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Sterile alpha motif and histidine-aspartic acid domain containing protein-1 (SAMHD1) is a deoxynucleoside triphosphate (dNTP) triphosphohydrolase central to cellular nucleotide pool homeostasis. Recent literature has also demonstrated how SAMHD1 can detoxify chemotherapy metabolites thereby controlling their clinical responses. To further understand SAMHD1 biology and to investigate the potential of targeting this enzyme as a neoadjuvant to existing chemotherapies we set out to discover selective small molecule-based inhibitors of SAMHD1. Here we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for further characterisation of the screen output. The identified hit compound TH6342 and its analogues, accompanied by their inactive negative control analogue TH7126, demonstrated specific, low μM potency in inhibiting the hydrolysis of both natural substrates and nucleotide analogue therapeutics, shown using complementary enzyme-coupled and direct enzymatic activity assays. Their mode of inhibition was subsequently detailed by coupling kinetic studies with thermal shift assays, where TH6342 and analogues were shown to engage with pre-tetrameric SAMHD1 and deter the oligomerisation and allosteric activation of SAMHD1 without occupying nucleotide binding pockets. We further outline the development and application of multiple cellular assays for assessing cellular target engagement and associated functional effects, including CETSA and an in-cell dNTP hydrolase activity assay, which highlighted future optimisation strategies of this chemotype. In summary, with a novel mode of inhibition, TH6342 and analogues broaden the set of tool compounds available in deciphering SAMHD1 enzymology and functions, and furthermore, the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development.Abstract Figure

Sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) is a (deoxy)guanosine triphosphate (dGTP/GTP)-activated deoxyribonucleoside triphosphate (dNTP) triphosphohydrolase involved in cellular dNTP homoeostasis. Mutations in SAMHD1 have been associated with the hyperinflammatory disease Aicardi–Goutieres syndrome (AGS). SAMHD1 also limits cells' permissiveness to infection with diverse viruses, including human immunodeficiency virus (HIV-1), and controls endogenous retroviruses. Increasing evidence supports the role of SAMHD1 as a tumor suppressor. However, SAMHD1 also can act as a resistance factor to nucleoside-based chemotherapies by hydrolyzing their active triphosphate metabolites, thereby reducing response of various malignancies to these anticancer drugs. Hence, informed cancer therapies must take into account the ambiguous properties of SAMHD1 as both an inhibitor of uncontrolled proliferation and a resistance factor limiting the efficacy of anticancer treatments. Here, we provide evidence that SAMHD1 is a double-edged sword for patients with acute myelogenous leukemia (AML). Our time-dependent analyses of The Cancer Genome Atlas (TCGA) AML cohort indicate that high expression of SAMHD1, even though it critically limits the efficacy of high-dose ara-C therapy, might be associated with more favorable disease progression.

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.).

Due to a polar or even charged binding interface, DNA-binding proteins are considered extraordinarily difficult targets for development of small-molecule ligands and only a handful of proteins have been targeted successfully to date. Recently, however, it has been shown that development of selective and efficient inhibitors of 8-oxoguanine DNA glycosylase is possible. Here, we describe the initial druggability assessment of DNA glycosylases in a computational setting and experimentally investigate several methods to target endonuclease VIII-like 1 (NEIL1) with small-molecule inhibitors. We find that DNA glycosylases exhibit good predicted druggability in both DNA-bound and -unbound states. Furthermore, we find catalytic sites to be highly flexible, allowing for a range of interactions and binding partners. One flexible catalytic site was rationalized for NEIL1 and further investigated experimentally using both a biochemical assay in the presence of DNA and a thermal shift assay in the absence of DNA.

DNA binding as an anti-inflammatory Mice that lack the gene encoding 8-oxoguanine DNA glycosylase 1 (OGG1) show resistance to inflammation. This enzyme binds to sites of oxidative DNA damage and initiates DNA base excision repair. Visnes et al. developed a small-molecule drug that acts as a potent and selective active-site inhibitor that stops OGG1 from recognizing its DNA substrate (see the Perspective by Samson). The drug inhibited DNA repair and modified OGG1 chromatin dynamics, which resulted in the inhibition of proinflammatory pathway genes. The drug was well tolerated by mice and suppressed lipopolysaccharide- and tumor necrosis factor–α–mediated neutrophilic inflammation in the lungs. Science , this issue p. 834 ; see also p. 748

Recently, we demonstrated that sterile α motif and HD domain containing protein 1 (SAMHD1) is a major barrier in acute myelogenous leukemia (AML) cells to the cytotoxicity of cytarabine (ara-C), the most important drug in AML treatment. Ara-C is intracellularly converted by the canonical dNTP synthesis pathway to ara-CTP, which serves as a substrate but not an allosteric activator of SAMHD1. Using an AML mouse model, we show here that wild type but not catalytically inactive SAMHD1 reduces ara-C treatment efficacy in vivo. Expanding the clinically relevant substrates of SAMHD1, we demonstrate that THP-1 CRISPR/Cas9 cells lacking a functional SAMHD1 gene showed increased sensitivity to the antimetabolites nelarabine, fludarabine, decitabine, vidarabine, clofarabine, and trifluridine. Within this Extra View, we discuss and build upon both these and our previously reported findings, and propose SAMHD1 is likely active against a variety of nucleoside analog antimetabolites present in anti-cancer chemotherapies. Thus, SAMHD1 may constitute a promising target to improve a wide range of therapies for both hematological and non-haematological malignancies.

// Anna Hagenkort 1, * , Cynthia B.J. Paulin 1, * , Matthieu Desroses 1, * , Antonio Sarno 2 , Elisee Wiita 1 , Oliver Mortusewicz 1 , Tobias Koolmeister 1 , Olga Loseva 1 , Ann-Sofie Jemth 1 , Ingrid Almlof 1 , Evert Homan 1 , Thomas Lundback 3 , Anna-Lena Gustavsson 3 , Martin Scobie 1 , Thomas Helleday 1 1 Division of Translational Medicine and Chemical Biology, Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden 2 Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim, Norway 3 Chemical Biology Consortium Sweden, Division of Translational Medicine and Chemical Biology, Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden * These authors have contributed equally to this work Correspondence to: Thomas Helleday, email: thomas.helleday@scilifelab.se Keywords: dUTPase, 5-Fluorouracil, DNA replication, combination therapy Received: July 07, 2016 Accepted: February 06, 2017 Published: February 28, 2017 ABSTRACT The antimetabolite 5-Fluorouracil (5-FU) is used in the treatment of various forms of cancer and has a complex mode of action. Despite 6 decades in clinical application the contribution of 5-FdUTP and dUTP [(5-F)dUTP] and 5-FUTP misincorporation into DNA and RNA respectively, for 5-FU-induced toxicity is still under debate. This study investigates DNA replication defects induced by 5-FU treatment and how (5-F)dUTP accumulation contributes to this effect. We reveal that 5-FU treatment leads to extensive problems in DNA replication fork progression, causing accumulation of cells in S-phase, DNA damage and ultimately cell death. Interestingly, these effects can be reinforced by either depletion or inhibition of the deoxyuridine triphosphatase (dUTPase, also known as DUT), highlighting the importance of (5-F)dUTP accumulation for cytotoxicity. With this study, we not only extend the current understanding of the mechanism of action of 5-FU, but also contribute to the characterization of dUTPase inhibitors. We demonstrate that pharmacological inhibition of dUTPase is a promising approach that may improve the efficacy of 5-FU treatment in the clinic.

Antimetabolites, in particular nucleobase and nucleoside analogues, are cytotoxic drugs that, starting from the small field of paediatric oncology, in combination with other chemotherapeutics, have revolutionised clinical oncology and transformed cancer into a curable disease. However, even though combination chemotherapy, together with radiation, surgery and immunotherapy, can nowadays cure almost all types of cancer, we still fail to achieve this for a substantial proportion of patients. The understanding of differences in metabolism, pharmacokinetics, pharmacodynamics, and tumour biology between patients that can be cured and patients that cannot, builds the scientific basis for rational therapy improvements. Here, we summarise current knowledge of how tumour-specific and patient-specific factors can dictate resistance to nucleobase/nucleoside analogues, and which strategies of re-sensitisation exist. We revisit well-established hurdles to treatment efficacy, like the blood-brain barrier and reduced deoxycytidine kinase activity, but will also discuss the role of novel resistance factors, such as SAMHD1. A comprehensive appreciation of the complex mechanisms that underpin the failure of chemotherapy will hopefully inform future strategies of personalised medicine.

The deoxycytidine analogue cytarabine (ara‐C) remains the backbone treatment of acute myeloid leukaemia (AML) as well as other haematological and lymphoid malignancies, but must be combined with other chemotherapeutics to achieve cure. Yet, the underlying mechanism dictating synergistic efficacy of combination chemotherapy remains largely unknown. The dNTPase SAMHD1, which regulates dNTP homoeostasis antagonistically to ribonucleotide reductase (RNR), limits ara‐C efficacy by hydrolysing the active triphosphate metabolite ara‐CTP. Here, we report that clinically used inhibitors of RNR, such as gemcitabine and hydroxyurea, overcome the SAMHD1‐mediated barrier to ara‐C efficacy in primary blasts and mouse models of AML, displaying SAMHD1‐dependent synergy with ara‐C. We present evidence that this is mediated by dNTP pool imbalances leading to allosteric reduction of SAMHD1 ara‐CTPase activity. Thus, SAMHD1 constitutes a novel biomarker for combination therapies of ara‐C and RNR inhibitors with immediate consequences for clinical practice to improve treatment of AML., SAMHD1 limits the efficacy of cytarabine by detoxifying cancer cells of its active metabolite, resulting in worse survival for acute myeloid leukaemia patients. We identify clinically used drugs capable of indirectly inactivating SAMHD1, which could lead to improved outcome for these patients.