Adsorption behavior of mercaptopurine and 6-thioguanine drugs on the B12N12, AlB11N12 and GaB11N12 nanoclusters, a comparative DFT study.

Lately, drug delivery systems established on nanostructures have become the most proficient to be studied. There are different studies suggested that the BN nanoclusters can be used as drug carriers and transport drugs in the target cell. Therefore, the interactions and adsorption behavior of Mercaptopurine (MC) and 6-thioguanine (TG) as anti-cancer drugs on the B12N12 (BN), AlB11N12 (AlBN) and GaB11N12 (GaBN) nanoclusters were studied by density functional theory (DFT) and quantum mechanics atoms in molecules (QMAIM) methods to find a new drug delivery system. Our results showed strong adsorption obtained in BN-MC/TG and AlBN-MC/TG complexes can be decomposed by the BN and AlBN indicating that these nanostructures are not suitable in drug efficiency of MC and TG drugs. Unlike the BN and AlBN nanoclusters, GaBN significantly makes the MC and TG drugs adsorption energetically favorable. The high solvation energy of GaBN when interacting with MC and TG drugs led it to applicability as nanocarriers for these drugs in the drug delivery systems. Furthermore, GaBN has a short recovery time for MC, and TG drugs desorption compared to BN and AlBN nanoclusters. It is predicted that the MC, and TG drugs over GaBN can be used as a drug delivery system.Communicated by Ramaswamy H. Sarma.

Design, synthesis and biological evaluation studies of novel small molecule ENPP1 inhibitors for cancer immunotherapy.

Ecto-nucleotide pyrophosphatase/phosphodiesterases 1 (ENPP1 or NPP1), is an attractive therapeutic target for various diseases, primarily cancer and mineralization disorders. The ecto-enzyme is located on the cell surface and has been implicated in the control of extracellular levels of nucleotide, nucleoside and (di) phosphate. Recently, it has emerged as a critical phosphodiesterase that hydrolyzes cyclic 2'3'- cGAMP, the endogenous ligand for STING (STimulator of INterferon Genes). STING plays an important role in innate immunity by activating type I interferon in response to cytosolic 2'3'-cGAMP. ENPP1 negatively regulates the STING pathway and hence its inhibition makes it an attractive therapeutic target for cancer immunotherapy. Herein, we describe the design, optimization and biological evaluation studies of a series of novel non-nucleotidic thioguanine based small molecule inhibitors of ENPP1. The lead compound 43 has shown good in vitro potency, stability in SGF/SIF/PBS, selectivity, ADME properties and pharmacokinetic profile and finally potent anti-tumor response in vivo. These compounds are a good starting point for the development of potentially effective cancer immunotherapy agents.

6-Thioguanine Inhibits Herpes Simplex Virus 1 Infection of Eyes.

Herpes simplex virus 1 (HSV-1) infects eye corneal tissues leading to herpetic stromal keratitis (HSK), which is one of the leading causes of blindness. Here in our study, we found that 6-thioguanine (6-TG), a once clinically approved medication for child acute myelogenous leukemia, inhibited multiple strains of HSV-1 infection in vitro and in vivo. 6-TG is more potent than acyclovir (ACV) and ganciclovir (GCV), with the 50% inhibitory concentration (IC50) of 6-TG at 0.104 µM with high stimulation index (SI) (SI = 6,475.48) compared to the IC50 of ACV at 1.253 µM and the IC50 of GCV at 1.257 µM. In addition, 6-TG at 500 µM topically applied to the eyes with HSV-1 infection significantly inhibits HSV-1 replication, alleviates virus-induced HSK pathogenesis, and improves eye conditions. More importantly, 6-TG is effective against ACV-resistant HSV-1 strains, including HSV-1/153 and HSV-1/blue. Knockdown of Rac1 with small interfering RNA (siRNA) negatively affected HSV-1 replication, suggesting that Rac1 facilitated HSV-1 replication. Following HSV-1 infection of human corneal epithelial cells (HCECs), endogenous Rac1 activity was upregulated by glutathione S-transferase (GST) pulldown assay. We further found that Rac1 was highly expressed in the corneal tissue of HSK patients compared to normal individuals. Mechanistic study showed that 6-TG inhibited HSV-1 replication by targeting Rac1 activity in HSV-1 infected cells, and the Rac1 is critical in the pathogenesis of HSK. Our results indicated that 6-TG is a promising therapeutic molecule for the treatment of HSK. IMPORTANCE We reported the discovery of 6-TG inhibition of HSV-1 infection and its inhibitory roles in HSK both in vitro and in vivo. 6-TG was shown to possess at least 10× more potent inhibitory activity against HSV-1 than ACV and GCV and, more importantly, inhibit ACV/GCV-resistant mutant viruses. Animal model studies showed that gel-formulated 6-TG topically applied to eyes locally infected with HSV-1 could significantly inhibit HSV-1 replication, alleviate virus-induced HSK pathogenesis, and improve eye conditions. Further study showed that HSV-1 infection upregulated Rac1 expression, and knockdown of Rac1 using siRNA markedly restricted HSV-1 replication, suggesting that Rac1 is required for HSV-1 replication. In addition, we also documented that Rac1 is highly expressed in corneal tissues from HSK patients, indicating that Rac1 is associated with HSK pathogenesis. In view of the high potency of 6-TG, low cytotoxicity, targeting a distinct therapeutic target, we suggest that 6-TG is a potential candidate for development as a therapeutic agent for HSK therapy.

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

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.

Thioguanine restoration through type I photosensitization-superoxide oxidation-glutathione reduction cycles.

UVA-induced deleterious effect of thiopurine prodrugs including azathioprine, 6-mercaptopurine and 6-thioguanine (6-TG) increases the risk of cancer development due to the incorporation of 6-TG in patients' DNA. The catalytic mechanism by which thiobases act as a sustained oxidant producer has yet to be explored, especially through the Type I electron transfer pathway that produces superoxide radicals (O2˙-). Under Fenton-like conditions O2˙- radicals convert to extremely reactive hydroxyl radicals (˙OH), thus carrying even higher risk of biological damage than that induced by the well-studied type II reaction. By monitoring 6-TG/UVA-induced photochemistry in mass spectra and superoxide radicals (O2˙-) via nitro blue tetrazolium (NBT) reduction, this work provides two new findings: (1) in the presence of reduced glutathione (GSH), the production of O2˙-via the type I reaction is enhanced 10-fold. 6-TG thiyl radicals are identified as the primary intermediate formed in the reaction of 6-TG with O2˙-. The restoration of 6-TG and concurrent generation of O2˙- occur via a 3-step-cycle: 6-TG type I photosensitization, O2˙- oxidation and GSH reduction. (2) In the absence of GSH, 6-TG thiyl radicals undergo oxygen addition and sulfur dioxide removal to form carbon radicals (C6) which further convert to thioether by reacting with 6-TG molecules. These findings help explain not only thiol-regulation in a biological system but chemoprevention of cancer.

Disulfiram and 6-Thioguanine synergistically inhibit the enzymatic activities of USP2 and USP21.

Disulfiram is a promising repurposed drug that, combining with radiation and chemotherapy, exhibits effective anticancer activities in several preclinical models. The cellular metabolites of disulfiram have been established, however, the intracellular targets of disulfiram remain largely unexplored. We have previously reported that disulfiram suppresses the coronaviral papain-like proteases through attacking their zinc-finger domains, suggesting an inhibitory function potentially on other proteases with similar catalytic structures. Ubiquitin-specific proteases (USPs) share a highly-conserved zinc-finger subdomain that structurally similar to the papain-like proteases and are attractive anticancer targets as upregulated USPs levels are found in a variety of tumors. Here, we report that disulfiram functions as a competitive inhibitor for both USP2 and USP21, two tumor-related deubiquitinases. In addition, we also observed a synergistic inhibition of USP2 and USP21 by disulfiram and 6-Thioguanine (6TG), a clinical drug for acute myeloid leukemia. Kinetic analyses revealed that both drugs exhibited a slow-binding mechanism, moderate inhibitory parameters, and a synergistically inhibitory effect on USP2 and USP21, suggesting the potential combinatory use of these two drugs for USPs-related tumors. Taken together, our study provides biochemical evidence for repurposing disulfiram and 6TG as a combinatory treatment in clinical applications.

Elucidating the binding mechanism of thione-containing mercaptopurine and thioguanine drugs to small gold clusters.

Density functional theory methods were employed to clarify the adsorption/desorption behaviors of the thione-containing mercaptopurine and thioguanine drugs on the gold surface using both small Au6 and Au8 clusters as model reactants. Structural features, thermodynamic parameters, bonding characteristics, and electronic properties of the resulting complexes were investigated using the Perdew-Burke-Ernzerhof (PBE) and LC-BLYP functionals along with correlation-consistent basis sets, namely cc-pVDZ-PP for gold and cc-pVTZ for non-metals. Computed results show that the drug molecules tend to anchor on the gold cluster at the S atom with binding energies around -34 to -40 kcal/mol (in vacuum) and - 28 to -32 kcal/mol (in aqueous solution). As compared to Au8 , Au6 undergoes a shorter recovery time and a larger change of energy gap that could be converted to an electrical signal for selective detection of the drugs. Furthermore, interactions between the drugs and gold clusters are reversible processes and a drug release mechanism was also proposed. Accordingly, the drugs are able to separate from the gold surface due to either a slight change of pH in tumor cells or the presence of cysteine residues in protein matrices.

Anticancer efficacy of 6-thioguanine loaded chitosan nanoparticles with or without curcumin.

6-Thioguanine encapsulated chitosan nanoparticles (6-TG-CNPs) has formulated by the ionic-gelation method. Morphologically, the 6-TG-CNPs were spherical and showed mean size, PDI, zeta potential, and entrapment efficiency of 261.63 ± 6.01 nm, 0.34 ± 0.10, +15.97 ± 0.46 mV and 44.27%, respectively. The IR spectra confirmed the 6-TG complex with chitosan. The in vitro drug release profile of 6-TG-CNPs revealed an increase in sustained-release (91.40 ± 1.08% at 48 h) at pH 4.8 compared to less sustained-release (73.96 ± 1.12% at 48 h) at pH 7.4. The MTT assay was conducted on MCF-7 and PA-1 cell lines at 48 h incubation to determine % cell viability. The IC50 values of 6-TG, 6-TG-CNPs, and curcumin for MCF-7 were 23.09, 17.82, and 15.73 µM, respectively. Likewise, IC50 values of 6-TG, 6-TG-CNPs, and curcumin for PA-1 were 5.81, 3.92, and 12.89 µM, respectively. A combination of 6-TG-CNPs (IC25) with curcumin (IC25) on PA-1 and MCF-7 showed % cell viability of 43.67 ± 0.02 and 49.77 ± 0.05, respectively. The in vitro cytotoxicity potential in terms of % cell viability, early apoptosis, G2/M phase arrest, and DNA demethylating activity of 6-TG-CNPs alone and combination with curcumin proved to be more effective than that of 6-TG on PA-1 cells.

Carbon and boron nanotubes as a template material for adsorption of 6-Thioguanine chemotherapeutic: a molecular dynamics and density functional approach.

The interaction of 6-Thioguanine molecule, an antitumor drug with carbon nanotube and boron nitride nanotube (BNNT) is investigated using molecular dynamics simulations. Based on the obtained results, the strongest negative van der Waals interaction is found between 6-TG and BNNT among the studied nanotubes, which indicated BNNT is a better nanocarrier of the 6-TG drug than CNT within biological systems. Moreover, the adsorption and electronic properties of the 6-Thioguanine interacted with boron-nitride nanotube has been studied within the framework of density functional theory calculations. The negative binding energy values denote that there is the favorable interaction between 6-TG drug and BNNT at the studied 6-TG/BNNT complexes. Also, the amounts of the binding energies indicated that the 6-Thioguanine molecule physically interacts with the surface of BNNT. The values of electron densities and their Laplacian have been analyzed using the Bader's theory of atoms in molecules to characterize the nature of the intermolecular interactions through the topological parameters. We hope that the results of this work may provide useful information about the nature of the nanotube-drug molecule interactions and highlight the ability of these materials to be used as an adsorbent enhancing delivery of drug to cancer cells. Communicated by Ramaswamy H. Sarma.

The adduct formation between the thioguanine-polyamine ligands and DNA with the AP site under UVA irradiated and non-irradiated conditions.

The AP sites are representative of DNA damage and known as an intermediate in the base excision repair (BER) pathway which is involved in the repair of damaged nucleobases by reactive oxygen species, UVA irradiation, and DNA alkylating agents. Therefore, it is expected that the inhibition or modulation of the AP site repair pathway may be a new type of anticancer drug. In this study, we investigated the effects of the thioguanine-polyamine ligands (SG-ligands) on the affinity and the reactivity for the AP site under UVA irradiated and non-irradiated conditions. The SG-ligands have a photo-reactivity with the A-F-C sequence where F represents a tetrahydrofuran AP site analogue. Interestingly, the SG-ligands promoted the ß-elimination of the AP site followed by the formation of a covalent bond with the ß-eliminated fragment without UVA irradiation.

Effective imprinting of an anticancer drug, 6-thioguanine, via mussel-inspired self-polymerization of dopamine over reduced graphene oxide.

Apart from being a vital catecholamine molecule responsible for the proper functioning of the central nervous system (CNS), hormonal and renal systems, dopamine (DA) has also been increasingly employed as a functional monomer in the fabrication of surface molecular imprinting polymers (MIPs) for valuable analytes. Herein, we demonstrate the effective imprinting of 6-thioguanine (6-TG), an anticancer drug, via mussel-inspired self-polymerization of dopamine conducted in a weakly alkaline solution over reduced graphene oxide (rGO). The polymerization of 6-TG resulted into a thin polydopamine (PDA) film of 8.4 nm thickness. Removal of 6-TG molecules from this imprinted PDA film created numerous cavities of 6-TG. The electrochemical investigation of MIP electrodes found an excellent electrocatalytic activity toward 6-TG with a significant decrease in the over-potential as compared to that of the bare glassy carbon electrode (GCE). This can be attributed to the graphene's distinct physical and chemical features such as subtle electronic characteristics, an attractive π-π interaction as well as the strong adsorptive capability of MIP films. This electrochemical sensor displayed a high selectivity owing to the specific imprinted cavities for adrenaline and worked well over a wide linear concentration range of adrenaline between 0.0015 and 50 µM with a detection limit (LOD) of 0.25 nM and good reproducibility and stability. Our system depicts excellent recoveries from 97.0% to 100.6% for two different samples of urine and thioguanine drugs. These results show the great potential of our system with multiple advantages, including convenient fabrication and optimization, high sensitivity and selectivity, high reproducibility and stability, and cost-effectiveness.

Thioguanine-based DENV-2 NS2B/NS3 protease inhibitors: Virtual screening, synthesis, biological evaluation and molecular modelling.

Dengue virus Type 2 (DENV-2) is predominant serotype causing major dengue epidemics. There are a number of studies carried out to find its effective antiviral, however to date, there is still no molecule either from peptide or small molecules released as a drug. The present study aims to identify small molecules inhibitor from National Cancer Institute database through virtual screening. One of the hits, D0713 (IC50 = 62 µM) bearing thioguanine scaffold was derivatised into 21 compounds and evaluated for DENV-2 NS2B/NS3 protease inhibitory activity. Compounds 18 and 21 demonstrated the most potent activity with IC50 of 0.38 µM and 16 µM, respectively. Molecular dynamics and MM/PBSA free energy of binding calculation were conducted to study the interaction mechanism of these compounds with the protease. The free energy of binding of 18 calculated by MM/PBSA is -16.10 kcal/mol compared to the known inhibitor, panduratin A (-11.27 kcal/mol), which corroborates well with the experimental observation. Results from molecular dynamics simulations also showed that both 18 and 21 bind in the active site and stabilised by the formation of hydrogen bonds with Asn174.

A sensitive colorimetric probe for detection of 6-thioguanine based on its protective effect on the silver nanoprisms.

In this work a non-aggregated colorimetric probe for detection of chemotherapeutic drug, 6-thioguanine (6-TG), is introduced. It is based on the protective effect of 6-TG on silver nanoprisms (AgNPRs) against the iodide-induced etching reaction. Iodide ions can attack the corners of AgNPRs and etch them, leading to the morphological transition from nanoprisms to nanodiscs. As a consequence, the solution color changes from blue to pink. However, in the presence of 6-TG, due to its protective effect on the corners of AgNPRs, I- ions cannot etch the prisms and the blue color of solution remains unchanged. Using this effect, selective sensor was designed for detection of 6-TG in the range of 2.5-500 µg L-1, with a detection limit of 0.95 µg L-1. Since with varying the concentration of 6-TG in this range, the color variation from pink to blue can be easily observed, the designed sensing scheme can be used as a colorimetric probe. The method was used for analysis of human plasma samples.

Assessment of the chitosan-functionalized graphene oxide as a carrier for loading thioguanine, an antitumor drug and effect of urea on adsorption process: Combination of DFT computational and molecular dynamics simulation studies.

In this study, the interaction thioguanine (TG) anticancer drug with the functionalized graphene oxide (GO) nanosheet surface is theoretically studied in both gas phase and separately in physiological media using the density functional theory (DFT) calculations. DFT calculations indicated the adsorption and solvation energies are negative for f-GONS/TG complexes which propose the adsorption process of TG molecule onto the f-GONS surface is possible from the energetic viewpoint. QTAIM calculations confirm the nature of partially covalent-partially electrostatic between drug and nanosheet. These results are sorely relevant that an approach for loading of TG molecule is the chemical modification of GO using covalent functionalization which can serve as a nanocarrier to load drug molecules. Moreover, to understand the effect of urea on the nature of the interaction between TG and f-GONS, molecular dynamics (MD) simulation was employed. The results indicated that in the presence of urea the adsorption process gets affected and leads to instability of system, while the affinity of the TG for adsorption onto GO surface is increased in pure water. Communicated by Ramaswamy H. Sarma.

Heavy-Atom-Substituted Nucleobases in Photodynamic Applications: Substitution of Sulfur with Selenium in 6-Thioguanine Induces a Remarkable Increase in the Rate of Triplet Decay in 6-Selenoguanine.

Sulfur substitution of carbonyl oxygen atoms of DNA/RNA nucleobases promotes ultrafast intersystem crossing and near-unity triplet yields that are being used for photodynamic therapy and structural-biology applications. Replacement of sulfur with selenium or tellurium should significantly red-shift the absorption spectra of the nucleobases without sacrificing the high triplet yields. Consequently, selenium/tellurium-substituted nucleobases are thought to facilitate treatment of deeper tissue carcinomas relative to the sulfur-substituted analogues, but their photodynamics are yet unexplored. In this contribution, the photochemical relaxation mechanism of 6-selenoguanine is elucidated and compared to that of the 6-thioguanine prodrug. Selenium substitution leads to a remarkable enhancement of the intersystem crossing lifetime both to and from the triplet manifold, resulting in an efficiently populated, yet short-lived triplet state. Surprisingly, the rate of triplet decay in 6-selenoguanine increases by 835-fold compared to that in 6-thioguanine. This appears to be an extreme manifestation of the classical heavy-atom effect in organic photochemistry, which challenges conventional wisdom.

A sensitive turn on fluorescent probe for detection of biothiols using MnO2@carbon dots nanocomposites.

Presently, the combination of carbon quantum dots (CQDs) and metal oxide nanostructures in one frame are being considered for the sensing of purine compounds. In this work, a combined system of CQDs and MnO2 nanostructures was used for the detection of anticancer drugs, 6-Thioguanine (6-TG) and 6-Mercaptopurine (6-MP). The CQDs were synthesized through microwave synthesizer and the MnO2 nanostructures (nanoflowers and nanosheets) were synthesized using facile hydrothermal technique. The CQDs exhibited excellent fluorescence emission at 420nm when excited at 320nm wavelength. By combining CQDs and MnO2 nanostructures, quenching of fluorescence was observed which was attributed to fluorescence resonance energy transfer (FRET) mechanism, where CQDs act as electron donor and MnO2 act as acceptor. This fluorescence quenching behaviour disappeared on the addition of 6-TG and 6-MP due to the formation of Mn-S bond. The detection limit for 6-TG (0.015µM) and 6-MP (0.014µM) was achieved with the linear range of concentration (0-50µM) using both MnO2 nanoflowers and nanosheets. Moreover, the as-prepared fluorescence-sensing technique was successfully employed for the detection of bio-thiol group in enapril drug. Thus a facile, cost-effective and benign chemistry approach for biomolecule detection was designed.

Thiopurine Drugs Repositioned as Tyrosinase Inhibitors.

Drug repositioning is the application of the existing drugs to new uses and has the potential to reduce the time and cost required for the typical drug discovery process. In this study, we repositioned thiopurine drugs used for the treatment of acute leukaemia as new tyrosinase inhibitors. Tyrosinase catalyses two successive oxidations in melanin biosynthesis: the conversions of tyrosine to dihydroxyphenylalanine (DOPA) and DOPA to dopaquinone. Continuous efforts are underway to discover small molecule inhibitors of tyrosinase for therapeutic and cosmetic purposes. Structure-based virtual screening predicted inhibitor candidates from the US Food and Drug Administration (FDA)-approved drugs. Enzyme assays confirmed the thiopurine leukaemia drug, thioguanine, as a tyrosinase inhibitor with the inhibitory constant of 52 µM. Two other thiopurine drugs, mercaptopurine and azathioprine, were also evaluated for their tyrosinase inhibition; mercaptopurine caused stronger inhibition than thioguanine did, whereas azathioprine was a poor inhibitor. The inhibitory constant of mercaptopurine (16 µM) was comparable to that of the well-known inhibitor kojic acid (13 µM). The cell-based assay using B16F10 melanoma cells confirmed that the compounds inhibit mammalian tyrosinase. Particularly, 50 µM thioguanine reduced the melanin content by 57%, without apparent cytotoxicity. Cheminformatics showed that the thiopurine drugs shared little chemical similarity with the known tyrosinase inhibitors.

Construction of 6-thioguanine and 6-mercaptopurine carriers based on ßcyclodextrins and gold nanoparticles.

As a novel strategy to overcome some of the therapeutic disadvantages of 6-thioguanine (TG) and 6-mercaptopurine (MP), we propose the inclusion of these drugs in ßcyclodextrin (ßCD) to form the complexes ßCD-TG and ßCD-MP, followed by subsequent interaction with gold nanoparticles (AuNPs), generating the ternary systems: ßCD-TG-AuNPs and ßCD-MP-AuNPs. This modification increased their solubility and improved their stability, betting by a site-specific transport due to their nanometric dimensions, among other advantages. The formation of the complexes was confirmed using powder X-ray diffraction, thermogravimetric analysis and one and two-dimensional NMR. A theoretical study using DFT and molecular modelling was conducted to obtain the more stable tautomeric species of TG and MP in solution and confirm the proposed inclusion geometries. The deposition of AuNPs onto ßCD-TG and ßCD-MP via sputtering was confirmed by UV-vis spectroscopy. Subsequently, the ternary systems were characterized by TEM, FE-SEM and EDX to directly observe the deposited AuNPs and evaluate their sizes, size dispersion, and composition. Finally, the in vitro permeability of the ternary systems was studied using parallel artificial membrane permeability assay (PAMPA).

DNA-thioguanine nucleotide concentration and relapse-free survival during maintenance therapy of childhood acute lymphoblastic leukaemia (NOPHO ALL2008): a prospective substudy of a phase 3 trial.

BACKGROUND: Adjustment of mercaptopurine and methotrexate maintenance therapy of acute lymphoblastic leukaemia by leucocyte count is confounded by natural variations. Cytotoxicity is primarily mediated by DNA-incorporated thioguanine nucleotides (DNA-TGN). The aim of this study was to establish whether DNA-TGN concentrations in blood leucocytes during maintenance therapy are associated with relapse-free survival. METHODS: In this substudy of the NOPHO ALL2008 phase 3 trial done in 23 hospitals in seven European countries (Denmark, Estonia, Finland, Iceland, Lithuania, Norway, and Sweden), we analysed data from centralised and blinded analyses of 6-mercaptopurine and methotrexate metabolites in blood samples from patients with non-high-risk childhood acute lymphoblastic leukaemia. Eligible patients were aged 1·0-17·9 years; had been diagnosed with non-high-risk precursor B-cell or T-cell leukaemia; had been treated according to the Nordic Society of Pediatric Hematology and Oncology ALL2008 protocol; and had reached maintenance therapy in first remission. Maintenance therapy was (mercaptopurine 75 mg/m2 once per day and methotrexate 20 mg/m2 once per week, targeted to a leucocyte count of 1·5-3·0 × 109 cells per L). We measured DNA-TGN and erythrocyte concentrations of TGN nucleotides, methylated mercaptopurine metabolites, and methotrexate polyglutamates. The primary objective was the association of DNA-TGN concentrations and 6-mercaptopurine and methotrexate metabolites with relapse-free survival. The secondary endpoint was the assessment of DNA-TGN concentration and 6-mercaptopurine and methotrexate metabolites during maintenance therapy phase 2. FINDINGS: Between Nov 26, 2008 and June 14, 2016, 1509 patients from the NOPHO ALL2008 study were assessed for eligibility in the DNA-TGN substudy, of which 918 (89%) of 1026 eligible patients had at least one DNA-TGN measurement and were included in the analyses. Median follow-up was 4·6 years (IQR 3·1-6·1). Relapse-free survival was significantly associated with DNA-TGN concentration (adjusted hazard ratio 0·81 per 100 fmol/µg DNA increase, 95% CI 0·67-0·98; p=0·029). In patients with at least five blood samples, erythrocyte concentrations of TGN, methylated mercaptopurine metabolites, and methotrexate polyglutamates were associated with DNA-TGN concentration (all p<0·0001). INTERPRETATION: Our results suggest the need for intervention trials to identify clinically applicable strategies for individualised drug dosing to increase DNA-TGN concentration, and randomised studies to investigate whether such strategies improve cure rates compared with current dose adjustments based on white blood cell counts. FUNDING: Danish Cancer Society, Childhood Cancer Foundation (Denmark), Childhood Cancer Foundation (Sweden), Nordic Cancer Union, Otto Christensen Foundation, University Hospital Rigshospitalet, and Novo Nordic Foundation.

Efficient Thymidine-Selective DNA Interstrand Photo-activated Crosslinking by the 6-Thioguanine Connected via an Ethylene-Linker to the 2'-Deoxyribose Unit.

Cross-linking is a widely-used technology in the studies of DNA, RNA and their complexes with proteins. Intrinsically active alkylating moieties and photo-activated agents are chemically or enzymatically incorporated into nucleic acids. Thionucleobases resemble the corresponding natural bases, and form cross-links by UVA irradiation. They form cross-links only with a site in close contact, thereby allowing identification of the contacts within the nucleic acids and/or between the nucleic acids and proteins in complex nucleoprotein assemblies. On the other hand, the thionucleobase forms a cross-link less efficiently for the reaction with the opposite natural base in the DNA duplex. In this study, 6-thioguanine was connected to 2'-deoxyribose through an ethylene linker at the 1'-position (Et-thioG). The linker was expected to bring the 6-thio group close to the nucleobase in the opposite strand. In a duplex in which the 2'-deoxy-6-thioguanosine (6-thio-dG) did not form a crosslink, Et-thioG efficiently formed crosslink with a high selectivity for T by UVA irradiation, but with a much lower efficiency for dA, dG, dC, 5-methyl-dC or dU. Interestingly, the yield of the photo-crosslinked product with dT was effectively improved in the presence of dithiothreitol or sodium hydrosulfide (NaSH) at a low UVA irradiation dose. The efficient and selective cross-link formation at a low UVA dose may be beneficial for the biological application of Et-thioG.