| 1. |
- Rehling, Daniel, et al.
(författare)
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Structural and biochemical investigation of class I ribonucleotide reductase from the hyperthermophile Aquifex aeolicus
- 2022
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Ingår i: Biochemistry. - : American Chemical Society (ACS). - 0006-2960 .- 1520-4995. ; 61:2, s. 92-106
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Tidskriftsartikel (refereegranskat)abstract
- Ribonucleotide reductase (RNR) is an essential enzyme with a complex mechanism of allosteric regulation found innearly all living organisms. Class I RNRs are composed of two proteins, a large α-subunit (R1) and a smaller β-subunit (R2) that exist as homodimers, that combine to form an active heterotetramer. Aquifex aeolicus is a hyperthermophilic bacterium with an unusual RNR encoding a 346-residue intein in the DNA sequence encoding its R2 subunit. We present the first structures of the A. aeolicus R1 and R2 (AaR1 and AaR2, respectively) proteins as well as the biophysical and biochemical characterization of active and inactive A. aeolicus RNR. While the active oligomeric state and activity regulation of A. aeolicus RNR are similar to those of other characterized RNRs, the X-ray crystal structures also reveal distinct features and adaptations. Specifically, AaR1 contains a β-hairpin hook structure at the dimer interface, which has an interesting π stacking interaction absent in other members of the NrdAh subclass, and its ATP cone houses two ATP molecules. We determined structures of two AaR2 proteins: one purified from a construct lacking the intein (AaR2) and a second purified from a construct including the intein sequence (AaR2_genomic). These structures in the context of metal content analysis and activity data indicate that AaR2_genomic displays much higher iron occupancy and activity compared to AaR2, suggesting that the intein is important for facilitating complete iron incorporation, particularly in the Fe2 site of the mature R2 protein, which may be important for the survival of A. aeolicus in low-oxygen environments.
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| 2. |
- Nishii, Rina, et al.
(författare)
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NUDT15 polymorphism influences the metabolism and therapeutic effects of acyclovir and ganciclovir
- 2021
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Ingår i: Nature Communications. - : Springer Science and Business Media LLC. - 2041-1723. ; 12:1
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Tidskriftsartikel (refereegranskat)abstract
- Nucleobase and nucleoside analogs (NNA) are widely used as anti-viral and anti-cancer agents, and NNA phosphorylation is essential for the activity of this class of drugs. Recently, diphosphatase NUDT15 was linked to thiopurine metabolism with NUDT15 polymorphism associated with drug toxicity in patients. Profiling NNA drugs, we identify acyclovir (ACV) and ganciclovir (GCV) as two new NNAs metabolized by NUDT15. NUDT15 hydrolyzes ACV and GCV triphosphate metabolites, reducing their effects against cytomegalovirus (CMV) in vitro. Loss of NUDT15 potentiates cytotoxicity of ACV and GCV in host cells. In hematopoietic stem cell transplant patients, the risk of CMV viremia following ACV prophylaxis is associated with NUDT15 genotype (P = 0.015). Donor NUDT15 deficiency is linked to graft failure in patients receiving CMV-seropositive stem cells (P = 0.047). In conclusion, NUDT15 is an important metabolizing enzyme for ACV and GCV, and NUDT15 variation contributes to inter-patient variability in their therapeutic effects.
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| 3. |
- Rehling, Daniel, et al.
(författare)
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Crystal structures of NUDT15 variants enabled by a potent inhibitor reveal the structural basis for thiopurine sensitivity
- 2021
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Ingår i: Journal of Biological Chemistry. - : Elsevier BV. - 0021-9258 .- 1083-351X. ; 296
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Tidskriftsartikel (refereegranskat)abstract
- The enzyme NUDT15 efficiently hydrolyzes the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and preemptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants because of their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared with TH1760, while showing no cytotoxicity on its own. This potent inhibitor also stabilized NUDT15, enabling analysis by X-ray crystallography. We have determined high-resolution structures of the clinically relevant NUDT15 variants Arg139Cys, Arg139His, Val18Ile, and V18_V19insGlyVal. These structures provide clear insights into the structural basis for the thiopurine intolerance phenotype observed in patients carrying these pharmacogenetic variants. These findings will aid in predicting the effects of new NUDT15 sequence variations yet to be discovered in the clinic.
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| 4. |
- Rehling, Daniel, 1985-
(författare)
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Structural Studies of Proteins involved in Nucleotide Metabolism : Studies of a ribonucleotide reductase from A. aeolicus and NUDT15
- 2021
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- This thesis is separated into two parts. The first part concerns ribonucleotide reductase from Aquifex aeolicus. A. aeolicus is a hyperthermophilic bacterium that thrives at extremely high temperatures of 80-95 °C. We present the X-ray crystal structures of both the R1 and R2 subunits of this protein, which represents the first structure of a class Ia ribonucleotide reductase from a hyperthermophile and the first structure of an R1 from the NrdAh phylogenetic subclass. Several novel features were seen in the R1 structure such as the simultaneous binding of two ATP molecules in the ATP-cone domain as well as a novel “β-hairpin hook” feature which spans the dimer interface of the R1 protein. The gene encoding the R2 protein contains a self-cleaving intein domain. We examined two constructs of this protein, one with the sequence of the intein removed at the DNA level and the wild-type construct. Both crystal structures were found to be identical, showing the efficient cleavage of the intein domain in the wild-type construct. The second part of this thesis concerns the NUDIX hydrolase NUDT15. The physiological function of NUDT15 is still unknown, however certain mutations in this gene are associated with thiopurine intolerance in patients. Thiopurines are chemotherapeutic drugs used in the treatment of diseases such as acute lymphoblastic leukemia, the most common type of childhood leukemia, and inflammatory bowel disease. Thiopurine drugs are converted by the cell to the active metabolite 6-thio-dGTP which can then act as a substrate for DNA polymerase. Incorporation of these anti-metabolites into DNA produces the desired cytotoxic effects. We show that NUDT15 breaks down the active metabolites of these drugs which leads to a lowered effective dose. The absence of a functioning NUDT15 protein in patients that have inactivating mutations in the gene coding for NUDT15 results in a drastically increased effective dose of these compounds. A normal dose of a thiopurine drug can lead to severe and possibly life-threatening complications in these patients. The role of NUDT15 in thiopurine metabolism is established by in vitro and cellular data as well as the X-ray crystal structure of NUDT15 in complex with 6-thio-GMP. Acyclovir and ganciclovir are two antiviral drugs whose mechanism of action is similar to that of thiopurines. These drugs are also metabolized to their tri-phosphorylated forms and are then preferentially incorporated into viral DNA. Here again, we use in vitro, cellular and structural data to show that NUDT15 breaks down the active metabolites of these drugs. Two separate and structurally distinct lines of potent inhibitors for NUDT15 were developed with support of crystallographic studies. We show that cells are sensitized to both thiopurine and antiviral treatments in the presence of these inhibitors. Binding of our inhibitors to NUDT15 provided substantial thermal stabilization. The stabilizing effect of inhibitor binding enabled us to solve structures of the four most clinically relevant NUDT15 variants, thus elucidating the structural basis for the thiopurine sensitivity phenotype.
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| 5. |
- Valerie, Nicholas C. K., et al.
(författare)
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NUDT15 Hydrolyzes 6-Thio-DeoxyGTP to Mediate the Anticancer Efficacy of 6-Thioguanine
- 2016
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Ingår i: Cancer Research. - 0008-5472 .- 1538-7445. ; 76:18, s. 5501-5511
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Tidskriftsartikel (refereegranskat)abstract
- Thiopurines are a standard treatment for childhood leukemia, but like all chemotherapeutics, their use is limited by inherent or acquired resistance in patients. Recently, the nucleoside diphosphate hydrolase NUDT15 has received attention on the basis of its ability to hydrolyze the thiopurine effector metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP, thereby limiting the efficacy of thiopurines. In particular, increasing evidence suggests an association between the NUDT15 missense variant, R139C, and thiopurine sensitivity. In this study, we elucidated the role of NUDT15 and NUDT15 R139C in thiopurine metabolism. In vitro and cellular results argued that 6-thio-dGTP and 6-thio-GTP are favored substrates for NUDT15, a finding supported by a crystallographic determination of NUDT15 in complex with 6-thio-GMP. We found that NUDT15 R139C mutation did not affect enzymatic activity but instead negatively influenced protein stability, likely due to a loss of supportive intramolecular bonds that caused rapid proteasomal degradation in cells. Mechanistic investigations in cells indicated that NUDT15 ablation potentiated induction of the DNA damage checkpoint and cancer cell death by 6-thioguanine. Taken together, our results defined how NUDT15 limits thiopurine efficacy and how genetic ablation via the R139C missense mutation confers sensitivity to thiopurine treatment in patients.
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| 6. |
- Zhang, Si Min, et al.
(författare)
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Development of a chemical probe against NUDT15
- 2020
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Ingår i: Nature Chemical Biology. - : Springer Science and Business Media LLC. - 1552-4450 .- 1552-4469. ; 16:10, s. 1120-1128
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Tidskriftsartikel (refereegranskat)abstract
- The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites, 6-thio-(d)GTP, thereby dictating the clinical response of this standard-of-care treatment for leukemia and inflammatory diseases. Nonetheless, its physiological roles remain elusive. Here, we sought to develop small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve NUDT15-dependent chemotherapeutics. Lead compound TH1760 demonstrated low-nanomolar biochemical potency through direct and specific binding into the NUDT15 catalytic pocket and engaged cellular NUDT15 in the low-micromolar range. We also employed thiopurine potentiation as a proxy functional readout and demonstrated that TH1760 sensitized cells to 6-thioguanine through enhanced accumulation of 6-thio-(d)GTP in nucleic acids. A biochemically validated, inactive structural analog, TH7285, confirmed that increased thiopurine toxicity takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.
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| 7. |
- Zhang, Si Min, et al.
(författare)
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NUDT15-mediated hydrolysis limits the efficacy of anti-HCMV drug ganciclovir
- 2021
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Ingår i: Cell Chemical Biology. - : Elsevier BV. - 2451-9456 .- 2451-9448. ; 28:12, s. 1693-1702
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Tidskriftsartikel (refereegranskat)abstract
- Ganciclovir (GCV) is the first-line therapy against human cytomegalovirus (HCMV), a widespread infection that is particularly dangerous for immunodeficient individuals. Closely resembling deoxyguanosine triphosphate, the tri-phosphorylated metabolite of GCV (GCV-TP) is preferentially incorporated by the viral DNA polymerase, thereby terminating chain extension and, eventually, viral replication. However, the treatment outcome of GCV varies greatly among individuals, therefore warranting better understanding of its metabolism. Here we show that NUDT15, a Nudix hydrolase known to metabolize thiopurine triphosphates, can similarly hydrolyze GCV-TP through biochemical studies and co-crystallization of the NUDT15/GCV-TP complex. More critically, GCV efficacy was potentiated in HCMV-infected cells following NUDT15 depletion by RNAi or inhibition by an in-house-developed, nanomolar NUDT15 inhibitor, TH8321, suggesting that pharmacological targeting of NUDT15 is a possible avenue to improve existing anti-HCMV regimens. Collectively, the data further implicate NUDT15 as a broad-spectrum metabolic regulator of nucleoside analog therapeutics, such as thiopurines and GCV.
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