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Nature Chemical Biology Oct 2020The NUDIX hydrolase NUDT15 was originally implicated in sanitizing oxidized nucleotides, but was later shown to hydrolyze the active thiopurine metabolites,...
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 small-molecule NUDT15 inhibitors to elucidate its biological functions and potentially to improve 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 also employed thiopurine potentiation as a proxy functional readout 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 takes place via direct NUDT15 inhibition. In conclusion, TH1760 represents the first chemical probe for interrogating NUDT15 biology and potential therapeutic avenues.
Topics: Binding Sites; Cell Line; Drug Design; Drug Development; Escherichia coli; Humans; Inorganic Pyrophosphatase; Models, Molecular; Protein Binding; Protein Conformation; Pyrophosphatases; Structure-Activity Relationship
PubMed: 32690945
DOI: 10.1038/s41589-020-0592-z -
Journal of Lipid Research Jan 2016The ectonucleotide pyrophosphatase/phosphodiesterase type 2, more commonly known as autotaxin (ATX), is an ecto-lysophospholipase D encoded by the human ENNP2 gene. ATX... (Review)
Review
The ectonucleotide pyrophosphatase/phosphodiesterase type 2, more commonly known as autotaxin (ATX), is an ecto-lysophospholipase D encoded by the human ENNP2 gene. ATX is expressed in multiple tissues and participates in numerous key physiologic and pathologic processes, including neural development, obesity, inflammation, and oncogenesis, through the generation of the bioactive lipid, lysophosphatidic acid. Overwhelming evidence indicates that altered ATX activity leads to oncogenesis and cancer progression through the modulation of multiple hallmarks of cancer pathobiology. Here, we review the structural and catalytic characteristics of the ectoenzyme, how its expression and maturation processes are regulated, and how the systemic integration of its pleomorphic effects on cells and tissues may contribute to cancer initiation, progression, and therapy. Additionally, the up-to-date spectrum of the most frequent ATX genomic alterations from The Cancer Genome Atlas project is reported for a subset of cancers.
Topics: Animals; Carcinogenesis; Cell Transformation, Neoplastic; Disease Progression; Humans; Neoplasms; Phosphoric Diester Hydrolases; Pyrophosphatases
PubMed: 25977291
DOI: 10.1194/jlr.R060020 -
Communications Biology Nov 2020Guanosine 3',5'-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years,...
Guanosine 3',5'-bis(pyrophosphate) (ppGpp) functions as a second messenger in bacteria to adjust their physiology in response to environmental changes. In recent years, the ppGpp-specific hydrolase, metazoan SpoT homolog-1 (Mesh1), was shown to have important roles for growth under nutrient deficiency in Drosophila melanogaster. Curiously, however, ppGpp has never been detected in animal cells, and therefore the physiological relevance of this molecule, if any, in metazoans has not been established. Here, we report the detection of ppGpp in Drosophila and human cells and demonstrate that ppGpp accumulation induces metabolic changes, cell death, and eventually lethality in Drosophila. Our results provide the evidence of the existence and function of the ppGpp-dependent stringent response in animals.
Topics: Animals; Bacteria; Drosophila melanogaster; Guanosine Pentaphosphate; Guanosine Tetraphosphate; Pyrophosphatases; Second Messenger Systems; Signal Transduction
PubMed: 33188280
DOI: 10.1038/s42003-020-01368-4 -
Bone Dec 2021Awareness for hypophosphatemic rickets has increased in the last years, based on the availability of specific medical treatments. Autosomal recessive hypophosphatemic... (Review)
Review
Awareness for hypophosphatemic rickets has increased in the last years, based on the availability of specific medical treatments. Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) is a rare form of hypophosphatemic rickets, which is known to develop in survivors of generalized arterial calcification of infancy (GACI). Both disorders are based on a deficiency of ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) and present with a high clinical variability and a lack of a phenotype-genotype association. ARHR2 is characterized by phosphate wasting due to elevated fibroblast growth factor 23 (FGF23) levels and might represent a response of the organism to minimize ectopic calcification in individuals with ENPP1-deficiency. This report reviews the recent clinical and preclinical data on this ultra-rare disease in childhood.
Topics: Familial Hypophosphatemic Rickets; Fibroblast Growth Factor-23; Fibroblast Growth Factors; Humans; Phosphates; Phosphoric Diester Hydrolases; Pyrophosphatases; Rickets, Hypophosphatemic
PubMed: 34252603
DOI: 10.1016/j.bone.2021.116111 -
Molecular Plant Pathology Mar 2021Plant viruses typically have highly condensed genomes, yet the plant-pathogenic viruses Cassava brown streak virus, Ugandan cassava brown streak virus, and Euphorbia... (Review)
Review
Plant viruses typically have highly condensed genomes, yet the plant-pathogenic viruses Cassava brown streak virus, Ugandan cassava brown streak virus, and Euphorbia ringspot virus are unusual in encoding an enzyme not yet found in any other virus, the "house-cleaning" enzyme inosine triphosphatase. Inosine triphosphatases (ITPases) are highly conserved enzymes that occur in all kingdoms of life and perform a house-cleaning function by hydrolysing the noncanonical nucleotide inosine triphosphate to inosine monophosphate. The ITPases encoded by cassava brown streak virus and Ugandan cassava brown streak virus have been characterized biochemically and are shown to have typical ITPase activity. However, their biological role in virus infection has yet to be elucidated. Here we review what is known of viral-encoded ITPases and speculate on potential roles in infection with the aim of generating a greater understanding of cassava brown streak viruses, a group of the world's most devastating viruses.
Topics: Manihot; Plant Diseases; Potyviridae; Pyrophosphatases; Viral Proteins; Inosine Triphosphatase
PubMed: 33471956
DOI: 10.1111/mpp.13021 -
Retrovirology Aug 2015Retroviruses are among the best studied viruses in last decades due to their pivotal involvement in cellular processes and, most importantly, in causing human diseases,... (Review)
Review
Retroviruses are among the best studied viruses in last decades due to their pivotal involvement in cellular processes and, most importantly, in causing human diseases, most notably-acquired immunodeficiency syndrome (AIDS) that is triggered by human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2, respectively). Numerous studied were conducted to understand the involvement of the three cardinal retroviral enzymes, reverse transcriptase, integrase and protease, in the life cycle of the viruses. These studies have led to the development of many inhibitors of these enzymes as anti-retroviral specific drugs that are used for routine treatments of HIV/AIDS patients. Interestingly, a fourth virus-encoded enzyme, the deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase) is also found in several major retroviral groups. The presence and the importance of this enzyme to the life cycle of retroviruses were usually overlooked by most retrovirologists, although the occurrence of dUTPases, particularly in beta-retroviruses and in non-primate retroviruses, is known for more than 20 years. Only more recently, retroviral dUTPases were brought into the limelight and were shown in several cases to be essential for viral replication. Therefore, it is likely that future studies on this enzyme will advance our knowledge to a level that will allow designing novel, specific and potent anti-dUTPase drugs that are effective in combating retroviral diseases. The aim of this review is to give concise background information on dUTPases in general and to summarize the most relevant data on retroviral dUTPases and their involvement in the replication processes and pathogenicity of the viruses, as well as in possibly-associated human diseases.
Topics: Animals; HIV Infections; HIV-1; HIV-2; Humans; Phylogeny; Pyrophosphatases; Retroviridae; Sequence Alignment; Virus Replication
PubMed: 26259899
DOI: 10.1186/s12977-015-0198-9 -
ChemMedChem Nov 2021Inhibition of membrane-bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan parasites. mPPases are absent...
Inhibition of membrane-bound pyrophosphatase (mPPase) with small molecules offer a new approach in the fight against pathogenic protozoan parasites. mPPases are absent in humans, but essential for many protists as they couple pyrophosphate hydrolysis to the active transport of protons or sodium ions across acidocalcisomal membranes. So far, only few nonphosphorus inhibitors have been reported. Here, we explore the chemical space around previous hits using a combination of screening and synthetic medicinal chemistry, identifying compounds with low micromolar inhibitory activities in the Thermotoga maritima mPPase test system. We furthermore provide early structure-activity relationships around a new scaffold having a pyrazolo[1,5-a]pyrimidine core. The most promising pyrazolo[1,5-a]pyrimidine congener was further investigated and found to inhibit Plasmodium falciparum mPPase in membranes as well as the growth of P. falciparum in an ex vivo survival assay.
Topics: Dose-Response Relationship, Drug; Humans; Molecular Structure; Pyrazoles; Pyrimidines; Pyrophosphatases; Structure-Activity Relationship
PubMed: 34459148
DOI: 10.1002/cmdc.202100392 -
Science (New York, N.Y.) Dec 2018Transient receptor potential (TRP) melastatin 2 (TRPM2) is a cation channel associated with numerous diseases. It has a C-terminal NUDT9 homology (NUDT9H) domain...
Transient receptor potential (TRP) melastatin 2 (TRPM2) is a cation channel associated with numerous diseases. It has a C-terminal NUDT9 homology (NUDT9H) domain responsible for binding adenosine diphosphate (ADP)-ribose (ADPR), and both ADPR and calcium (Ca) are required for TRPM2 activation. Here we report cryo-electron microscopy structures of human TRPM2 alone, with ADPR, and with ADPR and Ca NUDT9H forms both intra- and intersubunit interactions with the N-terminal TRPM homology region (MHR1/2/3) in the apo state but undergoes conformational changes upon ADPR binding, resulting in rotation of MHR1/2 and disruption of the intersubunit interaction. The binding of Ca further engages transmembrane helices and the conserved TRP helix to cause conformational changes at the MHR arm and the lower gating pore to potentiate channel opening. These findings explain the molecular mechanism of concerted TRPM2 gating by ADPR and Ca and provide insights into the gating mechanism of other TRP channels.
Topics: Adenosine Diphosphate Ribose; Calcium; Cryoelectron Microscopy; Humans; Ion Channel Gating; Protein Domains; Protein Multimerization; Pyrophosphatases; TRPM Cation Channels
PubMed: 30467180
DOI: 10.1126/science.aav4809 -
Experimental Dermatology Apr 2022Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes...
Pseudoxanthoma elasticum (PXE; OMIM 264800) is a rare heritable multisystem disorder, characterized by ectopic mineralization affecting elastic fibres in the skin, eyes and the cardiovascular system. Skin findings often lead to early diagnosis of PXE, but currently, no specific treatment exists to counteract the progression of symptoms. PXE belongs to a group of Mendelian calcification disorders linked to pyrophosphate metabolism, which also includes generalized arterial calcification of infancy (GACI) and arterial calcification due to CD73 deficiency (ACDC). Inactivating mutations in ABCC6, ENPP1 and NT5E are the genetic cause of these diseases, respectively, and all of them result in reduced inorganic pyrophosphate (PP ) concentration in the circulation. Although PP is a strong inhibitor of ectopic calcification, oral supplementation therapy was initially not considered because of its low bioavailability. Our earlier work however demonstrated that orally administered pyrophosphate inhibits ectopic calcification in the animal models of PXE and GACI, and that orally given Na P O is absorbed in humans. Here, we report that gelatin-encapsulated Na H P O has similar absorption properties in healthy volunteers and people affected by PXE. The sodium-free K H P O form resulted in similar uptake in healthy volunteers and inhibited calcification in Abcc6 mice as effectively as its sodium counterpart. Novel pyrophosphate compounds showing higher bioavailability in mice were also identified. Our results provide an important step towards testing oral PP in clinical trials in PXE, or potentially any condition accompanied by ectopic calcification including diabetes, chronic kidney disease or ageing.
Topics: Animals; Dietary Supplements; Diphosphates; Humans; Mice; Mutation; Phosphoric Diester Hydrolases; Pseudoxanthoma Elasticum; Pyrophosphatases; Vascular Calcification
PubMed: 34758173
DOI: 10.1111/exd.14498 -
MBio Dec 2022Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule...
Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP. IP dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg or Mn; IP, 5-IP, or 1,5-IP) and the other which was ligand-free. The ligand-free enzyme adopts an "open" conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP or 5-IP engagement above the nucleotide favors adoption of a "closed" conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3'-OH and of Glu248 with adenine-. Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize -benzyl-ATP as phosphate donor. The inositol pyrophosphate signaling molecule 1,5-IP modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular IP levels are determined by Asp1, a bifunctional enzyme composed of an N-terminal kinase and a C-terminal pyrophosphatase domain. Here, we present a series of crystal structures of the Asp1 kinase domain, in a ligand-free state and in complexes with nucleotides ADPNP, ADP, and ATP, divalent cations magnesium and manganese, and inositol polyphosphates IP, 5-IP, and 1,5-IP. Substrate binding elicits a switch from open to closed conformations, entailing a disordered-to-ordered transition and a rearrangement or movement of two peptide segments that form a binding site for the phospho-acceptor. Our structures, along with structure-guided mutagenesis, fortify understanding of the mechanism and substrate specificity of Asp1 kinase, and they extend and complement structural and functional studies of the orthologous human kinase PPIP5K2.
Topics: Humans; Adenosine Diphosphate; Adenosine Triphosphate; Diphosphates; Inositol Phosphates; Multifunctional Enzymes; Phosphotransferases (Phosphate Group Acceptor); Pyrophosphatases; Schizosaccharomyces; Schizosaccharomyces pombe Proteins
PubMed: 36468882
DOI: 10.1128/mbio.03087-22