Crystal structures of NUDT15 variants enabled by a potent inhibitor reveal the structural basis for thiopurine sensitivity

Rehling, Daniel; Zhang, Si Min; Jemth, Ann-Sofie; Koolmeister, Tobias; Throup, Adam; Wallner, Olov; Scaletti, Emma Rose; Moriyama, Takaya; Nishii, Rina; Davies, Jonathan; Desroses, Matthieu; Rudd, Sean G.; Scobie, Martin; Homan, Evert; Yang, Jun J.; Helleday, Thomas; Stenmark, Pål

doi:10.1016/j.jbc.2021.100568

Cited by 9 publications

(18 citation statements)

References 32 publications

(50 reference statements)

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“…NUDT15 deactivates the nucleoside analog antimetabolite, thiopurine, by hydrolyzing the active triphosphates and limiting DNA damage-induced toxicity 16, 28 . Several destabilizing variants of NUDT15 have been associated with clinical thiopurine intolerance 28 and can be stabilized towards thermal denaturation by NUDT15 inhibitors (NUDT15i) in vitro 29 , including R139C, which is known to be rapidly degraded by the proteasome 16 . When cells expressing an HA-tagged R139C variant were incubated with thioguanine, we saw increased incorporation-related DNA damage but, interestingly, also an accumulation of HA-R139C ( Fig.…”

Section: Resultsmentioning

confidence: 99%

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Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM

Valerie

Sanjiv

Mortusewicz

et al. 2022

Preprint

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Cellular target engagement technologies are reforming drug discovery by enabling quantification of intracellular drug binding; however, concomitant assessment of drug-associated phenotypes has proven challenging. We have developed cellular target engagement by accumulation of mutant (CeTEAM) as a platform that can seamlessly evaluate drug-target interactions and phenotypic responses in a single multiparametric experiment. In the presence of binding ligand, accumulation of an initially unstable target protein acts as a biosensor that permits holistic assessment of drug pharmacology under physiological conditions. We demonstrate this proof-of-concept by uncoupling target binding from divergent cellular activities of MTH1 inhibitors, repurposing the R139C variant to dissect complex NUDT15-thiopurine interactions, and profiling the live-cell dynamics of DNA trapping by PARP inhibitors. Further, PARP1-derived drug biosensors facilitated multimodal ex vivo analysis of drug-target engagement and non-invasive tracking of drug binding in live animals. CeTEAM empowers real-time, comprehensive characterization of target engagement by bridging drug binding events and their biological consequences.

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Section: Resultsmentioning

confidence: 99%

“…3c). An enzymecoupled malachite green inhibition assay determined that NSC56456 and TH8234 similarly inhibited dGTP hydrolysis (IC50 = 355 and 375 nM, respectively), albeit less potently than optimized NUDT15i [29][30][31] , while TH8228 was completely inactive (Fig. 3d).…”

Section: Illuminating Complex Nudt15-thiopurine Interactions With The...mentioning

confidence: 96%

Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM

Valerie

Sanjiv

Mortusewicz

et al. 2022

Preprint

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“…Among the different NUTD15 variants identified up to date, we selected for our purposes two well characterized mutants, both with substitutions in the position of Arg139 by a cysteine (R139C) and histidine (R139H). This decision was based in the clinical relevance of these mutants in thiopurine therapy and the availability of experimental data to validate and compare our computational results (Table 1) (Yang and others 2014; Rehling and others 2021; Cargnin and others 2018).…”

Section: Resultsmentioning

confidence: 99%

“…NUDIXs substrate specificity has been the object of extensive research, and recent studies have proved that NUTD15 is able to hydrolyze metabolites of thiopurines drugs like 6-thio-deoxyguanosine triphosphate (Rehling and others 2021; Valerie and others 2016). Azathioprine, mercaptopurine and thioguanine belong to the group of thiopurine antimetabolites, a kind of drugs that, after their bioactivation inside the cell into thioguanosine triphosphate (TGTP), inhibit several enzymes belonging to the purine biosynthesis and produce misincorporation into DNA and RNA (Figure 1) (Karran and Attard 2008).…”

Section: Introductionmentioning

confidence: 99%

Molecular dynamics simulations reveal the impact of NUTD15 variants in structural conformation and dynamics

Gómez-Rubio¹,

García-Marín

2022

Preprint

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NUDT15 or MTH2 is a member of NUDIX protein family that catalyze the hydrolysis of nucleotides and deoxynucleotides, including thioguanine analogues. NUTD15 has been reported as a DNA sanitizer in humans, and more recent studies have proved that genomic variants are related to a poor prognosis in inmoplastic and immunologic diseases with thioguanines. Despite of this, the role of NUTD15 in physiology and molecular biology is quite unclear, as well as the mechanism of action of this enzyme. The existence of clinically relevant variants has prompted the study of this enzymes, whose capacity to bound and hydrolyze thioguanine nucleotides is still poor understood. By using a combination of biomolecular modelling techniques together with molecular dynamics we have studied the monomeric wild type NUTD15, as well as two important variants R139C and R139H. Our findings reveal not only how nucleotide binding stabilizes the enzyme, but also how two loops are responsible for keeping the enzyme in a packed close conformation. Mutations in α2 helix affect a network of hydrophobic and π-interactions that are responsible of active site enclosing.

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“…While polymorphisms in TPMT alone explain around 40% of thiopurine-induced ADRs (Schaeffeler et al 2019 ), predictions can be further improved by including the missense variant p.R139C in NUDT15 (c.415C > T; rs116855232) (Yang et al 2015b , 2014 ). Mechanistically, this variant destabilizes the protein structure, thereby resulting in lower enzymatic activity (Rehling et al 2021 ). p.R139C defines NUDT15*3 and is moreover part of NUDT15*2 in combination with the inframe deletion variant (rs746071566), in both cases resulting in a loss of gene product function.…”

Section: Tpmt and Nudt15mentioning

confidence: 99%

Population pharmacogenomics: an update on ethnogeographic differences and opportunities for precision public health

Zhou

Lauschke

2021

Hum Genet

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Both safety and efficacy of medical treatment can vary depending on the ethnogeographic background of the patient. One of the reasons underlying this variability is differences in pharmacogenetic polymorphisms in genes involved in drug disposition, as well as in drug targets. Knowledge and appreciation of these differences is thus essential to optimize population-stratified care. Here, we provide an extensive updated analysis of population pharmacogenomics in ten pharmacokinetic genes (CYP2D6, CYP2C19, DPYD, TPMT, NUDT15 and SLC22A1), drug targets (CFTR) and genes involved in drug hypersensitivity (HLA-A, HLA-B) or drug-induced acute hemolytic anemia (G6PD). Combined, polymorphisms in the analyzed genes affect the pharmacology, efficacy or safety of 141 different drugs and therapeutic regimens. The data reveal pronounced differences in the genetic landscape, complexity and variant frequencies between ethnogeographic groups. Reduced function alleles of CYP2D6, SLC22A1 and CFTR were most prevalent in individuals of European descent, whereas DPYD and TPMT deficiencies were most common in Sub-Saharan Africa. Oceanian populations showed the highest frequencies of CYP2C19 loss-of-function alleles while their inferred CYP2D6 activity was among the highest worldwide. Frequencies of HLA-B*15:02 and HLA-B*58:01 were highest across Asia, which has important implications for the risk of severe cutaneous adverse reactions upon treatment with carbamazepine and allopurinol. G6PD deficiencies were most frequent in Africa, the Middle East and Southeast Asia with pronounced differences in variant composition. These variability data provide an important resource to inform cost-effectiveness modeling and guide population-specific genotyping strategies with the goal of optimizing the implementation of precision public health.

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Crystal structures of NUDT15 variants enabled by a potent inhibitor reveal the structural basis for thiopurine sensitivity

Cited by 9 publications

References 32 publications

Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM

Coupling cellular drug-target engagement to downstream pharmacology with CeTEAM

Molecular dynamics simulations reveal the impact of NUTD15 variants in structural conformation and dynamics

Population pharmacogenomics: an update on ethnogeographic differences and opportunities for precision public health

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