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.
Deregulated redox metabolism in cancer leads to oxidative damage to cellular components including deoxyribonucleoside triphosphates (dNTPs). Targeting dNTP pool sanitizing enzymes, such as MTH1, is a highly promising anticancer strategy. The MTH2 protein, known as NUDT15, is described as the second human homologue of bacterial MutT with 8-oxo-dGTPase activity. We present the first NUDT15 crystal structure and demonstrate that NUDT15 prefers other nucleotide substrates over 8-oxo-dGTP. Key structural features are identified that explain different substrate preferences for NUDT15 and MTH1. We find that depletion of NUDT15 has no effect on incorporation of 8-oxo-dGTP into DNA and does not impact cancer cell survival in cell lines tested. NUDT17 and NUDT18 were also profiled and found to have far less activity than MTH1 against oxidized nucleotides. We show that NUDT15 is not a biologically relevant 8-oxo-dGTPase, and that MTH1 is the most prominent sanitizer of the cellular dNTP pool known to date.
To sustain their proliferation, cancer cells become dependent on one-carbon metabolism to support purine and thymidylate synthesis. Indeed, one of the most highly upregulated enzymes during neoplastic transformation is MTHFD2, a mitochondrial methylenetetrahydrofolate dehydrogenase and cyclohydrolase involved in one-carbon metabolism. Because MTHFD2 is expressed normally only during embryonic development, it offers a disease-selective therapeutic target for eradicating cancer cells while sparing healthy cells. Here we report the synthesis and preclinical characterization of the first inhibitor of human MTHFD2. We also disclose the first crystal structure of MTHFD2 in complex with a substrate-based inhibitor and the enzyme cofactors NAD and inorganic phosphate. Our work provides a rationale for continued development of a structural framework for the generation of potent and selective MTHFD2 inhibitors for cancer treatment. .
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.
The highly poisonous botulinum neurotoxins, produced by the bacterium Clostridium botulinum, act on their hosts by a high-affinity association to two receptors on neuronal cell surfaces as the first step of invasion. The glycan motifs of gangliosides serve as initial coreceptors for these protein complexes, whereby a membrane protein receptor is bound. Herein we set out to characterize the carbohydrate minimal binding epitope of the botulinum neurotoxin serotype A. By means of ligand-based NMR spectroscopy, X-ray crystallography, computer simulations, and isothermal titration calorimetry, a screening of ganglioside analogues together with a detailed characterization of various carbohydrate ligand complexes with the toxin were accomplished. We show that the representation of the glycan epitope to the protein affects the details of binding. Notably, both branches of the oligosaccharide GD1a can associate to botulinum neurotoxin serotype A when expressed as individual trisaccharides. It is, however, the terminal branch of GD1a as well as this trisaccharide motif alone, corresponding to the sialyl-Thomsen-Friedenreich antigen, that represents the active ligand epitope, and these compounds bind to the neurotoxin with a high degree of predisposition but with low affinities. This finding does not correlate with the oligosaccharide moieties having a strong contribution to the total affinity, which was expected to be the case. We here propose that the glycan part of the ganglioside receptors mainly provides abundance and specificity, whereas the interaction with the membrane itself and protein receptor brings about the strong total binding of the toxin to the neuronal membrane.
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