucleotide hydrolase that breaks down nucleoside triphosphates into nucleoside monophosphates . Its significance in research stems from its critical role in thiopurine metabolism, where it hydrolyzes the active thiopurine metabolites, 6-thio-(d)GTP, preventing their incorporation into DNA .

NUDT15 has gained prominence in pharmacogenetic research because:

• Polymorphisms in the NUDT15 gene are strongly associated with thiopurine-induced myelosuppression in patients with inflammatory bowel disease and acute lymphoblastic leukemia

• Patients homozygous for defective NUDT15 alleles can tolerate only approximately 8% of standard thiopurine dosages

• NUDT15 deficiency explains approximately 22% of variance in mercaptopurine tolerance

• Unlike TPMT variants (another cause of thiopurine toxicity) which are rare in Asian populations, NUDT15 genetic variation is substantially over-represented in Asians, making it their predominant genetic cause for thiopurine toxicity

• What techniques can NUDT15 antibodies be applied to in laboratory research?

NUDT15 antibodies can be utilized in multiple experimental techniques:

When selecting an antibody, researchers should verify reactivity with their species of interest, as some antibodies (like ABIN6144931) are reactive with human, mouse, and rat samples, while others may have more limited species reactivity .

• How does NUDT15 function in thiopurine metabolism and what is the relevance of studying its activity?

NUDT15 plays a crucial role in inactivating thiopurine active metabolites through enzymatic hydrolysis:

• Biochemical mechanism: NUDT15 hydrolyzes 6-thio-dGTP to 6-thio-dGMP, preventing incorporation of thioguanine nucleotides (TGNs) into DNA

• Therapeutic impact: This hydrolysis reduces the cytotoxic effects of thiopurines by decreasing DNA-TG levels

• Clinical relevance: NUDT15 deficiency leads to excessive accumulation of DNA-TG in a dose-dependent manner, resulting in severe hematopoietic toxicity

Research using NUDT15 antibodies helps elucidate:

• Protein expression levels in different tissues

• Correlation between NUDT15 expression and thiopurine sensitivity

• Validation of genetic findings with protein-level verification

In preclinical models, NUDT15-knockout mice exposed to mercaptopurine demonstrated severe leukopenia, rapid weight loss, earlier toxicity-related death, and bone marrow hypocellularity compared to wild-type mice, highlighting the enzyme's critical protective role .

Intermediate Research Questions

• How can researchers validate NUDT15 antibody specificity in their experiments?

Validating NUDT15 antibody specificity requires a multi-faceted approach:

• Positive and negative controls:

  • Use recombinant NUDT15 protein as a positive control

  • Include NUDT15 knockout or knockdown samples as negative controls

• Cross-reactivity assessment:

  • Test antibody reactivity in cell lines with confirmed NUDT15 expression levels

  • Verify specificity using siRNA or shRNA-mediated knockdown of NUDT15

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide (e.g., amino acids 1-164 for ABIN6144931)

  • Loss of signal confirms specificity for the target epitope

• Western blot analysis:

  • Confirm band appears at the expected molecular weight (~18-20 kDa)

  • Verify consistent results across multiple NUDT15 antibodies with different epitopes

• Correlation with genetic status:

  • Compare protein expression between samples with known NUDT15 genotypes

  • Homozygous variant samples should show altered protein expression

• What are the most common NUDT15 variants and how do they affect protein detection?

The major NUDT15 variants and their potential impact on antibody detection:

For comprehensive detection of NUDT15 variants:

• Use antibodies targeting conserved regions of the protein

• Consider multiple antibodies targeting different epitopes

• Correlate antibody detection with genotyping results, particularly for the common p.Arg139Cys variant that shows severely reduced enzymatic activity

The crystal structures of these variants have been determined using TH7755 (a potent NUDT15 inhibitor) to stabilize the proteins, providing insights into structural changes that may affect antibody binding .

Advanced Research Questions

• How can researchers use NUDT15 antibodies to study inhibitor binding and development?

NUDT15 antibodies are instrumental in validating small molecule inhibitors through multiple methodological approaches:

• Cellular Thermal Shift Assay (CETSA):

  • Methodology: Treat cells with inhibitor compounds at varying concentrations, heat to different temperatures, and analyze NUDT15 stability by Western blot

  • Application: The potent inhibitor TH7755 was shown to increase the apparent aggregation temperature (Tagg) of NUDT15 by ~4°C at 10μM, indicating strong binding

  • Quantification: Calculate thermal shift (ΔTagg) compared to vehicle control

• Isothermal Dose Response Fingerprint CETSA (ITDRF CETSA):

  • Methodology: Maintain constant temperature while varying inhibitor concentration

  • Example findings: TH7755 stabilized cellular NUDT15 starting at 1μM with increasing stabilization at higher concentrations

  • Advantage: Provides dose-dependent binding information

• Drug Affinity Responsive Target Stability (DARTS):

  • Methodology: Treat protein extracts with inhibitor then partially digest with proteases

  • Analysis: Protection from proteolysis indicates binding of inhibitor to target

  • Verification: Compare with structurally similar but inactive analogs as controls

• Structure-activity relationship studies:

  • Compare binding of multiple inhibitor candidates using consistent antibody-based detection methods

  • Correlate binding affinity with functional inhibition in cellular models

  • Example: TH7755 demonstrated improved cellular target engagement compared to previous inhibitor TH1760

• What methodological approaches can be used to study NUDT15's role in thiopurine sensitivity using antibodies?

To investigate NUDT15's role in thiopurine sensitivity, researchers can employ several antibody-dependent methodologies:

• Correlation studies between protein expression and drug sensitivity:

  • Quantify NUDT15 protein levels by Western blot across cell lines

  • Determine IC50 values for thiopurines in the same cell lines

  • Calculate Pearson or Spearman correlation coefficients between expression and sensitivity

• Knockdown validation experiments:

  • Generate NUDT15 knockdown models using siRNA/shRNA

  • Confirm knockdown efficiency by Western blot with NUDT15 antibodies

  • Measure changes in thiopurine sensitivity and active metabolite levels

  • Results from previous studies: NUDT15 knockdown cells showed increased levels of TGTP and DNA-TG, with higher susceptibility to thioguanine-induced apoptosis

• Immunoprecipitation studies:

  • Immunoprecipitate NUDT15 from cells before and after thiopurine treatment

  • Analyze binding partners and post-translational modifications

  • Investigate changes in protein complex formation during drug metabolism

• Subcellular localization analysis:

  • Use immunofluorescence with NUDT15 antibodies to track protein localization

  • Compare localization between wild-type and variant forms

  • Examine changes in localization upon thiopurine treatment

• Combined protein and metabolite analysis:

  • Correlate NUDT15 protein levels (by immunoblotting) with DNA-TG levels (by mass spectrometry)

  • Example protocol: Yang et al. demonstrated that NUDT15-deficient mice accumulated higher DNA-TG levels in an MP dose-dependent manner

• How can NUDT15 antibodies be used in combination with genetic analysis to identify patients at risk for thiopurine toxicity?

A comprehensive approach combining antibody-based detection and genetic analysis provides deeper insights into NUDT15-mediated thiopurine toxicity:

• Genotype-protein expression correlation:

  • Methodology: Collect peripheral blood mononuclear cells from patients with known NUDT15 genotypes

  • Analysis: Quantify NUDT15 protein expression by Western blot

  • Expected results: Patients with homozygous variant genotypes (e.g., *3/*3) should show significantly reduced protein levels compared to wild-type (*1/*1)

  • Research finding: Patients with the *3/*3 genotype (p.Arg139Cys homozygous) tolerated only 8% of standard thiopurine doses

• Functional validation of novel variants:

  • Identify novel NUDT15 variants through genetic screening

  • Express recombinant variant proteins in vitro

  • Detect variant protein stability and expression using NUDT15 antibodies

  • Measure enzymatic activity to classify variants as damaging or benign

  • Example from research: Moriyama et al. used massively parallel variant characterization to classify 1,152 deleterious NUDT15 variants out of 3,097 possible missense variants

• Tissue-specific expression analysis:

  • Perform immunohistochemistry on tissue biopsies from patients with different NUDT15 genotypes

  • Quantify expression differences that may contribute to tissue-specific toxicity

  • Correlate with clinical outcomes

• Personalized therapy monitoring:

  • Methodology: Collect samples during thiopurine therapy

  • Analysis: Monitor NUDT15 protein levels and DNA-TG accumulation

  • Application: Adjust dosing based on combined genetic and protein data

• What are the considerations for using NUDT15 antibodies in studies of cancer tissues with potential NUDT15 copy number alterations?

Recent findings indicate NUDT15 copy number variations in cancer may affect antibody-based studies:

• Copy number validation:

  • Recent research shows that over 60% of ovarian cancers have copy number losses in NUDT15

  • Researchers should validate copy number status using techniques like FISH or qPCR before interpreting antibody staining results

• Calibration considerations:

  • Establish control samples with known NUDT15 copy numbers

  • Use digital image analysis for precise quantification of immunohistochemistry

  • Normalize expression data to account for copy number variations

• Heterogeneity assessment:

  • Perform regional sampling within tumors

  • Employ dual staining for NUDT15 and tumor markers

  • Account for tumor-normal tissue mosaicism in analysis

• Interpretation guidelines:

  • Low NUDT15 expression may reflect genetic loss rather than transcriptional regulation

  • Compare protein expression with mRNA data when available

  • Consider functional consequences of reduced NUDT15 on thiopurine metabolism within tumor cells

• Therapeutic implications:

  • Tumor-specific NUDT15 deficiency might influence local thiopurine metabolism

  • This could potentially increase sensitivity to thiopurine therapy in NUDT15-deficient tumors

  • Studies using NUDT15 inhibitors like TH7755 suggest this approach may enhance thiopurine efficacy

• What methodological approaches can be used to investigate NUDT15 antibody cross-reactivity with other NUDIX family members?

The NUDIX family contains multiple structurally related enzymes that may create specificity challenges for antibodies:

• Sequence alignment analysis:

  • Compare epitope sequences of NUDT15 with other NUDIX family proteins (especially MTH1)

  • Identify regions of high homology that might lead to cross-reactivity

  • Select antibodies targeting unique regions of NUDT15

• Recombinant protein panel testing:

  • Express recombinant proteins for multiple NUDIX family members

  • Test antibody reactivity against the panel by Western blot

  • Quantify relative affinity for each protein

• Knockout/knockdown validation:

  • Generate NUDT15-specific knockouts using CRISPR-Cas9

  • Test for complete elimination of antibody signal

  • Any remaining signal might indicate cross-reactivity

  • Methodology example: The NUDT15 knockout mouse model created by Moriyama et al. can serve as an excellent negative control

• Immunoprecipitation-mass spectrometry:

  • Perform immunoprecipitation with NUDT15 antibody

  • Analyze pulled-down proteins by mass spectrometry

  • Identify any co-precipitated NUDIX family members

• Epitope mapping:

  • Use peptide arrays to precisely map antibody binding regions

  • Compare with known structures of NUDIX family proteins

  • Select antibodies with minimal potential for cross-reactivity

This methodical approach ensures accurate data interpretation in studies examining NUDT15 expression and function, particularly in systems where multiple NUDIX proteins are expressed.

Research Applications and Future Directions

• How can NUDT15 antibodies contribute to the development of companion diagnostics for thiopurine therapy?

NUDT15 antibody-based diagnostics could complement genetic testing in personalized medicine:

• Protein-level phenotyping:

  • While genetic testing identifies known variants, antibody-based assays detect functional NUDT15 protein levels

  • This approach could identify patients with reduced NUDT15 activity due to regulatory mechanisms rather than coding variants

  • Combined genetic and protein analysis provides comprehensive risk assessment

• Development of semi-quantitative IHC assays:

  • Standardize NUDT15 antibody-based immunohistochemistry

  • Develop scoring systems correlating with thiopurine sensitivity

  • Validate across multiple tissue types relevant to treatment (bone marrow, intestinal mucosa)

• Point-of-care testing possibilities:

  • Develop simplified antibody-based assays for rapid assessment

  • Could complement genetic testing in clinical decision-making

  • Particularly valuable in populations with high NUDT15 variant frequencies

• Monitoring during therapy:

  • Serial measurement of NUDT15 protein levels during treatment

  • Potential to identify acquired changes affecting thiopurine metabolism

  • Early detection of toxicity risk before clinical manifestation

Current clinical practice primarily relies on genetic testing of TPMT and NUDT15, with body's such as the FDA, NICE (UK) and GESA noting rather than recommending testing prior to initiating thiopurine therapy . Antibody-based approaches could strengthen the evidence base for mandatory testing.

• How can researchers use NUDT15 antibodies to investigate unexplained thiopurine toxicity cases?

Many patients experiencing thiopurine toxicity have no identified variants in TPMT or NUDT15 , suggesting other mechanisms could be involved:

• Post-translational modification analysis:

  • Methodology: Use phospho-specific or ubiquitin-specific NUDT15 antibodies

  • Rationale: Post-translational modifications might affect NUDT15 activity without genetic alterations

  • Application: Compare PTM patterns between toxicity cases and controls

• Protein-protein interaction studies:

  • Methodology: Co-immunoprecipitation with NUDT15 antibodies followed by mass spectrometry

  • Purpose: Identify novel interaction partners that modulate NUDT15 activity

  • Analysis: Compare interactome between toxicity cases and controls

• Alternative splicing investigation:

  • Methodology: Use domain-specific NUDT15 antibodies to detect splice variants

  • Rationale: Splice variants might affect function without changing common genetic markers

  • Application: Develop targeted assays for functionally significant isoforms

• Stability and degradation pathway analysis:

  • Methodology: Pulse-chase experiments with NUDT15 antibody detection

  • Purpose: Assess protein half-life and degradation rates

  • Application: Identify cases with accelerated NUDT15 turnover

• Subcellular localization studies:

  • Methodology: Subcellular fractionation with NUDT15 antibody detection

  • Rationale: Mislocalization could affect function without altering expression

  • Analysis: Compare nuclear/cytoplasmic ratios between samples