NUDT5 Human

What is NUDT5 and what are its primary functions in human cells?

NUDT5 belongs to the NUDIX (Nucleoside Diphosphate linked to X) hydrolase family that catalyzes the hydrolysis of nucleoside diphosphates. Structurally, NUDT5 shares similarities with bacterial enzymes but differs from other human NUDIX hydrolases like NUDT9 . Its primary functions include:

• Hydrolysis of ADP-ribose and related substrates

• Involvement in hormone signaling pathways, particularly in breast cancer cells

• Recently discovered role in mediating 6-thioguanine (6-TG) toxicity through a non-enzymatic function

Functionally, NUDT5 has been identified through genome-wide CRISPR loss-of-function screens as a crucial mediator of 6-TG toxicity across multiple cell lines, including HEK293T, HT29, and A375 cells .

How does NUDT5 contribute to hormone signaling in cancer cells?

NUDT5 plays a significant role in hormone signaling pathways, particularly in breast cancer cells. Research has demonstrated that:

• NUDT5 is involved in ADP-ribose metabolism, which affects nuclear ATP levels and consequently hormone-dependent gene regulation

• Targeted inhibition of NUDT5 blocks hormone signaling in breast cancer models

• The development of compounds like TH5427 has provided valuable tools to study NUDT5's role in hormone-dependent signaling pathways

Methodologically, researchers can investigate NUDT5's role in hormone signaling using targeted inhibitors like TH5427, which has been developed specifically to probe NUDT5 activity and ADP-ribose metabolism in cellular contexts .

What structural characteristics define human NUDT5?

Human NUDT5 has been characterized structurally through crystallographic studies that reveal:

• A dimeric structure with specific binding regions for substrates like ADPR

• Structural similarity to bacterial NUDIX enzymes but distinct differences from human NUDT9

• Crystal structures of NUDT5 in complex with inhibitors like TH1713 have been resolved at 2.2 Å resolution

The crystal structure data provides insights into the binding modality of NUDT5 inhibitors, showing that compounds like TH1713 occupy the same binding regions as ADPR within the active site of the NUDT5 dimer .

How do researchers distinguish between enzymatic and non-enzymatic functions of NUDT5?

Recent research has revealed that NUDT5 possesses distinct enzymatic and non-enzymatic functions, particularly in the context of 6-TG toxicity. Methodological approaches to distinguish these functions include:

• Enzymatic inhibition vs. protein depletion comparison:

  • Treatment with enzymatic inhibitors like TH5427 fails to rescue cells from 6-TG-induced toxicity

  • In contrast, degradation of NUDT5 protein via PROTACs (dNUDT5) effectively rescues cell viability in a dose-dependent manner

• Mutation studies:

  • Reconstitution experiments in NUDT5 knockout cells with either wild-type or catalytically inactive E112Q NUDT5 mutant

  • Both variants show comparable sensitivity to 6-TG treatment, similar to parental cells, confirming that the protein's presence rather than its catalytic activity mediates 6-TG toxicity

These approaches have conclusively demonstrated that cell death mediated by 6-TG depends on a novel, non-enzymatic function of NUDT5, highlighting the importance of protein degradation approaches in uncovering protein functions beyond enzymatic activity .

What methodologies are optimal for developing and validating NUDT5 inhibitors?

Development and validation of NUDT5 inhibitors follows a comprehensive workflow:

• Initial screening:

  • High-throughput screening of chemical libraries (e.g., 72,000 compounds) using modified malachite green assays

  • Assessment of screening performance using z′ factors (average values of 0.87 and 0.85 reported)

• Biophysical validation:

  • Differential scanning fluorimetry (DSF) to verify thermal stabilization of NUDT5 (e.g., 5°C shift in melting temperature with TH1533 and TH1713 at 20 μM)

  • Crystallographic studies to confirm binding modes and inform structure-based design

• Cellular target engagement:

  • Cellular thermal shift assay (CETSA) to verify binding in cellular environments

  • Progressive CETSA screening funnel to prioritize compounds:
    a) Biochemical inhibition (IC₅₀ < 100 nM)
    b) Thermostabilization in cell lysates (HT-CETSA)
    c) Target engagement in intact cells
    d) Isothermal dose-response fingerprint CETSA (ITDRF CETSA)

This methodological pipeline ensures selection of compounds that genuinely engage NUDT5 under increasingly stringent biological conditions without bias toward predefined phenotypic responses .

What are the applications of Targeted Protein Degradation (TPD) in studying NUDT5 function?

Targeted Protein Degradation (TPD) has emerged as a powerful approach to study NUDT5 with several key applications:

• Development of NUDT5-specific PROTACs:

  • Design of bifunctional molecules linking NUDT5 ligands to E3 ligase (CRBN) recruiting moieties

  • Optimization of linker chemistry (e.g., tetrasubstituted urea linker motif in dNUDT5)

  • Achievement of potent degradation (DC₅₀ = 0.3-0.5 nM) across multiple cell lines

• Mechanistic studies:

  • Confirmation of degradation mechanisms through rescue experiments using neddylation inhibitors, proteasome inhibitors, and NUDT5 inhibitors

  • Verification of ternary complex formation between NUDT5, PROTAC, and CRBN

• Selectivity assessment:

  • Global proteome analysis showing exquisite selectivity with no other proteins significantly degraded

  • Development of negative controls (e.g., dNUDT5nc) through N-methylation of the imide ring to attenuate CRBN binding

• Functional validation:

  • Dose-dependent rescue of cell viability in 6-TG-treated cells

  • Comparison of degraders vs. inhibitors to dissect domain-specific or scaffolding roles

This TPD approach has been instrumental in uncovering the non-enzymatic role of NUDT5 in 6-TG-mediated cell death, highlighting its value as a complementary strategy to genetic loss-of-function studies .

What crystallographic data is available for NUDT5-inhibitor complexes and how does it inform structure-based drug design?

Crystallographic studies have provided valuable insights into NUDT5-inhibitor interactions. Key crystallographic data includes:

These structural data reveal that:

• Inhibitors occupy the same binding regions as ADPR within the active site of the NUDT5 dimer

• The binding pose of degraders like dNUDT5 is analogous to that of inhibitors like TH5427

• Structure-guided optimization can inform development of more potent and selective compounds

This structural information has directly facilitated the rational design of improved NUDT5 modulators with enhanced potency and cellular activity.

How should researchers design experiments to evaluate the role of NUDT5 in 6-TG response?

To comprehensively evaluate NUDT5's role in 6-TG response, researchers should implement a multi-modal experimental design:

• Genetic perturbation:

  • CRISPR knockout of NUDT5 in relevant cell lines (e.g., HAP1, HL-60, HCT116)

  • Reconstitution with wild-type or mutant NUDT5 variants to assess functional requirements

• Pharmacological intervention:

  • Parallel assessment of enzymatic inhibitors (e.g., TH5427, MRK-952) and protein degraders (e.g., dNUDT5)

  • Dose-response studies with 6-TG treatment (typically 72h exposure)

• Mechanistic validation:

  • Control experiments with inactive derivatives (e.g., dNUDT5nc)

  • Assessment across multiple cell lines to confirm generalizability of findings

  • Cell viability assays in naive cells to confirm specificity of observed effects

This comprehensive approach has successfully revealed that NUDT5 knockout confers a four-fold increase in 6-TG IC₅₀ values, yet enzymatic inhibition fails to recapitulate this effect—demonstrating the non-enzymatic nature of NUDT5's role in 6-TG toxicity .

What cellular assays are most informative for studying NUDT5 degradation kinetics?

For robust characterization of NUDT5 degradation kinetics, researchers should employ multiple complementary assays:

• Western blot analysis:

  • Concentration-dependent degradation assessment (e.g., DC₅₀ determination)

  • Time-course experiments (sustained degradation over 72h)

  • Hook effect characterization at high concentrations (>300 nM)

• Ternary complex formation:

  • NanoBRET assays to confirm target engagement in live cells

  • Co-immunoprecipitation studies to verify interaction between NUDT5 and E3 ligase machinery

• Degradation mechanism validation:

  • Rescue experiments using:
    a) Neddylation inhibitors
    b) Proteasome inhibitors
    c) NUDT5 inhibitors
    d) CRBN knockout cells

• Global proteome analysis:

  • Mass spectrometry-based proteomics at multiple timepoints (6h, 24h)

  • Assessment of degradation selectivity across the entire proteome

These methodologies have successfully characterized dNUDT5 as a highly selective NUDT5 degrader with DC₅₀ values of 0.3-0.5 nM and sustained activity over 72 hours .

How do researchers address data inconsistencies between genetic and pharmacological NUDT5 modulation?

When confronted with discrepancies between genetic knockout and pharmacological inhibition of NUDT5, researchers should implement a systematic approach:

• Validation of tool quality:

  • Confirm inhibitor target engagement using biophysical techniques (DSF, CETSA)

  • Verify biochemical activity in enzymatic assays

  • Assess cellular permeability and stability

• Exploration of alternative mechanisms:

  • Develop orthogonal modalities (e.g., PROTACs vs. inhibitors)

  • Test multiple chemical scaffolds to rule out compound-specific effects

  • Consider protein scaffolding vs. enzymatic functions

• Functional reconstitution:

  • Generate catalytically inactive mutants (e.g., E112Q)

  • Perform rescue experiments in knockout backgrounds

  • Compare phenotypic responses between approaches

This strategy successfully resolved the paradox of NUDT5 in 6-TG response, where genetic knockout conferred resistance, yet enzymatic inhibition failed to recapitulate this effect—ultimately revealing NUDT5's non-enzymatic role in this context .

What are the implications of NUDT5's role in 6-TG response for leukemia treatment?

The discovery of NUDT5's non-enzymatic role in 6-TG toxicity has significant implications for leukemia treatment:

• Therapeutic resistance mechanisms:

  • NUDT5 status may influence patient response to 6-TG therapy

  • Altered NUDT5 expression could contribute to treatment resistance

  • Depletion of NUDT5 protein is antagonistic to NUDT15 inhibition, suggesting distinct modes of action

• Potential biomarkers:

  • NUDT5 expression levels might serve as predictive biomarkers for 6-TG response

  • Assessment in leukemia patient samples could inform treatment stratification

• New therapeutic strategies:

  • Development of NUDT5 degraders as potential sensitizers or resistance modulators

  • Combination approaches targeting both enzymatic and non-enzymatic functions

These findings are particularly relevant given that 6-TG is widely used in leukemia treatment, and understanding its mechanism of action and resistance pathways is crucial for optimizing therapeutic outcomes .

How might NUDT5 degraders be optimized for potential therapeutic applications?

Optimization of NUDT5 degraders for therapeutic applications requires addressing several key parameters:

• Potency and selectivity:

  • Achievement of sub-nanomolar DC₅₀ values (0.3-0.5 nM demonstrated)

  • Verification of selectivity through global proteome analysis

  • Optimization of degradation kinetics for sustained effect

• Physicochemical properties:

  • Optimization of linker chemistry (tetrasubstituted urea linker motif shows promise)

  • Balance of solubility and permeability considerations

  • Consideration of hook effect limitations at high concentrations

• Pharmacological characteristics:

  • Assessment across diverse cell types (A549, MCF7, HAP1, etc.)

  • Evaluation of on-target toxicity (degraders themselves showed no impact on cell viability)

  • Development of appropriate negative controls (e.g., dNUDT5nc)

• Structural optimization:

  • Utilization of co-crystal structures to inform rational design

  • Fine-tuning of binding pose and protein-protein interactions

  • Enhancement of ternary complex formation efficiency

These optimization strategies have successfully yielded NUDT5 degraders with promising characteristics for potential therapeutic applications, particularly in contexts where modulation of NUDT5's non-enzymatic functions is desired .

What are the most promising directions for future NUDT5 research based on current findings?

Based on recent discoveries, several promising research directions for NUDT5 emerge:

• Mechanistic elucidation:

  • Detailed characterization of NUDT5's non-enzymatic functions

  • Identification of protein-protein interactions mediating 6-TG toxicity

  • Investigation of potential interplay between NUDT5 and NUDT15 pathways

• Expanded therapeutic applications:

  • Exploration of NUDT5 modulation in hormone-dependent cancers

  • Investigation of combination approaches with standard-of-care therapies

  • Assessment of NUDT5 status across diverse cancer types

• Advanced tool development:

  • Creation of degraders with improved pharmacological properties

  • Development of bifunctional molecules with dual inhibitory/degradation capabilities

  • Generation of cell-type specific degradation systems

• Translational validation:

  • Evaluation in patient-derived models

  • Correlation of NUDT5 expression with treatment outcomes

  • Assessment of genetic variants affecting NUDT5 function or expression

These research directions build upon the foundation of recent discoveries revealing NUDT5's unexpected non-enzymatic functions and the powerful approaches now available to modulate this protein for both research and potential therapeutic applications .

What are the optimal conditions for NUDT5 crystallization and structure determination?

Based on successful crystallographic studies of NUDT5, the following conditions have proven effective:

• Crystallization parameters:

  • Space group: C 1 2 1 (for both NUDT5-TH1713 and NUDT5-TH5427 complexes)

  • Cell dimensions vary by complex:

    • NUDT5-TH1713: a=111.5, b=39.3, c=98.72 Å; α=90, β=122.2, γ=90°

    • NUDT5-TH5427: a=100.6, b=40.1, c=104.1 Å; α=90, β=113.4, γ=90°

• Data collection:

  • Resolution ranges: 41.8–2.2 Å (NUDT5-TH1713) and 46.2–2.6 Å (NUDT5-TH5427)

  • Completeness: >96% for both structures

  • I/σI values: 9.7 and 15.6 respectively

• Refinement:

  • Number of reflections: 18,626 (NUDT5-TH1713) and 11,883 (NUDT5-TH5427)

  • Careful consideration of protein-ligand interactions, particularly in the ADPR binding site

These parameters have successfully yielded high-quality structures that reveal the binding modality of inhibitors and inform structure-based drug design efforts .

How should researchers implement CETSA for evaluating NUDT5 target engagement?

Implementation of Cellular Thermal Shift Assay (CETSA) for NUDT5 target engagement requires careful optimization:

• Initial thermal stability assessment:

  • Differential scanning fluorimetry (DSF) to confirm inhibitor binding (e.g., 5°C shift in Tm observed with TH1533 and TH1713 at 20 μM)

• CETSA protocol optimization:

  • Temperature gradient determination (83°C identified as optimal for NUDT5 aggregation)

  • Evaluation in both intact cells and cell lysates

  • Western blot detection with thermostable SOD1 as loading control

• Technical considerations:

  • Cell washing steps (two PBS washes) to remove excess compound before heating

  • Assessment of membrane integrity at high temperatures

  • Temperature selection where nearly all protein has aggregated in untreated samples

• Progressive CETSA implementation:

  • HT-CETSA (high-throughput CETSA) for initial screening of compounds that engage NUDT5 in cell lysates

  • Transition to intact cell assessment for promising candidates

  • ITDRF CETSA (isothermal dose-response fingerprint CETSA) to determine relative potency

This methodological approach has successfully guided medicinal chemistry campaigns by selecting compounds based on their ability to engage NUDT5 under increasingly stringent biological conditions .

What are the critical parameters for designing effective NUDT5 PROTACs?

Design of effective NUDT5 PROTACs requires optimization of several critical parameters:

• Warhead selection:

  • Use of established NUDT5 ligands with confirmed binding modes

  • Retention of key interactions within the ADPR binding site

  • Consideration of exit vectors for linker attachment

• E3 ligase recruitment:

  • Selection of appropriate E3 ligase ligand (CRBN ligand demonstrated success)

  • Prevention of steric clashes in ternary complex formation

  • Strategic N-methylation of the imide ring in negative controls (dNUDT5nc) to attenuate CRBN binding

• Linker optimization:

  • Exploration of various linker chemistries (tetrasubstituted urea linker motif showed superior results)

  • Balancing flexibility and rigidity for optimal ternary complex formation

  • Length optimization to facilitate productive protein-protein interactions

• Validation considerations:

  • Confirmation of target engagement (NanoBRET assays)

  • Verification of ternary complex formation

  • Assessment of degradation efficiency and kinetics

  • Evaluation of hook effect at high concentrations