Identification and evaluation of small-molecule inhibitors against the dNTPase SAMHD1 via a comprehensive screening funnel.

SAMHD1 is a dNTP triphosphohydrolase governing nucleotide pool homeostasis and can detoxify chemotherapy metabolites controlling their clinical responses. To understand SAMHD1 biology and investigate the potential of targeting SAMHD1 as neoadjuvant to current chemotherapies, we set out to discover selective small-molecule inhibitors. Here, we report a discovery pipeline encompassing a biochemical screening campaign and a set of complementary biochemical, biophysical, and cell-based readouts for rigorous characterization of the screen output. The identified small molecules, TH6342 and analogs, accompanied by inactive control TH7126, demonstrated specific, low µM potency against both physiological and oncology-drug-derived substrates. By coupling kinetic studies with thermal shift assays, we reveal the inhibitory mechanism of TH6342 and analogs, which engage pre-tetrameric SAMHD1 and deter oligomerization and allosteric activation without occupying nucleotide-binding pockets. Altogether, our study diversifies inhibitory modes against SAMHD1, and the discovery pipeline reported herein represents a thorough framework for future SAMHD1 inhibitor development.

Pharmacological targeting of MTHFD2 suppresses acute myeloid leukemia by inducing thymidine depletion and replication stress.

The folate metabolism enzyme MTHFD2 (methylenetetrahydrofolate dehydrogenase/cyclohydrolase) is consistently overexpressed in cancer but its roles are not fully characterized, and current candidate inhibitors have limited potency for clinical development. In the present study, we demonstrate a role for MTHFD2 in DNA replication and genomic stability in cancer cells, and perform a drug screen to identify potent and selective nanomolar MTHFD2 inhibitors; protein cocrystal structures demonstrated binding to the active site of MTHFD2 and target engagement. MTHFD2 inhibitors reduced replication fork speed and induced replication stress followed by S-phase arrest and apoptosis of acute myeloid leukemia cells in vitro and in vivo, with a therapeutic window spanning four orders of magnitude compared with nontumorigenic cells. Mechanistically, MTHFD2 inhibitors prevented thymidine production leading to misincorporation of uracil into DNA and replication stress. Overall, these results demonstrate a functional link between MTHFD2-dependent cancer metabolism and replication stress that can be exploited therapeutically with this new class of inhibitors.

Computational and Experimental Druggability Assessment of Human DNA Glycosylases.

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.

Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation.

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-α-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor κB and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.

Nucleobase and Nucleoside Analogues: Resistance and Re-Sensitisation at the Level of Pharmacokinetics, Pharmacodynamics and Metabolism.

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.

With me or against me: Tumor suppressor and drug resistance activities of SAMHD1.

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-Goutières 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.

SAMHD1 protects cancer cells from various nucleoside-based antimetabolites.

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.

dUTPase inhibition augments replication defects of 5-Fluorouracil.

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.

Targeting SAMHD1 with the Vpx protein to improve cytarabine therapy for hematological malignancies.

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.