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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 the first small-molecule NUDT15 inhibitors to elucidate its biological functions, and potentially for improving 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 further employed thiopurine potentiation as a proxy functional read-out 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 is via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.

Herold, Nikolas et al., The cytostatic deoxycytidine analog cytarabine (ara-C) is the most active agent available against acute myelogenous leukemia (AML). Together with anthracyclines, ara-C forms the backbone of AML treatment for children and adults. In AML, both the cytotoxicity of ara-C in vitro and the clinical response to ara-C therapy are correlated with the ability of AML blasts to accumulate the active metabolite ara-C triphosphate (ara-CTP), which causes DNA damage through perturbation of DNA synthesis. Differences in expression levels of known transporters or metabolic enzymes relevant to ara-C only partially account for patient-specific differential ara-CTP accumulation in AML blasts and response to ara-C treatment. Here we demonstrate that the deoxynucleoside triphosphate (dNTP) triphosphohydrolase SAM domain and HD domain 1 (SAMHD1) promotes the detoxification of intracellular ara-CTP pools. Recombinant SAMHD1 exhibited ara-CTPase activity in vitro, and cells in which SAMHD1 expression was transiently reduced by treatment with the simian immunodeficiency virus (SIV) protein Vpx were dramatically more sensitive to ara-C-induced cytotoxicity. CRISPR-Cas9-mediated disruption of the gene encoding SAMHD1 sensitized cells to ara-C, and this sensitivity could be abrogated by ectopic expression of wild-type (WT), but not dNTPase-deficient, SAMHD1. Mouse models of AML lacking SAMHD1 were hypersensitive to ara-C, and treatment ex vivo with Vpx sensitized primary patient-derived AML blasts to ara-C. Finally, we identified SAMHD1 as a risk factor in cohorts of both pediatric and adult patients with de novo AML who received ara-C treatment. Thus, SAMHD1 expression levels dictate patient sensitivity to ara-C, providing proof-of-concept that the targeting of SAMHD1 by Vpx could be an attractive therapeutic strategy for potentiating ara-C efficacy in hematological malignancies., This work was supported by grants from the Swedish Children’s Cancer Foundation (TJ2016-0040 (to N.H.); 2015-0005 (to J.-I.H.); PR2015-0009 (to D.G.); and PR2013-0002 and PR2014-0048 (both to T.H.)), the Swedish Cancer Society (CAN 2016/837 to J.W.; CAN 2014/814 to S.L.; CAN 2015/768 to D.G.; CAN 2013/396 to J.-I.H.; and CAN 2012/770 and CAN 2015/255 to T.H.), the Swedish Research Council (2014-1839 to M.U.; 2015-02498 to S.L.; 2012-2037 to D.G.; and 2012-5935 and 2013-3791 to T.H.), Radiumhemmet’s Research Foundations (154242 to G.R. and 144063 to D.G.), the Knut and Alice Wallenberg Foundation (KAW2014.0273 to T.H.), the Swedish Pain Relief Foundation (SSF/01-05 to T.H.), the Torsten and Ragnar Söderberg Foundation (to T.H.), the David and Astrid Hagelén Foundation (C24702193 to B.D.G.P.) and the Stockholm County Council (ALF project) (20150353 to S.L. and 20150016 to J.-I.H.). This work was supported by the German Research Foundation (DFG) (SCHA1950/1-1 to T.S.) and partially through the Federal Ministry of Education and Research of Germany (BMBF)–supported Immunoquant project (0316170 C to T.S.) and HIVERA: EURECA project (01KI1307B to T.S.). S.G.R. is supported by an EMBO Long-Term Fellowship (ALTF-605-2014). Chemical Biology Consortium Sweden is funded by the Swedish Research Council, Science for Life Laboratories and Karolinska Institutet (829-2009-6241 to H.A. and T.L.).

Intracerebroventricular injection of angiotensin IV, a ligand of insulin-regulated aminopeptidase (IRAP), has been shown to improve cognitive functions in several animal models. Consequently, IRAP is considered a potential target for treatment of cognitive disorders. To identify nonpeptidic IRAP inhibitors, we adapted an established enzymatic assay based on membrane preparations from Chinese hamster ovary cells and a synthetic peptide-like substrate for high-throughput screening purposes. The 384-well microplate-based absorbance assay was used to screen a diverse set of 10,500 compounds for their inhibitory capacity of IRAP. The assay performance was robust with Z'-values ranging from 0.81 to 0.91, and the screen resulted in 23 compounds that displayed greater than 60% inhibition at a compound concentration of 10 μM. After hit confirmation experiments, purity analysis, and promiscuity investigations, three structurally different compounds were considered particularly interesting as starting points for the development of small-molecule-based IRAP inhibitors. After resynthesis, all three compounds confirmed low μM activity and were shown to be rapidly reversible. Additional characterization included activity in a fluorescence-based orthogonal assay and in the presence of a nonionic detergent and a reducing agent, respectively. Importantly, the characterized compounds also showed inhibition of the human ortholog, prompting our further interest in these novel IRAP inhibitors.

With a diverse network of substrates, NUDIX hydrolases have emerged as a key family of nucleotide-metabolizing enzymes. NUDT5 (also called NUDIX5) has been implicated in ADP-ribose and 8-oxo-guanine metabolism and was recently identified as a rheostat of hormone-dependent gene regulation and proliferation in breast cancer cells. Here, we further elucidate the physiological relevance of known NUDT5 substrates and underscore the biological requirement for NUDT5 in gene regulation and proliferation of breast cancer cells. We confirm the involvement of NUDT5 in ADP-ribose metabolism and dissociate a relationship to oxidized nucleotide sanitation. Furthermore, we identify potent NUDT5 inhibitors, which are optimized to promote maximal NUDT5 cellular target engagement by CETSA. Lead compound, TH5427, blocks progestin-dependent, PAR-derived nuclear ATP synthesis and subsequent chromatin remodeling, gene regulation and proliferation in breast cancer cells. We herein present TH5427 as a promising, targeted inhibitor that can be used to further study NUDT5 activity and ADP-ribose metabolism., NUDIX hydrolases are an important family of nucleotide-metabolizing enzymes. Here, the authors identify potent, small molecule inhibitors of NUDT5, which is implicated in ADP-ribose and 8-oxo-guanine metabolism, and confirm its role in gene regulation and proliferation in breast cancer cells.

Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery., Drugs therapeutic efficacy relies on their capability of binding the relevant targets in a physiological environment, which has so far been hard to measure. Here, the authors present a compound library screen based on a target engagement assay that reports on protein stability upon ligands binding in cell.

A safe and efficient therapy for patients with the bone marrow failure syndrome Diamond-Blackfan Anemia (DBA) is urgently needed. To identify novel drug candidates for DBA, a small molecule screen was performed using c-Kit positive E14.5 fetal liver cells from a DBA mouse model in which the DBA phenotype is induced upon doxycycline (DOX)-inducible silencing of Rps19, the most frequently mutated gene in DBA (Jaako et al. PMID: 21989989). Test compounds were added 24 hours after cell seeding and DOX addition. After four days the number of live metabolically active cells in each well was estimated based on quantitation of intracellular ATP. Fifteen commercial annotated small-molecule libraries (3 800 molecules) and 10 500 selected compounds from a diverse compound library were screened. Between the screens we identified 20 molecules that reproducibly increased proliferation of rps19-deficient erythroid progenitors 4-8 fold in a concentration-dependent manner. Hits from annotated libraries included inhibitors of TGFb receptor, DYRK and Casein kinases. The most potent hits however were compounds in a series of thienopyridines, with an unknown target profile, but with a core structure suggesting kinase inhibition activity. Database searches revealed that the structure-activity relationships (SAR) of 12 active and 10 inactive analogues were similar to that of a series of thienopyridines previously reported as bone anabolic agents by unknown mechanism (Saito et al. PMID: 23453217). The most potent analogues described by Saito et al. as bone anabolic agents were synthesized, and the compounds rescue also RPS19-deficient erythroid cell proliferation in a potent manner (EC50= 20-50 nM). In an attempt to identify the molecular target of these compounds, six actives including thienopyridines 15k and 15w were subjected to kinase profiling against 468 kinases (DiscoverX). Three of the compounds targeted cyclin- dependent kinases CDK8 and its paralog CDK19, and in particular the most potent molecule (15w) is a highly selective CDK8 inhibitor. To confirm whether CDK8 inhibition underlies the rescue of the DBA phenotype, structurally unrelated and potent CDK8 inhibitors including CCT-251545, Senexin A, Senexin B, and Sel120-34A were also evaluated. All tested CDK8-inhibitors rescue proliferation and erythroid maturation of c-kit+ cells from the DBA mouse in a concentration-dependent manner. To further investigate the potential of CDK8 as a therapeutic target in DBA several CDK8 inhibitors were evaluated in erythroid cultures of primary DBA patient cells. CDK8 inhibitors 15w, Senexin B and Sel120-34A increase erythroid progenitor proliferation 5-10 fold of CD34+ peripheral blood cells from three DBA patients (RPS19, RPS26, RPL35a mutations), and increase the fraction of transferrin receptor (CD71) and glycophorin A positive cells in culture. Healthy CD34+ peripheral blood treated with CDK8 inhibitors show no increase in proliferation. Finally, we show that bone marrow failure and anemia in the DBA mouse model is partially rescued (RBC and Hemoglobin levels, p Disclosures Rzymski: Selvita S.A.: Employment, Equity Ownership. Johansson:LU Holding: Patents & Royalties. Lundbäck:LU Holding: Patents & Royalties. Mazan:Selvita S.A.: Employment. Majewska:Selvita S.A.: Employment. Brzózka:Selvita S.A.: Employment, Equity Ownership, Membership on an entity's Board of Directors or advisory committees. Flygare:LU Holding: Patents & Royalties: Patent.

In Mycobacterium tuberculosis the sulfate activating complex provides a key branching point in sulfate assimilation. The complex consists of two polypeptide chains, CysD and CysN. CysD is an ATP sulfurylase that, with the energy provided by the GTPase activity of CysN, forms adenosine-5’-phosphosulfate (APS) which can then enter the reductive branch of sulfate assimilation leading to the biosynthesis of cysteine. The CysN polypeptide chain also contains an APS kinase domain (CysC) that phosphorylates APS leading to 3’-phosphoadenosine-5’-phosphosulfate, the sulfate donor in the synthesis of sulfolipids. We have determined the crystal structures of CysC from M. tuberculosis as a binary complex with ADP, and as ternary complexes with ADP and APS and the ATP mimic AMP-PNP and APS, respectively, to resolutions of 1.5 Å, 2.1 Å and 1.7 Å, respectively. CysC shows the typical APS kinase fold, and the structures provide comprehensive views of the catalytic machinery, conserved in this enzyme family. Comparison to the structure of the human homolog show highly conserved APS and ATP binding sites, questioning the feasibility of the design of specific inhibitors of mycobacterial CysC. Residue Cys556 is part of the flexible lid region that closes off the active site upon substrate binding. Mutational analysis revealed this residue as one of the determinants controlling lid closure and hence binding of the nucleotide substrate.

Nature 508, 215–221 (2014); doi:10.1038/nature13181 In this Article, the structure of compound TH650 (4) in Fig. 4a was drawn incorrectly; the correct structure is shown as Fig. 1 to this Corrigendum. Preparative, spectroscopic and biological data associated with this compound are as reported in theArticle, and the error does not influence any of the reported data or interpretations.

Thermal shift assays are used to study thermal stabilization of proteins upon ligand binding. Such assays have been used extensively on purified proteins in the drug discovery industry and in academia to detect interactions. Recently, we published a proof-of-principle study describing the implementation of thermal shift assays in a cellular format, which we call the cellular thermal shift assay (CETSA). The method allows studies of target engagement of drug candidates in a cellular context, herein exemplified with experimental data on the human kinases p38α and ERK1/2. The assay involves treatment of cells with a compound of interest, heating to denature and precipitate proteins, cell lysis, and the separation of cell debris and aggregates from the soluble protein fraction. Whereas unbound proteins denature and precipitate at elevated temperatures, ligand-bound proteins remain in solution. We describe two procedures for detecting the stabilized protein in the soluble fraction of the samples. One approach involves sample workup and detection using quantitative western blotting, whereas the second is performed directly in solution and relies on the induced proximity of two target-directed antibodies upon binding to soluble protein. The latter protocol has been optimized to allow an increased throughput, as potential applications require large numbers of samples. Both approaches can be completed in a day.

The TWEAK-Fn14 pathway is upregulated in models of inflammation, autoimmune diseases, and cancer. Both TWEAK and Fn14 show increased expression also in the CNS in response to different stimuli, particularly astrocytes, microglia, and neurons, leading to activation of NF-κB and release of proinflammatory cytokines. Although neutralizing antibodies against these proteins have been shown to have therapeutic efficacy in animal models of inflammation, no small-molecule therapeutics are yet available. Here, we describe the development of a novel homogeneous time-resolved fluorescence (HTRF)-based screening assay together with several counterassays for the identification of small-molecule inhibitors of this protein-protein interaction. Recombinant HIS-TWEAK and Fn14-Fc proteins as well as FLAG-TWEAK and Fn14-FLAG proteins and an anti-Fn14 antibody were used to establish and validate these assays and to screen a library of 60 000 compounds. Two HTRF counterassays with unrelated proteins in the same assay format, an antiaggregation assay and a redox assay, were applied to filter out potential false-positive compounds. The novel assay and associated screening cascade should be useful for the discovery of small-molecule inhibitors of the TWEAK-Fn14 protein interaction.

Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bind in the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

Diamond-Blackfan anemia (DBA) is a bone marrow failure syndrome associated with ribosomal protein (RP) deficiency caused by mutations in RP genes. 25% of patients carry mutations in the RPS19 gene. A salient feature of DBA is proliferative arrest of erythroid progenitors. It is still not clear how defects in RPs, which are essential to all cell types, affect the erythroid compartment more severely than the others. Today, DBA patients are treated with glucocorticoids and/or blood transfusions, often for long periods, causing severe adverse effects. We hypothesize that novel drugs with more disease-specific therapeutic mechanisms can be developed and that such drugs will be superior to current therapies. Towards this aim we have developed a method for screening chemical libraries to identify novel drug candidates. Use of DBA patient cells is not feasible as their limited availability severely limits the size of the applied screening library. Immortalized cell lines are also less suitable since the disease mechanism involves TP53 activation and most cell lines have perturbations in the TP53 pathway. Hence, primary c-Kit+ E14.5-15.5 fetal liver erythroid progenitor cells from a mouse model of DBA with doxycycline inducible expression of rps19-shRNA were used (Jaako et. al. Blood, 2011). The DBA phenotype is induced by adding doxycycline to the culture medium which reduces erythroid proliferation >80%. Rescue of this proliferation defect is a simple and relevant readout for large-scale screening. Assay development in 96 well microtiter plates allowed rational liquid handling. Firstly, the proliferation readout method was optimized. Use of CellTiter-Glö Luminescent Cell Viability Assay generated data with superior linear correlation with cell numbers compared to Prestobluë and high content screening microscopy approaches. To enable large-scale screening, the assay was further optimized for cell numbers per well, media composition, culture duration, doxycycline concentration and timing of induction and of addition of test chemicals. We also controlled for evaporation during incubation to significantly reduce plate edge effects. During these optimizations, luminescence readout from uninduced cells was set to represent 100% rescue and readout from doxycycline-induced controls to represent 0% recue. This allows reliable normalization between plates and gives toxic chemicals a negative value and chemicals rescuing proliferation a positive value with 100% meaning complete rescue. To objectively quantify how changes of different parameters improved variability in both induced and uninduced cells, we calculated the Z factor as described by Zhang et. al. (JBS, 1999). The Z factor takes into consideration both the range and variability of data to calculate the suitability of an assay for high throughput screening. It is represented as: (SD: Standard Deviation). Where Z value is meaningful in the range of -1 < Z ² 1. The larger the value of Z, the higher is the data quality and a Z-factor above 0.5 is considered very robust. After assay development we arrived at the conditions shown in the table, resulting in the Z factor of 0.7:Culture medium100 ng/ml mSCF, 2 U/ml Epo, 100 nM dexamethasone in serum-free medium. (doxycycline: 0.5 μg/ml).Culture days4 (addition of test chemicals 24 hours after doxycycline induction)Cells per well2000 (murine cKit+ fetal liver cells with inducible rps19-shRNA)ReadoutLuciferase based ATP detection (CellTiter-Glo¨). For screening involving small molecule based libraries we added the compounds 24 hours after cell seeding and doxycycline addition, which allows for the induction of the proliferation perturbed phenotype. We have pharmacologically validated the assay by testing and quantifying the impact of IL-3, a cytokine known to have a positive effect on erythroid progenitors. IL-3 had a 25% rescue value in our assay. Ongoing experiments (>11,000 compounds screened to date) show that more than 50% of compounds with >20% rescue score in the screen could be confirmed to rescue proliferation in dose-dependent experiments. In conclusion, we have established a robust scalable assay for screening molecules that rescue proliferation arrest caused by Rps19-deficiency in mouse erythroid progenitors. We are currently using this assay to screen small molecule libraries in our search for novel tool compounds for DBA research and drug candidates for DBA treatment. Disclosures: No relevant conflicts of interest to declare.