Pyrophosphatase Activity Research Articles

Characterization of a non-nudix pyrophosphatase points to interplay between flavin and NAD(H) homeostasis in Saccharomyces cerevisiae.

The flavin cofactors FMN and FAD are required for a wide variety of biological processes, however, little is known about their metabolism. Here, we report the cloning and biochemical characterization of the Saccharomyces cerevisiae pyrophosphatase Fpy1p. Genetic and functional studies suggest that Fpy1p may play a key role in flavin metabolism and is the first-reported non-Nudix superfamily enzyme to display FAD pyrophosphatase activity. Characterization of mutant yeast strains found that deletion of fpy1 counteracts the adverse effects that are caused by deletion of flx1, a known mitochondrial FAD transporter. We show that Fpy1p is capable of hydrolyzing FAD, NAD(H), and ADP-ribose. The enzymatic activity of Fpy1p is dependent upon the presence of K+ and divalent metal cations, with similar kinetic parameters to those that have been reported for Nudix FAD pyrophosphatases. In addition, we report that the deletion of fpy1 intensifies the FMN-dependence of null mutants of the riboflavin kinase Fmn1p, demonstrate that fpy1 mutation abolishes the decreased fitness resulting from the deletion of the flx1 ORF, and offer a possible mechanism for the genetic interplay between fpy1, flx1 and fmn1.

Cezomycin Is Activated by CalC to Its Ester Form for Further Biosynthesis Steps in the Production of Calcimycin in Streptomyces chartreusis NRRL 3882.

Calcimycin, N-demethyl calcimycin, and cezomycin are polyether divalent cation ionophore secondary metabolites produced by Streptomyces chartreusis A thorough understanding of the organization of their encoding genes, biosynthetic pathway(s), and cation specificities is vitally important for their efficient future production and therapeutic use. So far, this has been lacking, as has information concerning any biosynthetic relationships that may exist between calcimycin and cezomycin. In this study, we observed that when a Cal- (calB1 mutant) derivative of a calcimycin-producing strain of S. chartreusis (NRRL 3882) was grown on cezomycin, calcimycin production was restored. This suggested that calcimycin synthesis may have resulted from postsynthetic modification of cezomycin rather than from a de novo process through a novel and independent biosynthetic mechanism. Systematic screening of a number of Cal-S. chartreusis mutants lacking the ability to convert cezomycin to calcimycin allowed the identification of a gene, provisionally named calC, which was involved in the conversion step. Molecular cloning and heterologous expression of the CalC protein along with its purification to homogeneity and negative-staining electron microscopy allowed the determination of its apparent molecular weight, oligomeric forms in solution, and activity. These experiments allowed us to confirm that the protein possessed ATP pyrophosphatase activity and was capable of ligating coenzyme A (CoA) with cezomycin but not 3-hydroxyanthranilic acid. The CalC protein's apparent Km and kcat for cezomycin were observed to be 190 μM and 3.98 min-1, respectively, and it possessed the oligomeric form in solution. Our results unequivocally show that cezomycin is postsynthetically modified to calcimycin by the CalC protein through its activation of cezomycin to a CoA ester form.IMPORTANCE Calcimycin is a secondary metabolite divalent cation-ionophore that has been studied in the context of human health. However, detail is lacking with respect to both calcimycin's biosynthesis and its biochemical/biophysical properties as well as information regarding its, and its analogues', divalent cation binding specificities and other activities. Such knowledge would be useful in understanding how calcimycin and related compounds may be effective in modifying the calcium channel ion flux and might be useful in influencing the homeostasis of magnesium and manganese ions for the cure or control of human and bacterial infectious diseases. The results presented here unequivocally show that CalC protein is essential for the production of calcimycin, which is essentially a derivative of cezomycin, and allow us to propose a biosynthetic mechanism for calcimycin's production.

Inhibition of nucleotide pyrophosphatase/phosphodiesterase 1: implications for developing a calcium pyrophosphate deposition disease modifying drug.

Calcium pyrophosphate deposition (CPPD) is associated with osteoarthritis and is the cause of a common inflammatory articular disease. Ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (eNPP1) is the major ecto-pyrophosphatase in chondrocytes and cartilage-derived matrix vesicles (MVs). Thus, eNPP1 is a principle contributor to extracellular pyrophosphate levels and a potential target for interventions aimed at preventing CPPD. Recently, we synthesized and described a novel eNPP1-specific inhibitor, SK4A, and we set out to evaluate whether this inhibitor attenuates nucleotide pyrophosphatase activity in human OA cartilage. Cartilage tissue, chondrocytes and cartilage-derived MVs were obtained from donors with OA undergoing arthroplasty. The effect of SK4A on cell viability was assayed by the XTT method. eNPP1 expression was evaluated by western blot. Nucleotide pyrophosphatase activity was measured by a colorimetric assay and by HPLC analysis of adenosine triphosphate (ATP) levels. ATP-induced calcium deposition in cultured chondrocytes was visualized and quantified with Alizarin red S staining. OA chondrocytes expressed eNPP1 in early passages, but this expression was subsequently lost upon further passaging. Similarly, significant nucleotide pyrophosphatase activity was only detected in early-passage chondrocytes. The eNPP1 inhibitor, SK4A, was not toxic to chondrocytes and stable in culture medium and human plasma. SK4A effectively inhibited nucleotide pyrophosphatase activity in whole cartilage tissue, in chondrocytes and in cartilage-derived MVs and reduced ATP-induced CPPD. Nucleotide analogues such as SK4A may be developed as potent and specific inhibitors of eNPP1 for the purpose of lowering extracellular pyrophosphate levels in human cartilage with the aim of preventing and treating CPPD disease.

MiR-122 does not impact recognition of the HCV genome by innate sensors of RNA but rather protects the 5' end from the cellular pyrophosphatases, DOM3Z and DUSP11.

Hepatitis C virus (HCV) recruits two molecules of the liver-specific microRNA-122 (miR-122) to the 5′ end of its genome. This interaction promotes viral RNA accumulation, but the precise mechanism(s) remain incompletely understood. Previous studies suggest that miR-122 is able to protect the HCV genome from 5′ exonucleases (Xrn1/2), but this protection is not sufficient to account for the effect of miR-122 on HCV RNA accumulation. Thus, we investigated whether miR-122 was also able to protect the viral genome from innate sensors of RNA or cellular pyrophosphatases. We found that miR-122 does not play a protective role against recognition by PKR, RIG-I-like receptors, or IFITs 1 and 5. However, we found that knockdown of both the cellular pyrophosphatases, DOM3Z and DUSP11, was able to rescue viral RNA accumulation of subgenomic replicons in the absence of miR-122. Nevertheless, pyrophosphatase knockdown increased but did not restore viral RNA accumulation of full-length HCV RNA in miR-122 knockout cells, suggesting that miR-122 likely plays an additional role(s) in the HCV life cycle, beyond 5′ end protection. Overall, our results support a model in which miR-122 stabilizes the HCV genome by shielding its 5′ terminus from cellular pyrophosphatase activity and subsequent turnover by exonucleases (Xrn1/2).

ITPA Activity in Children Treated by Azathioprine: Relationship to the Occurrence of Adverse Drug Reactions and Inflammatory Response.

Azathioprine (AZA), a thiopurine drug, is widely used in the treatment of children with immunological diseases such as inflammatory bowel disease (IBD) and autoimmune hepatitis (AIH); however, interindividual variability in the occurrence of adverse drug reactions (ADRs) and drug response is observed. This study investigated (i) the relationships between inosine triphosphate pyrophosphatase (ITPA) activity, an enzyme involved in thiopurine metabolism, and the occurrence of ADRs in children with immunological disease on AZA therapy, and (ii) the relationship between ITPA activity and the inflammatory activity observed in children with IBD. ITPA and TPMT activities were determined in 106 children with immunological disease on AZA therapy. Markers of hepatotoxicity, myelotoxicity, pancreatitis and inflammation as well as clinical information were retrospectively collected during regular medical visits. No significant association was found between ITPA activity and hepatotoxicity or clinical ADRs such as cutaneous reactions, arthralgia, flulike symptoms and gastrointestinal disorders. Concerning myelotoxicity, a significant relation was observed between ITPA activity and RBC mean corpuscular volume (MCV; p=0.003). This observation may be related to the significant relationship found between high ITPA activity and the increase in γ-globulin level reflecting inflammation (p=0.005). In our study, ITPA activity was not associated with occurrence of ADRs, but a relationship between high ITPA activity and γ-globulin, a marker of inflammation, was found in children with IBD. Therefore, measurement of ITPA activity may help to identify children with IBD predisposed to residual inflammation on AZA therapy. Further prospective studies are needed to confirm this result.

Copper nanoclusters capped with tannic acid as a fluorescent probe for real-time determination of the activity of pyrophosphatase.

The authors show that copper nanoclusters capped with tannic acid (TA-CuNCs) are viable fluorescent probes for the determination of the activity of pyrophosphatase (PPase). The fluorescence of the TA-CuNCs is quenched by Fe(III) but restored on subsequant addition of pyrophosphate (PPi). If, however, PPi is split by PPase into two phosphate ions, the fluorescence of the TA-CuNCs is quenched again. Under optimized conditions, fluorescence intensity linearly correlates with the activity of PPase in the range from 0.50 to 18.0U L-1 with a detection limit of 0.19U L-1. The method was employed to the determination of PPase in spiked human serum samples and gave satisfactory results. Graphical abstract Schematic illustration of fluorometric assay for PPase activity. It is based onthe finding that pyrophosphate (PPi) canrestore the Fe3+-quenched TA-CuNC fluorescence. PPi is formed by enzymatic action ofPPasei.

The role of anion in the impact of tetraethylammonium salts on soil phosphatase activities

Quaternary ammonium salts (QAS) are increasingly commonly used in the chemical industry. The diverse usage of these compounds results in high possibility of their penetration to the environment. The aim of this study was to compare the effect of three salts with tetraethylammonium cation with different anions such as chloride [TEA][Cl], bromide [TEA][Br], and iodine [TEA][I] on the activity of phosphatases in loamy sand. Organic carbon and total nitrogen content in the soil were 8.71 and 0.97 g·kg-1, respectively. The salts were applied to soil at doses of 0, 0.05, 0.50, and 5.00 mmol·kg-1. The samples were adjusted to 60% maximum water holding capacity, and they were incubated at a temperature of 20°C. Activities of acid phosphomonoesterase, alkaline phopshomonoesterase, phosphotriesterase, and inorganic pyrophosphatase were assayed on day 1, 7, 14, 28, 56, and 112. The obtained results demonstrated that the presence of [TEA] in the soil at the low dose (0.05 mmol·kg-1) may stimulate the activity of soil phosphatases. Higher doses of the soil had mostly inhibitory effect on phosphatases activity. Among the determined enzymes, phosphomonoesterases were the most sensitive to salts containing [TEA] cation. A considerable contribution of the anion type on the formation of activity of the determined enzymes of phosphorus compound changes. The strongest effect characterized tetraethylammonium bromide [TEA][Br].

Apple S-RNase triggers inhibition of tRNA aminoacylation by interacting with a soluble inorganic pyrophosphatase in growing self-pollen tubes invitro.

Apple exhibits S-RNase-based self-incompatibility (SI), in which S-RNase plays a central role in rejecting self-pollen. It has been proposed that the arrest of pollen growth in SI of Solanaceae plants is a consequence of the degradation of pollen rRNA by S-RNase; however, the underlying mechanism in Rosaceae is still unclear. Here, we used S2 -RNase as a bait to screen an apple pollen cDNA library and characterized an apple soluble inorganic pyrophosphatase (MdPPa) that physically interacted with S-RNases. When treated with self S-RNases, apple pollen tubes showed a marked growth inhibition, as well as a decrease in endogenous soluble pyrophosphatase activity and elevated levels of inorganic pyrophosphate (PPi). In addition, S-RNase was found to bind to two variable regions of MdPPa, resulting in a noncompetitive inhibition of its activity. Silencing of MdPPa expression led to a reduction in pollen tube growth. Interestingly, tRNA aminoacylation was inhibited in self S-RNase-treated or MdPPa-silenced pollen tubes, resulting in the accumulation of uncharged tRNA. Furthermore, we provide evidence showing that this disturbance of tRNA aminoacylation is independent of RNase activity. We propose an alternative mechanism differing from RNA degradation to explain the cytotoxicity of the S-RNase apple SI process.

Combined effect of low-molecular-weight organic acids and creosote on phosphatase activities in sandy soil

This paper assesses the impact of creosote and low-molecular-weight organic acids (LMWOAs) on the activity of acid phosphomonoesterase, alkaline phosphomonoesterase, phosphotriesterase, and inorganic pyrophosphatase in soil. The experiment was carried out on loamy sand samples with organic carbon content of 8.71 g · kg -1 , with the following variable factors: dosages of creosote: 0, 0.5%, and 2.5%; type of LMWOAs: oxalic acid, tartaric acid, and citric acid in the amount of 50 mmol · kg -1 of soil; days of experiment: 1, 7, 14, 28, 56, 112. Obtained results showed that contamination with creosote caused decrease in the activity of soil phosphatases. The observed effect did not always increase with increase in the dosage of the pollutant. Among the assayed phosphatases, the biggest changes were noted in the activity of phosphomonoesterases. Application of LMWOAs to contaminated soil mainly effected the inhibition of phosphatase, especially the activity of acid phosphomonoesterase. Comparison of the effects of LMWOAs showed that the citric acid was the least toxic to soil phosphatases.

ExciTides: NTP-derived probes for monitoring pyrophosphatase activity based on excimer-to-monomer transitions.

We describe a new type of nucleotide-derived fluorescent probe designed for monitoring pyrophosphatase activity based on excimer-to-monomer transitions, called ExciTide. The nucleotides were designed with two self-interacting dye moieties and synthesised using copper-catalysed azide-alkyne cycloaddition click chemistry. We applied these probes for enzyme activity monitoring and inhibitor evaluation. Some of the probes permeated into living cells, yielding interesting prospects for future applications.

A high-throughput method for orthophosphate determination of thermostable membrane-bound pyrophosphatase activity

Membrane-bound pyrophosphatases (mPPases) are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates coupled to the active transport of protons or sodium ions across membranes.

Molecular cloning and functional characterisation of an H+-pyrophosphatase from Iris lactea

Tonoplast H+-pyrophosphatases (VPs) mediate vacuolar Na+ sequestration, a process important for salt tolerance of plants. The function of VP in the highly drought- and salt-tolerant perennial Iris lactea under salt stress is unclear. Here, we isolated IlVP from I. lactea and investigated its function in transgenic tobacco. IlVP was found to comprise 771 amino acid residues and showed 88% similarity with Arabidopsis AtVP1. IlVP was mainly expressed in shoots and was up-regulated by salt stress. Overexpression of IlVP enhanced growth of transgenic tobacco plants compared with wild-type (WT) plants exposed to salt stress. Transgenic plants accumulated higher quantities of Na+ and K+ in leaves, stems, and roots under salt stress, which caused higher leaf relative water content and decreased cell membrane damage compared with WT plants. Overall, IlVP encoding a tonoplast H+-pyrophosphatase can reduce Na+ toxicity in plant cells through increased sequestration of ions into vacuoles by enhanced H+-pyrophosphatase activity.

Purification and Characterization of an Alkaline Phosphatase Induced by Phosphorus Starvation in Common Bean (Phaseolus vulgaris L.) Roots

Two phosphatase isoforms from roots of the common bean (Phaseolus vulgaris L.) showed an increase in activity in response to phosphate deficiency. One of them (APIII) was chosen for further purification through ionic exchange chromatography and preparative electrophoresis. The estimated molecular mass of APIII was 35 kDa by both SDS-PAGE and gel filtration analyses, suggesting a monomeric form of the active enzyme. The phosphatase was classified as an alkaline phosphatase based on the requirement of pH 8 for optimum catalysis. It not only exhibited broad substrate specificity, with the most activity against pyrophosphate, but also effectively catalyzed the hydrolysis of polyphosphate, glucose-1-phosphate and phosphoenolpyruvate. Activity was completely inhibited by molybdate, vanadate and phosphate but was only partially inhibited by fluoride. Although divalent cations were not essential for the pyrophosphatase activity of this enzyme, the hydrolysis of pyrophosphate increased substantially in the presence of Mg2+.

Electrochemical strategy for pyrophosphatase detection Based on the peroxidase-like activity of G-quadruplex-Cu2+ DNAzyme

A new simple and highly sensitive electrochemical method for pyrophosphatase (PPase) activity detection was developed based on the peroxidase-like activity of G-quadruplex-Cu2+ DNAzyme. In the absence of PPase, Cu2+ could coordinate with pyrophosphate (PPi) to form Cu2+-PPi compound. While in the presence of PPase, it could destroy the coordinate compound because PPase catalyzed the hydrolysis of PPi into inorganic phosphate and produced free Cu2+, which then could be coupled with G-rich DNA to form G-quadruplex-Cu2+ DNAzyme. The formation of a mimic enzyme (G-quadruplex-Cu2+ DNAzyme) was immobilized on the surface of screen-printed gold electrode (SPGE). Using 3, 3′, 5, 5′-tetramethylbenzidine (TMB) as a redox mediator and H2O2 as an enzyme substrate, the DNAzyme catalyzed the reduction of H2O2 to generate quantitative chronoamperometric signal. The catalytic activity of G-quadruplex-Cu2+ DNAzyme for TMB-H2O2 reaction was proportional to the activity of PPase, based on which, a simple and sensitive turn-on electrochemical method for PPase activity was thus developed for the first time. The chronoamperometric intensity of the system had a linear relationship with the PPase activities in the range of 1.0–50.0mU/mL and the detection limit could be down to 0.6mU/mL (S/N = 3). This proposed method was selective, cost-effective and convenient without any labels or complicated operations, which was furthermore applied to screen the inhibitor for PPase with high efficiency.

A new method to investigate the catalytic mechanism of YhdE pyrophosphatase by using a pyrophosphate fluorescence probe

YhdE is a Maf (multicopy associated filamentation) proteins from Escherichia coli which exhibits pyrophosphatase activity towards selected nucleotides, although its catalytic mechanism remains unclear. Herein we used a novel fluorescence probe (4-isoACBA–Zn(II) complex) to characterize the enzymatic properties of YhdE and its mutant, establishing a new method for assaying pyrophosphatase catalytic function. Our results reveal for the first time that the new fluorescence sensor confers high sensitivity and specificity and pyrophosphate (PPi) is the direct catalytic product of YhdE. Crystal structures of a mutant in the active-site loop (YhdE_E33A) show conformational flexibility implicated in the catalytic mechanism of YhdE. ITC experiments and computational docking further reveal that Asp70 and substrate dTTP coordinate Mn2+. Quantum mechanics calculations indicate that YhdE hydrolysis appears to follow a stepwise pathway in which a water molecule first attacks the α-phosphorus atom in the substrate, followed by the release of PPi from the pentavalent intermediate.

Inhibition of Escherichia coli Inorganic Pyrophosphatase by Fructose-1-phosphate.

Pyrophosphate regulates vital cellular reactions, and its level in E. coli cells is under the ultimate control of inorganic pyrophosphatase. The mechanisms involved in the regulation of pyrophosphatase activity still need to be elucidated. The present study demonstrated that fructose-1-phosphate inhibits pyrophosphatase activity by a mechanism not involving competition with substrate for binding to the active site. The inhibition constant governing the binding of the inhibitor to the enzyme-substrate complex is 1.1mM. Substitutions of Lys112, Lys115, Lys148, and Arg43 in the regulatory site completely or partially abolished the inhibition. Thus, Fru-1-P is a physiological inhibitor of pyrophosphatase that acts via a regulatory site in this enzyme.

Triphosphate Tunnel Metalloenzyme Function in Senescence Highlights a Biological Diversification of This Protein Superfamily.

The triphosphate tunnel metalloenzyme (TTM) superfamily comprises a group of enzymes that hydrolyze organophosphate substrates. They exist in all domains of life, yet the biological role of most family members is unclear. Arabidopsis (Arabidopsis thaliana) encodes three TTM genes. We have previously reported that AtTTM2 displays pyrophosphatase activity and is involved in pathogen resistance. Here, we report the biochemical activity and biological function of AtTTM1 and diversification of the biological roles between AtTTM1 and 2 Biochemical analyses revealed that AtTTM1 displays pyrophosphatase activity similar to AtTTM2, making them the only TTMs characterized so far to act on a diphosphate substrate. However, knockout mutant analysis showed that AtTTM1 is not involved in pathogen resistance but rather in leaf senescence. AtTTM1 is transcriptionally up-regulated during leaf senescence, and knockout mutants of AtTTM1 exhibit delayed dark-induced and natural senescence. The double mutant of AtTTM1 and AtTTM2 did not show synergistic effects, further indicating the diversification of their biological function. However, promoter swap analyses revealed that they functionally can complement each other, and confocal microscopy revealed that both proteins are tail-anchored proteins that localize to the mitochondrial outer membrane. Additionally, transient overexpression of either gene in Nicotiana benthamiana induced senescence-like cell death upon dark treatment. Taken together, we show that two TTMs display the same biochemical properties but distinct biological functions that are governed by their transcriptional regulation. Moreover, this work reveals a possible connection of immunity-related programmed cell death and senescence through novel mitochondrial tail-anchored proteins.

Silencing PPA1 inhibits human epithelial ovarian cancer metastasis by suppressing the Wnt/β-catenin signaling pathway.

Inorganic pyrophosphatase (PPA1) activity is a key determinant of cellular inorganic pyrophosphate levels, and its expression is correlated with growth of several solid tumors. To investigate this relationship, we first examined PPA1 expression in human epithelial ovarian cancer (EOC) samples, and found that PPA1 was overexpressed in tumors from EOC patients. Higher PPA1 levels correlated with advanced grades, stages, and poor survival in EOC patients. Examination of PPA1 function in EOC revealed that silencing PPA1 inhibited EOC migration, epithelial-mesenchymal transition (EMT), and metastasis in vitro and in vivo. In addition, PPA1 may promote the dephosphorylation and translocation of β-catenin. These results demonstrate that silencing PPA1 inhibits EOC metastasis by suppressing the Wnt/β-catenin signaling pathway. Strategies for downregulating PPA1 may have therapeutic potential for the prevention and treatment of EOC.

Vγ9Vδ2 T cell activation by strongly agonistic nucleotidic phosphoantigens.

Human Vγ9Vδ2 T cells can sense through their TCR tumor cells producing the weak endogenous phosphorylated antigen isopentenyl pyrophosphate (IPP), or bacterially infected cells producing the strong agonist hydroxyl dimethylallyl pyrophosphate (HDMAPP). The recognition of the phosphoantigen is dependent on its binding to the intracellular B30.2 domain of butyrophilin BTN3A1. Most studies have focused on pyrophosphate phosphoantigens. As triphosphate nucleotide derivatives are naturally co-produced with IPP and HDMAPP, we analyzed their specific properties using synthetic nucleotides derived from HDMAPP. The adenylated, thymidylated and uridylated triphosphate derivatives were found to activate directly Vγ9Vδ2 cell lines as efficiently as HDMAPP in the absence of accessory cells. These antigens were inherently resistant to terminal phosphatases, but apyrase, when added during a direct stimulation of Vγ9Vδ2 cells, abrogated their stimulating activity, indicating that their activity required transformation into strong pyrophosphate agonists by a nucleotide pyrophosphatase activity which is present in serum. Tumor cells can be sensitized with nucleotide phosphoantigens in the presence of apyrase to become stimulatory, showing that this can occur before their hydrolysis into pyrophosphates. Whereas tumors sensitized with HDMAPP rapidly lost their stimulatory activity, sensitization with nucleotide derivatives, in particular with the thymidine derivative, induced long-lasting stimulating ability. Using isothermal titration calorimetry, binding of some nucleotide derivatives to BTN3A1 intracellular domain was found to occur with an affinity similar to that of IPP, but much lower than that of HDMAPP. Thus, nucleotide phosphoantigens are precursors of pyrophosphate antigens which can deliver strong agonists intracellularly resulting in prolonged and strengthened activity.

MTH1 as a Chemotherapeutic Target: The Elephant in the Room.

Many tumors sustain elevated levels of reactive oxygen species (ROS), which drive oncogenic signaling. However, ROS can also trigger anti-tumor responses, such as cell death or senescence, through induction of oxidative stress and concomitant DNA damage. To circumvent the adverse consequences of elevated ROS levels, many tumors develop adaptive responses, such as enhanced redox-protective or oxidatively-generated damage repair pathways. Targeting these enhanced oxidative stress-protective mechanisms is likely to be both therapeutically effective and highly specific to cancer, as normal cells are less reliant on such mechanisms. In this review, we discuss one such stress-protective protein human MutT Homolog1 (MTH1), an enzyme that eliminates 8-oxo-7,8-dihydro-2’-deoxyguanosine triphosphate (8-oxodGTP) through its pyrophosphatase activity, and is found to be elevated in many cancers. Our studies, and subsequently those of others, identified MTH1 inhibition as an effective tumor-suppressive strategy. However, recent studies with the first wave of MTH1 inhibitors have produced conflicting results regarding their cytotoxicity in cancer cells and have led to questions regarding the validity of MTH1 as a chemotherapeutic target. To address the proverbial "elephant in the room" as to whether MTH1 is a bona fide chemotherapeutic target, we provide an overview of MTH1 function in the context of tumor biology, summarize the current literature on MTH1 inhibitors, and discuss the molecular contexts likely required for its efficacy as a therapeutic target.