Search

ENPP1 acts as a negative regulator of the cGAS-STING pathway through the hydrolysis of 2'3'-cGAMP. Inhibitors of ENPP1 are regarded as promising agents for stimulating the immune response in cancer immunotherapy. This study describes the identification and optimization of imidazo[1,2- a ]pyrazine derivative 7 as a highly potent and selective ENPP1 inhibitor. Compound 7 demonstrated substantial inhibitory activity against ENPP1 with an IC 50 value of 5.70 or 9.68 nM while showing weak inhibition against ENPP2 and ENPP3. Furthermore, compound 7 was shown to enhance the mRNA expression of cGAMP-induced STING pathway downstream target genes, such as IFNB1 , CXCL10 , and IL 6. In vivo pharmacokinetic and antitumor studies showed promising results, with 7 not only exhibiting efficient pharmacokinetic properties but also enhancing the antitumor efficacy of the anti-PD-1 antibody. Treatment with 7 (80 mg/kg) combined with anti-PD-1 antibody achieved a tumor growth inhibition rate of 77.7% and improved survival in a murine model.

The human gastric pathogen Helicobacter pylori chronically affects the gastric mucosal layer of approximately half of world's population. The emergence of resistant strains urges the need for identification of novel and selective drug against new molecular targets. A ubiquitous enzyme, Deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), is considered as first line of defense against uracil mis-incorporation into DNA, and essential for genome integrity. Lack of dUTPase triggers an elevated recombination frequency, DNA breaks and ultimately cell death. Hence, dUTPase can be considered as a promising target for development of novel lead inhibitor compounds in H. pylori treatment. Herein, we report the generation of three-dimensional model of the target protein using comparative modelling and its validation. To identify dUTPase inhibitors, a high throughput virtual screening approach utilizing Knowledge-based inhibitors and DrugBank database was implemented. Top ranked compounds were scrutinized based on investigations of the protein-ligand interaction fingerprints, molecular interaction maps and binding affinities and the drug potentiality. The best ligands were studied further for complex stability and intermolecular interaction profiling with respect to time under 100 ns classical molecular dynamic stimulation, establishing significant stability in dynamic states as observed from RMSD and RMSF parameters and interactions with the catalytic site residues. The binding free energy calculation computed using MM-GBSA method from the MD simulation trajectories demonstrated that our molecules possess strong binding affinity towards the Helicobacter pylori dUTPase protein. We conclude that our proposed molecules may be potential lead molecules for effective inhibition against the H. pylori dUTPase protein subject to experimental validation.Communicated by Ramaswamy H. Sarma.

The STING (stimulator of interferon genes) pathway is one of the pathways that regulate innate immunity, and the extracellular hydrolytic enzyme ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as its dominant negative regulator. Since activation of the innate immune system is a promising strategy for the treatment of various infectious diseases and cancers, ENPP1 inhibitors have attracted great attention as candidate drugs. We have previously identified small-molecule ENPP1 inhibitors having a [1,2,4]triazolo[1,5-a]pyrimidine scaffold by means of chemical screening using a fluorescence probe, TG-mAMP. In this study, we evaluated the structure-activity relationships of the hit and lead compounds in detail, and succeeded in developing compounds that strongly and selectively inhibit ENPP1 not only in vitro, but also in cellular systems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1) is the dominant hydrolase of 2',3'-cyclic GMP-AMP (cGAMP). Inhibition of ENPP1 contributes to increased cGAMP concentration and stimulator of interferon gene (STING) activation, with the potential to boost immune response against cancer. ENPP1 is a promising therapeutic target in tumor immunotherapy. To date, orally bioavailable ENPP1 inhibitors with highly potent activity under physiological conditions have been rarely reported. Herein, we report our effort in the design and synthesis of two different series of ENPP1 inhibitors, and in the identification of a highly potent ENPP1 inhibitor 27 (IC 50  = 1.2 nM at pH 7.5), which significantly enhanced the cGAMP-mediated STING activity in THP-1 cells. Phosphonate compound 27 has good preclinical pharmacokinetic profiles with low plasma clearance rate in mouse, rat, and dog. It has been developed as bis-POM prodrug 36 which successfully improves the oral bioavailability of 27. In the Pan02 syngeneic mouse model of pancreatic cancer, orally administered 36 showed synergistic effect in combination with radiotherapy., Competing Interests: Declaration of competing interest The authors are all employees of Haihe Biopharma. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The zoonosis caused by Nocardia is increasing seriously. But commonly used antibiotic drugs often lead to resistance. N. seriolae dUTPase ( Ns dUTPase) plays a key role in the proliferation of Nocardia , and was regarded as a potent drug target. However, there was little report about the Ns dUTPase inhibitors. In this study, we discovered a series of novel Ns dUTPase inhibitors to fight against Nocardia . The first crystal structure of Ns dUTPase was released, and a structure-based computational design was performed. Compounds 4b and 12b exhibited promising activities towards Ns dUTPase (IC 50  = 0.99 μM and 0.7 μM). In addition, they showed satisfied anti- Nocardia activity (MIC value ranges from 0.5 to 2 mg/L) and low cytotoxicity, which were better than approved drugs oxytetracycline and florfenicol. Molecular modelling study indicated that hydrophobic interaction might be the main contribution for ligand binding. Our results suggested that Ns dUTPase inhibitors might be a useful way to repress Nocardia .

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

Recent studies in colorectal cancer patients (CRC) have shown that increased resistance to thymidylate synthase (TS) inhibitors such as 5-fluorouracil (5-FU), reduce the efficacy of standard of care (SoC) treatment regimens. The nucleotide pool cleanser dUTPase is highly expressed in CRC and is an attractive target for potentiating anticancer activity of chemotherapy. The purpose of the current work was to investigate the activity of P 1 , P 4 -di(2',5'-dideoxy-5'-selenouridinyl)-tetraphosphate (P 4 -SedU 2 ), a selenium-modified symmetrically capped dinucleoside with prodrug capabilities that is specifically activated by dUTPase. Using mechanochemistry, P 4 -SedU 2 and the corresponding selenothymidine analogue P 4 -SeT 2 were prepared with a yield of 19% and 30% respectively. The phosphate functionality facilitated complexation with the amphipathic cell-penetrating peptide RALA to produce nanoparticles (NPs). These NPs were designed to deliver P 4 -SedU 2 intracellularly and thereby maximise in vivo activity. The NPs demonstrated effective anti-cancer activity and selectivity in the HCT116 CRC cell line, a cell line that overexpresses dUTPase; compared to HT29 CRC cells and NCTC-929 fibroblast cells which have reduced levels of dUTPase expression. In vivo studies in BALB/c SCID mice revealed no significant toxicity with respect to weight or organ histology. Pharmacokinetic analysis of blood serum showed that RALA facilitates effective delivery and rapid internalisation into surrounding tissues with NPs eliciting lower plasma C max than the equivalent injection of free P 4 -SedU 2 , translating the in vitro findings. Tumour growth delay studies have demonstrated significant inhibition of growth dynamics with the tumour doubling time extended by >2weeks. These studies demonstrate the functionality and action of a new pro-drug nucleotide for CRC., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

An extensive range of new biologically active morpholine based thiosemicarbazones derivatives 3a-r were synthesized, characterized by spectral techniques and evaluated as inhibitors of ENPP isozymes. Most of the novel thiosemicarbazones exhibit potent inhibition towards NPP1 and NPP3 isozymes. Compound 3 h was potent inhibitor of NPP1 with IC 50 value of 0.55 ± 0.02. However, the most powerful inhibitor of NPP3 was 3e with an IC 50 value of 0.24 ± 0.02. Furthermore, Lineweaver-Burk plot for compound 3 h against NPP1 and for compound 3e against NPP3 was devised through enzymes kinetics studies. Molecular docking and in silico studies was also done for analysis of interaction pattern of all newly synthesized compounds. The results were further validated by molecular dynamic (MD) simulation where the stability of conformational transformation of the best protein-ligand complex (3e) were justified on the basis of RMSD and RMSF analysis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Ecto-nucleotide pyrophosphatase/phosphodiesterases 1 (ENPP1 or NPP1), is an attractive therapeutic target for various diseases, primarily cancer and mineralization disorders. The ecto-enzyme is located on the cell surface and has been implicated in the control of extracellular levels of nucleotide, nucleoside and (di) phosphate. Recently, it has emerged as a critical phosphodiesterase that hydrolyzes cyclic 2'3'- cGAMP, the endogenous ligand for STING (STimulator of INterferon Genes). STING plays an important role in innate immunity by activating type I interferon in response to cytosolic 2'3'-cGAMP. ENPP1 negatively regulates the STING pathway and hence its inhibition makes it an attractive therapeutic target for cancer immunotherapy. Herein, we describe the design, optimization and biological evaluation studies of a series of novel non-nucleotidic thioguanine based small molecule inhibitors of ENPP1. The lead compound 43 has shown good in vitro potency, stability in SGF/SIF/PBS, selectivity, ADME properties and pharmacokinetic profile and finally potent anti-tumor response in vivo. These compounds are a good starting point for the development of potentially effective cancer immunotherapy agents., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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., (© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH.)

Geraniol, citronellol and their esters are high-value acyclic monoterpenes used in food technology, perfumery and cosmetics. A major source of these compounds is the essential oil of rose-scented geraniums of the genus Pelargonium. We provide evidence that their biosynthesis mainly takes place in the cytosol of glandular trichomes via geranyl monophosphate (GP) through the action of a Nudix hydrolase. Protein preparations could convert geranyl diphosphate (GDP) to geraniol in in vitro assays, a process which could be blocked by inorganic phosphatase inhibitors, suggesting a two-step conversion of GDP to geraniol. Pelargonium graveolens chemotypes enriched in either geraniol or (-)-citronellol accumulate GP or citronellyl monophosphate (CP), respectively, the presumed precursors to their monoterpenoid end products. Geranyl monophosphate was highly enriched in isolated glandular trichomes of lines producing high amounts of geraniol. In contrast, (-)-isomenthone-rich lines are depleted in these prenyl monophosphates and monoterpene alcohols and instead feature high levels of GDP, the precursor to plastidic p-menthane biosynthesis. A Nudix hydrolase cDNA from Pelargonium glandular trichomes, dubbed PgNdx1, encoded a cytosolic protein capable of hydrolyzing GDP to GP with a K M of about 750 nm but is only weakly active towards farnesyl diphosphate. In citronellol-rich lines, GDP, GP and CP were detected in nearly equimolar amounts, while citronellyl diphosphate was absent, suggesting that citronellol biosynthesis may proceed by reduction of GP to CP in this species. These findings highlight the cytosol as a compartment that supports monoterpene biosynthesis and expands the roles of Nudix hydrolases in the biosynthesis of plant volatiles., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

The innate immune response to cancer is initiated by cytosolic DNA, where it binds to cGAS and triggers type I interferon (IFN) expression via the STING receptor, leading to activation of tumor-specific T cells. Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) has been identified as the primary enzyme responsible for degrading cGAMP, and therefore it is under intense investigation as a therapeutic target for cancer immunotherapy. ENPP1 hydrolyzes cGAMP to produce AMP and GMP, and hydrolyzes ATP and other nucleotides to monophosphates and pyrophosphate. We developed a robust, high-throughput screening (HTS)-compatible enzymatic assay method for ENPP1 using the Transcreener AMP 2 /GMP 2 Assay, a competitive fluorescence polarization (FP) immunoassay that enables direct detection of AMP and GMP in a homogenous format. The monoclonal antibody used in the Transcreener AMP 2 /GMP 2 Assay showed more than 104-fold selectivity for AMP and GMP versus cGAMP, and 3000-fold selectivity for AMP over ATP, indicating that the assay can be used for detection at initial velocity with either substrate. A working concentration of 100 pM ENPP1 was determined as optimal with a 60 min reaction period, enabling screening with very low quantities of enzyme. A Z' value of 0.72 was determined using ATP as substrate, indicating a high-quality assay. Consistent with previous studies, we found that ENPP1 preferred ATP as a substrate when compared with other nucleotides like GTP, ADP, and GDP. ENPP1 showed a 20-fold selectivity for 2'3'cGAMP compared with 2'3'c-diGMP and showed no activity with 3'3'c-diAMP. The Transcreener AMP 2 /GMP 2 Assay should prove to be a valuable tool for the discovery of ENPP1 lead molecules.

Ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs) together with nucleoside triphosphate diphosphohydrolases (NTPDases) and alkaline phosphatases (APs) are nucleotidases located at the surface of the cells. NPP1 and NPP3 are important members of NPP family that are known as druggable targets for a number of disorders such as impaired calcification, type 2 diabetes, and cancer. Sulfonylurea derivatives have been reported as antidiabetic and anticancer agents, therefore, we synthesized and investigated series of sulfonylurea derivatives 1a-m possessing pyrrolo[2,3-b]pyridine core as inhibitors of NPP1 and NPP3 isozymes that are over-expressed in cancer and diabetes. The enzymatic evaluation highlighted compound 1a as selective NPP1 inhibitor, however, 1c was observed as the most potent inhibitor of NPP1 with an IC 50 value of 0.80 ± 0.04 μM. Compound 1l was found to be the most potent and moderately selective inhibitor of NPP3 (IC 50  = 0.55 ± 0.01 μM). Furthermore, in vitro cytotoxicity assays of compounds 1a-m against MCF-7 and HT-29 cancer cell lines exhibited compound 1c (IC 50  = 4.70 ± 0.67 μM), and 1h (IC 50  = 1.58 ± 0.20 μM) as the most cytotoxic compounds against MCF-7 and HT-29 cancer cell lines, respectively. Both of the investigated compounds showed high degree of selectivity towards cancer cells than normal cells (WI-38). Molecular docking studies of selective and potent enzyme inhibitors revealed promising mode of interactions with important binding sites residues of both isozymes i.e., Thr256, His380, Lys255, Asn277 residues of NPP1 and His329, Thr205, and Leu239 residues of NPP3. In addition, the most potent antiproliferative agent, compound 1h, doesn't produce hypoglycemia as a side effect when injected to mice. This is an additional merit of the promising compound 1h., Competing Interests: Declaration of competing interest None., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

Bacterial infections caused by multi-drug-resistant Gram-negative pathogens pose a serious threat to public health. Gram-negative bacteria are characterized by the enrichment of lipid A-anchored lipopolysaccharide (LPS) or lipooligosaccharide (LOS) in the outer leaflet of their outer membrane. Constitutive biosynthesis of lipid A via the Raetz pathway is essential for bacterial viability and fitness in the human host. The inhibition of early-stage lipid A enzymes such as LpxC not only suppresses the growth of Pseudomonas aeruginosa , Klebsiella pneumoniae , Enterobacter spp., and other clinically important Gram-negative pathogens but also sensitizes these bacteria to other antibiotics. The inhibition of late-stage lipid A enzymes such as LpxH is uniquely advantageous because it has an extra mechanism of bacterial killing through the accumulation of toxic lipid A intermediates, rendering LpxH inhibition additionally lethal to Acinetobacter baumannii . Because essential enzymes of the Raetz pathway have never been exploited by commercial antibiotics, they are excellent targets for the development of novel antibiotics against multi-drug-resistant Gram-negative infections.This Account describes the ongoing research on characterizing the structure and inhibition of LpxC and LpxH, the second and fourth enzymes of the Raetz pathway of lipid A biosynthesis, in the laboratories of Dr. Pei Zhou and Dr. Jiyong Hong at Duke University. Our studies have elucidated the molecular basis of LpxC inhibition by the first broad-spectrum inhibitor, CHIR-090, as well as the mechanism underlying its spectrum of activity. Such an analysis has provided a molecular explanation for the broad-spectrum antibiotic activity of diacetylene-based LpxC inhibitors. Through the structural and biochemical investigation of LpxC inhibition by diacetylene LpxC inhibitors and the first nanomolar LpxC inhibitor, L-161,240, we have elucidated the intrinsic conformational and dynamics difference in individual LpxC enzymes near the active site. A similar approach has been taken to investigate LpxH inhibition, leading to the establishment of the pharmacophore model of LpxH inhibitors and subsequent structural elucidation of LpxH in complex with its first reported small-molecule inhibitor based on a sulfonyl piperazine scaffold.Intriguingly, although our crystallographic analysis of LpxC- and LpxH-inhibitor complexes detected only a single inhibitor conformation in the crystal lattice, solution NMR studies revealed the existence of multiple ligand conformations that together delineate a cryptic ligand envelope expanding the ligand-binding footprint beyond that observed in the crystal structure. By harnessing the ligand dynamics information and structural insights, we demonstrate the feasibility to design potent LpxC and LpxH inhibitors by merging multiple ligand conformations. Such an approach has enabled us to rationally design compounds with significantly enhanced potency in enzymatic assays and outstanding antibiotic activities in vitro and in animal models of bacterial infection. We anticipate that continued efforts with structure and ligand dynamics-based lead optimization will ultimately lead to the discovery of LpxC- and LpxH-targeting clinical antibiotics against a broad range of Gram-negative pathogens.

Rationale: Pulmonary arterial hypertension (PAH) is a life-threatening condition characterized by abnormally elevated pulmonary pressures and right ventricular failure. Excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) is one of the most important drivers of vascular remodeling in PAH, for which available treatments have limited effectiveness. Objectives: To gain insights into the mechanisms leading to the development of the disease and identify new actionable targets. Methods: Protein expression profiling was conducted by two-dimensional liquid chromatography coupled to tandem mass spectrometry in isolated PASMCs from controls and patients with PAH. Multiple molecular, biochemical, and pharmacologic approaches were used to decipher the role of NUDT1 (nudrix hyrolase 1) in PAH. Measurements and Main Results: Increased expression of the detoxifying DNA enzyme NUDT1 was detected in cells and tissues from patients with PAH and animal models. In vitro , molecular or pharmacological inhibition of NUDT1 in PAH-PASMCs induced accumulation of oxidized nucleotides in the DNA, irresolvable DNA damage (comet assay), disruption of cellular bioenergetics (Seahorse), and cell death (terminal deoxynucleotidyl transferase dUTP nick end labeling assay). In two animal models with established PAH (i.e., monocrotaline and Sugen/hypoxia-treated rats), pharmacological inhibition of NUDT1 using (S)-Crizotinib significantly decreased pulmonary vascular remodeling and improved hemodynamics and cardiac function. Conclusions: Our results indicate that, by overexpressing NUDT1, PAH-PASMCs hijack persistent oxidative stress in preventing incorporation of oxidized nucleotides into DNA, thus allowing the cell to escape apoptosis and proliferate. Given that NUDT1 inhibitors are under clinical investigation for cancer, they may represent a new therapeutic option for PAH.

Extracellular ATP mediates proinflammatory and antiproliferative effects via activation of P2 nucleotide receptors. In contrast, its metabolite, the nucleoside adenosine, is strongly immunosuppressive and enhances tumor proliferation and metastasis. The conversion of ATP to adenosine is catalyzed by ectonucleotidases, which are expressed on immune cells and typically upregulated on tumor cells. In the present study, we identified sulfopolysaccharides from brown and red sea algae to act as potent dual inhibitors of the main ATP-hydrolyzing ectoenzymes, ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and ecto-nucleoside triphosphate diphosphohydrolase-1 (NTPDase1, CD39), showing nano- to picomolar potency and displaying a non-competitive mechanism of inhibition. We showed that one of the sulfopolysaccharides tested as a representative example reduced adenosine formation at the surface of the human glioblastoma cell line U87 in a concentration-dependent manner. These natural products represent the most potent inhibitors of extracellular ATP hydrolysis known to date and have potential as novel therapeutics for the immunotherapy of cancer.

African swine fever, caused by the African swine fever virus (ASFV), is among the most significant swine diseases. There are currently no effective treatments against ASFV. ASFV contains a gene encoding a dUTPase (E165R), which is required for viral replication in swine macrophages, making it an attractive target for inhibitor development. However, the full structural details of the ASFV dUTPase and those of the comparable swine enzyme are not available, limiting further insights. Herein, we determine the crystal structures of ASFV dUTPase and swine dUTPase in both their ligand-free and ligand-bound forms. We observe that the swine enzyme employs a classical dUTPase architecture made up of three-subunit active sites, whereas the ASFV enzyme employs a novel two-subunit active site. We then performed a comparative analysis of all dUTPase structures uploaded in the Protein Data Bank (PDB), which showed classical and non-classical types were mainly determined by the C-terminal β-strand orientation, and the difference was mainly related to the four amino acids behind motif IV. Thus, our study not only explains the reason for the structural diversity of dUTPase but also reveals how to predict dUTPase type, which may have implications for the dUTPase family. Finally, we tested two dUTPase inhibitors developed for the Plasmodium falciparum dUTPase against the swine and ASFV enzymes. One of these compounds inhibited the ASFV dUTPase at low micromolar concentrations (K d  = 15.6 μM) and with some selectivity (∼2x) over swine dUTPase. In conclusion, our study expands our understanding of the dUTPase family and may aid in the development of specific ASFV inhibitors., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Deficiency in DNA repair proteins confers susceptibility to DNA damage, making cancer cells vulnerable to various cancer chemotherapies. 5-Fluorouracil (5-FU) is an anticancer nucleoside analog that both inhibits thymidylate synthase (TS) and causes DNA damage via the misincorporation of FdUTP and dUTP into DNA under the conditions of dTTP depletion. However, the role of the DNA damage response to its antitumor activity is still unclear. To determine which DNA repair pathway contributes to DNA damage caused by 5-FU and uracil misincorporation, we examined cancer cells treated with 2'-deoxy-5-fluorouridine (FdUrd) in the presence of TAS-114, a highly potent inhibitor of dUTPase that restricts aberrant base misincorporation. Addition of TAS-114 increased FdUTP and dUTP levels in HeLa cells and facilitated 5-FU and uracil misincorporation into DNA, but did not alter TS inhibition or 5-FU incorporation into RNA. TAS-114 showed synergistic potentiation of FdUrd cytotoxicity and caused aberrant base misincorporation, leading to DNA damage and induced cell death even after short-term exposure to FdUrd. Base excision repair (BER) and homologous recombination (HR) were found to be involved in the DNA repair of 5-FU and uracil misincorporation caused by dUTPase inhibition in genetically modified chicken DT40 cell lines and siRNA-treated HeLa cells. These results suggested that BER and HR are major pathways that protect cells from the antitumor effects of massive incorporation of 5-FU and uracil. Further, dUTPase inhibition has the potential to maximize the antitumor activity of fluoropyrimidines in cancers that are defective in BER or HR., (© 2020 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Identification of potent anticancer agents with high selectivity and low toxicity remains on the way to human health. Pyridazine featuring advantageous physicochemical properties and antitumor potential usually is regarded as a central core in numerous anticancer derivatives. There are several approved pyridazine-based drugs in the market and analogues currently going through different clinical phases or registration statuses, suggesting pyridazine as a promising drug-like scaffold. The current review is intended to provide a comprehensive and updated overview of pyridazine derivatives as potential anticancer agents. In particular, we focused on their structure-activity relationship (SAR) studies, design strategies, binding modes and biological activities in the hope of offering novel insights for further rational design of more active and less toxic anticancer drugs., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

The enzyme NUDT15 efficiently hydrolyzes the active metabolites of thiopurine drugs, which are routinely used for treating cancer and inflammatory diseases. Loss-of-function variants in NUDT15 are strongly associated with thiopurine intolerance, such as leukopenia, and preemptive NUDT15 genotyping has been clinically implemented to personalize thiopurine dosing. However, understanding the molecular consequences of these variants has been difficult, as no structural information was available for NUDT15 proteins encoded by clinically actionable pharmacogenetic variants because of their inherent instability. Recently, the small molecule NUDT15 inhibitor TH1760 has been shown to sensitize cells to thiopurines, through enhanced accumulation of 6-thio-guanine in DNA. Building upon this, we herein report the development of the potent and specific NUDT15 inhibitor, TH7755. TH7755 demonstrates a greatly improved cellular target engagement and 6-thioguanine potentiation compared with TH1760, while showing no cytotoxicity on its own. This potent inhibitor also stabilized NUDT15, enabling analysis by X-ray crystallography. We have determined high-resolution structures of the clinically relevant NUDT15 variants Arg139Cys, Arg139His, Val18Ile, and V18_V19insGlyVal. These structures provide clear insights into the structural basis for the thiopurine intolerance phenotype observed in patients carrying these pharmacogenetic variants. These findings will aid in predicting the effects of new NUDT15 sequence variations yet to be discovered in the clinic., Competing Interests: Conflict of interest The authors declare no competing financial interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cancer cells initiate an innate immune response by synthesizing and exporting the small-molecule immunotransmitter cGAMP, which activates the anti-cancer Stimulator of Interferon Genes (STING) pathway in the host. An extracellular enzyme, ectonucleotide pyrophosphatase phosphodiesterase 1 (ENPP1), hydrolyzes cGAMP and negatively regulates this anti-cancer immune response. Small-molecule ENPP1 inhibitors are much needed as tools to study the basic biology of extracellular cGAMP and as investigational cancer immunotherapy drugs. Here, we surveyed structure-activity relationships around a series of cell-impermeable and thus extracellular-targeting phosphonate inhibitors of ENPP1. In addition, we solved the crystal structure of an exemplary phosphonate inhibitor to elucidate the interactions that drive potency. This study yielded several best-in-class inhibitors with K i  < 2 nM and excellent physicochemical and pharmacokinetic properties. Finally, we demonstrate that an ENPP1 inhibitor delays tumor growth in a breast cancer mouse model. Together, we have developed ENPP1 inhibitors that are excellent tool compounds and potential therapeutics., Competing Interests: Declaration of Interests M.S. and L.L. are scientific cofounders of Angarus Therapeutics, which has exclusive licensing rights to patents PCT/US2018/50,018 and PCT/US2020/015968. J.A.C., V.B., M.S., and L.L. are inventors on patents PCT/US2018/50,018 and PCT/US2020/015968., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Ectonucleotidases are a broad family of ectoenzymes that play a crucial role in purinergic cell signaling. Ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs) belong to this group and are important drug targets. In particular, NPP1 and NPP3 are known to be druggable targets for treatment of impaired calcification disorders (including pathological aortic calcification) and cancer, respectively. In this study, we investigated a series of sulfonate and sulfamate derivatives of benzofuran and benzothiophene as potent and selective inhibitors of NPP1 and NPP3. Compounds 1c, 1g, 1n, and 1s are the most active NPP1 inhibitors (IC 50 values in the range 0.12-0.95 µM). Moreover, compounds 1e, 1f, 1j, and 1l are the most potent inhibitors of NPP3 (IC 50 ranges from 0.12 to 0.95 µM). Compound 1d, 1f and 1t are highly selective inhibitors of NPP1 over NPP3, whereas compounds 1m and 1s are found to be highly selective towards NPP3 over NPP1. Structure-activity relationship (SAR) study has been discussed in detailed. With the aid of molecular docking studies, a common binding mode of these compounds and suramin (the standard inhibitor) was revealed, where the sulfonate group acts as a cation-binding moiety that comes in close contact with the zinc ion of the active site. Moreover, cytotoxic evaluation against MCF-7 and HT-29 cancer cell lines revealed that compound 1r is the most cytotoxic towards MCF-7 cell line with IC 50 value of 0.19 µM. Compound 1r is more potent and selective against cancer cells than normal cells (WI-38) as compared to doxorubicin., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Introduction TAS-114 is a potent inhibitor of deoxyuridine triphosphatase, which is a gatekeeper protein preventing uracil and 5-fluorouracil (5-FU) misincorporation into DNA. TAS-114 has been suggested to enhance the antitumor activity of 5-FU. This randomized, phase 2 study investigated TAS-114 plus S-1 (TAS-114/S-1) vs. S-1 in non-small-cell lung cancer (NSCLC) patients. Methods Patients with advanced NSCLC, previously treated with ≥ 2 regimens, were randomized 1:1 to receive TAS-114 (400 mg)/S-1 (30 mg/m 2 ) or S-1 (30 mg/m 2 ). Progression-free survival (PFS, independent central review) was the primary endpoint. Secondary endpoints included disease control rate (DCR), overall survival (OS), overall response rate (ORR), and safety. Results In total, 127 patients received treatment. Median PFS was 3.65 and 4.17 months in the TAS-114/S-1 and S-1 groups, respectively (hazard ratio [HR] 1.16, 95% confidence interval [CI] 0.71-1.88; P = 0.2744). DCR was similar between groups (TAS-114/S-1 80.3%, S-1 75.9%) and median OS was 7.92 and 9.82 months for the TAS-114/S-1 and S-1 groups, respectively (HR 1.31, 95% CI 0.80-2.14; P = 0.1431). The ORR was higher in the TAS-114/S-1 group than the S-1 group (19.7% vs. 10.3%), and more patients with tumor shrinkage were observed in the TAS-114/S-1 group. Incidence rates of anemia, skin toxicities, and Grade ≥ 3 treatment-related adverse events were higher in the TAS-114/S-1 group compared with the monotherapy group. Conclusions Although the TAS-114/S-1 combination improved the response rate, this did not translate into improvements in PFS. Clinical Trial Registration No. NCT02855125 (ClinicalTrials.gov) registered on 4 August 2016.

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.

A series of six compounds (1a-f) possessing pyridine-pyrazole-benzenethiourea or pyridine-pyrazole-benzenesulfonamide scaffold were synthesized. The target compounds were screened to evaluate their inhibitory effect on human nucleotide pyrophosphatase/phosphodiesterase 1 and -3 (ENPP1 and ENPP3) isoenzymes. Compounds 1c-e were the most potent inhibitors of ENPP1 with sub-micromolar IC 50 values (0.69, 0.18, and 0.40 µM, respectively. Moreover, compound 1b was the most potent inhibitor of ENPP3 (IC 50  = 0.21 µM). They were much more potent than the reference standard inhibitor, suramin (IC 50 values against ENPP1 and -3 were 7.77 and 0.89 µM, respectively). Furthermore, all the six compounds were investigated for cytotoxic effect against cancerous cell lines (HeLa, MCF-7, and 1321N1) and normal cell line (BHK-21). Compound 1e was active against all the three cancer cell lines, however, showed preferential cytotoxicity against MCF-7 (IC 50  = 16.05 µM), which is comparable to the potency of cisplatin. All the tested compounds exhibited low or negligible cytotoxic effect against the normal cells. They have the merit of superior selectivity towards cancer cells than normal cells compared to cisplatin. The relative selectivity and potency of the inhibitors was justified by molecular docking studies. All the docked structures showed considerable binding interactions with amino acids residues of active sites of ENPP isoenzymes., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier Inc.)

The UDP-2,3-diacylglucosamine pyrophosphate hydrolase LpxH is an essential lipid A biosynthetic enzyme that is conserved in the majority of gram-negative bacteria. It has emerged as an attractive novel antibiotic target due to the recent discovery of an LpxH-targeting sulfonyl piperazine compound (referred to as AZ1) by AstraZeneca. However, the molecular details of AZ1 inhibition have remained unresolved, stymieing further development of this class of antibiotics. Here we report the crystal structure of Klebsiella pneumoniae LpxH in complex with AZ1. We show that AZ1 fits snugly into the L -shaped acyl chain-binding chamber of LpxH with its indoline ring situating adjacent to the active site, its sulfonyl group adopting a sharp kink, and its N -CF 3 -phenyl substituted piperazine group reaching out to the far side of the LpxH acyl chain-binding chamber. Intriguingly, despite the observation of a single AZ1 conformation in the crystal structure, our solution NMR investigation has revealed the presence of a second ligand conformation invisible in the crystalline state. Together, these distinct ligand conformations delineate a cryptic inhibitor envelope that expands the observed footprint of AZ1 in the LpxH-bound crystal structure and enables the design of AZ1 analogs with enhanced potency in enzymatic assays. These designed compounds display striking improvement in antibiotic activity over AZ1 against wild-type K. pneumoniae , and coadministration with outer membrane permeability enhancers profoundly sensitizes Escherichia coli to designed LpxH inhibitors. Remarkably, none of the sulfonyl piperazine compounds occupies the active site of LpxH, foretelling a straightforward path for rapid optimization of this class of antibiotics., Competing Interests: Competing interest statement: P.Z. and J.H. are inventors of a provisional patent covering the designed LpxH inhibitors.

Overexpression of ecto-nucleotide pyrophosphatase-1 (NPP1) is associated with diseases such as calcium pyrophosphate dihydrate deposition disease, calcific aortic valve disease, and type 2 diabetes. In this context, NPP1 inhibitors are potential drug candidates for the treatment of these diseases. The present study focuses on the analysis of the structure-activity relationship of NPP1 inhibitors based on acyclic uracil-nucleotides. For this purpose, we synthesized acyclic uridine-monophosphate analogs, 10-11, uridine-diphosphate analogs, 12-14, and uridine-P α , α -dithio-triphosphate analogs, 15-17. We evaluated their inhibitory activity and selectivity towards NPP1, -3, NTPDase1, -2, -3, and -8, and P2Y 2,4,6 receptors. Analogs 16 and 17 were the most selective and potent NPP1 inhibitors (Ki 0.94 and 0.73 μM, respectively) among the tested molecules. Analogs 10-17 had only minute effect on uracil-nucleotide responding P2Y 2,4,6 receptors. Analog 17 (100 μM) displayed 96% inhibition of NPPase activity in osteoarthritic human chondrocytes. Analogs 14-17 displayed weak inhibitory effect on alkaline phosphatase activity at equimolar concentrations in human chondrocytes. All tested analogs showed no toxicity at human chondrocytes. We concluded that ribose-ring to chain transformation, as well as the type of the nucleobase, are parameters of minor significance to NPP1 inhibition, whereas the major parameter is P α -dithio-substitution. In addition, the length of the phosphate chain also significantly affects inhibition. Overall, the experimental results were well reproduced by molecular docking. A correlation was observed between the activities of the compounds and the number of H-bonds and salt bridges formed between the inhibitors and NPP1 binding site residues. Uracil-N1-(methoxy)ethyl-β-P α,α -dithio, P β,γ -methylene tri-phosphate, 17, was identified as the most potent, selective, and non-toxic NPP1 inhibitor among the tested analogs, and may be used as a lead structure for further drug development., (Copyright © 2019. Published by Elsevier Masson SAS.)

Nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) inhibitors have been suggested as a potential treatment for calcium pyrophosphate dihydrate (CPPD) deposition disease. Here, we targeted the development of improved NPP1 inhibitors based on acyclic mimics of Pα,α-phosphorodithioate-substituted adenine nucleotides, 7-10. The latter were obtained in a facile two-step synthesis from adenine-(methoxy)ethanol. Among analogs 7-10, adenine-(methoxy)ethoxy-Pα,α-dithio-triphosphate, 8, was the most potent NPP1 inhibitor both with purified enzyme (IC50 0.645 μM) and in osteoarthritic human chondrocytes (IC50 0.033 μM). Furthermore, it efficaciously (10-fold vs. control) inhibited ATP-induced CPPD in human articular chondrocytes. Importantly, 8 was a highly selective NPP1 inhibitor which showed only minor inhibition of NPP3, CD39 and CD73, and did not inhibit TNAP (tissue nonspecific alkaline phosphatase) activity in human chondrocytes. Furthermore, 8 did not activate P2Y1,2,6 receptors. Analog 8 was not toxic to cultured chondrocytes at 100 μM. Therefore, 8 may be suitable for further development as a drug candidate for the treatment of CPPD arthritis and other NPP1-related diseases.

Membrane-bound pyrophosphatases (mPPases) are dimeric enzymes that occur in bacteria, archaea, plants, and protist parasites. These proteins cleave pyrophosphate into two orthophosphate molecules, which is coupled with proton and/or sodium ion pumping across the membrane. Since no homologous proteins occur in animals and humans, mPPases are good candidates in the design of potential drug targets. Here we present a detailed protocol to screen for mPPase inhibitors utilizing the molybdenum blue reaction in a 96 well plate system. We use mPPase from the thermophilic bacterium Thermotoga maritima (TmPPase) as a model enzyme. This protocol is simple and inexpensive, producing a consistent and robust result. It takes only about one hour to complete the activity assay protocol from the start of the assay until the absorbance measurement. Since the blue color produced in this assay is stable for a long period of time, subsequent assay(s) can be performed immediately after the previous batch, and the absorbance can be measured later for all batches at once. The drawback of this protocol is that it is done manually and thus can be exhausting as well as require good skills of pipetting and time keeping. Furthermore, the arsenite-citrate solution used in this assay contains sodium arsenite, which is toxic and should be handled with necessary precautions.

Pyrophosphate deficiency may explain the excessive vascular calcification found in children with Hutchinson-Gilford progeria syndrome (HGPS) and in a mouse model of this disease. The present study found that hydrolysis products of ATP resulted in a <9% yield of pyrophosphate in wild-type blood and aortas, showing that eNTPD activity (ATP → phosphate) was greater than eNPP activity (ATP → pyrophosphate). Moreover, pyrophosphate synthesis from ATP was reduced and pyrophosphate hydrolysis (via TNAP; pyrophosphate → phosphate) was increased in both aortas and blood obtained from mice with HGPS. The reduced production of pyrophosphate, together with the reduction in plasma ATP, resulted in marked reduction of plasma pyrophosphate. The combination of TNAP inhibitor levamisole and eNTPD inhibitor ARL67156 increased the synthesis and reduced the degradation of pyrophosphate in aortas and blood ex vivo, suggesting that these combined inhibitors could represent a therapeutic approach for this devastating progeroid syndrome. Treatment with ATP prevented vascular calcification in HGPS mice but did not extend longevity. By contrast, combined treatment with ATP, levamisole, and ARL67156 prevented vascular calcification and extended longevity by 12% in HGPS mice. These findings suggest a therapeutic approach for children with HGPS., Competing Interests: The author declares no competing interest.

Ectonucleotide pyrophosphatase/phosphodiesterase I (ENPP1) was identified several decades ago as a type II transmembrane glycoprotein with nucleotide pyrophosphatase and phosphodiesterase enzymatic activities, critical for purinergic signaling. Recently, ENPP1 has emerged as a critical phosphodiesterase that degrades the stimulator of interferon genes (STING) ligand, cyclic GMP-AMP (cGAMP). cGAMP or analogs thereof have emerged as potent immunostimulatory agents, which have potential applications in immunotherapy. This emerging role of ENPP1 has placed this "old" enzyme at the frontier of immunotherapy. This review highlights the roles played by ENPP1, the mechanism of cGAMP hydrolysis by ENPP1, and small molecule inhibitors of ENPP1 with potential applications in diverse disease states, including cancer.

A new series of raloxifene sulfonate/sulfamate derivatives were designed and synthesized. The target compounds were tested for inhibitory effect against nucleotide pyrophosphatase/phosphodiesterase-1 and -3 (NPP1 and NPP3) enzymes. Furthermore, all the ten target compounds were subjected to cytotoxic studies on various cancer cell lines, and the most potent derivatives were explored for their potency against these cancer cell lines as well as F180 fibroblasts to investigate the selectivity indexes. Compound 1f exerted the highest potency against HT-29 colon cancer cell line (IC 50  = 1.4 μM) with 8.43-fold selectivity towards HT-29 than F180 fibroblasts. Compound 1f exerted sub-micromolar IC 50 values against NPP1 and NPP3 (IC 50  = 0.29 μM and 0.71 μM, respectively). The most potent inhibitors were docked in developed homology model of NPP1 and crystal structure of NPP3. All the docked analogues manifested remarkable interactions within the active pocket of NPP1 and NPP3., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) is a type II transmembrane glycoprotein that is involved in bone metabolism and insulin resistance, hydrolyzes 2',3'-cGAMP (a STING ligand that promotes innate immunity), and is associated with cancer stemness in breast cancers and glioblastoma. Therefore, ENPP1 is considered a candidate therapeutic target and/or biomarker for early diagnosis of malignant tumors. In this study, we designed and synthesized a sensitive ENPP1 fluorescence probe, Tokyo Green (TG) mAMP. We used it to screen a chemical library for non-phosphate ENPP1 inhibitors. Structural optimization of a selected hit afforded a potent and specific ENPP1 inhibitor. We further found that ENPP1 mRNA expression in tissue samples from patients with triple-negative breast cancer was significantly inversely related to recurrence-free survival (RFS) and overall survival (OS), and TG-mAMP assay revealed a significant difference in ENPP1 activity between ENPP1 high-expressing and ENPP1 low-expressing samples. Our results suggest that TG-mAMP assay might be a rapid and inexpensive tool for predicting the prognosis of patients with malignant breast cancers.

EDTA is commonly used as an efficient chelator of metal ion enzyme cofactors. It is highly soluble, optically inactive and does not interfere with most chemicals used in standard buffers making EDTA a common choice to generate metal-free conditions for biochemical and biophysical investigations. However, the controversy in the literature on metal-free enzyme activities achieved using EDTA or by other means called our attention to a putative effect of EDTA beyond chelation. Here, we show that EDTA competes for the nucleotide binding site of the nucleotide hydrolase dUTPase by developing an interaction network within the active site similar to that of the substrate. To achieve these findings, we applied kinetics and molecular docking techniques using two different dUTPases. Furthermore, we directly measured the binding of EDTA to dUTPases and to two other dNTPases, the Taq polymerase and MutT using isothermal titration calorimetry. EDTA binding proved to be exothermic and mainly enthalpy driven with a submicromolar dissociation constant considerably lower than that of the enzyme:substrate or the Mg:EDTA complexes. Control proteins, including an ATPase, did not interact with EDTA. Our findings indicate that EDTA may act as a selective inhibitor against dNTP hydrolyzing enzymes and urge the rethinking of the utilization of EDTA in enzymatic experiments., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Cell-based screening is a powerful approach to identify novel chemical modulators and biological components of relevant biological processes. The canonical Wnt pathway is essential for normal embryonic development and tissue homeostasis, and its deregulation plays a crucial role in carcinogenesis. Therefore, the identification of new pathway members and regulators is of significant interest. By means of a cell-based assay monitoring Wnt signaling we identified the pyrrolocoumarin Pyrcoumin as inhibitor of canonical Wnt signaling. Target identification and validation revealed that Pyrcoumin is a competitive inhibitor of dCTP pyrophosphatase 1 (dCTPP1). We demonstrate a yet unknown interaction of dCTPP1 with ubiquitin carboxyl-terminal hydrolase (USP7) that is counteracted by dCTPP1 inhibitors. These findings indicate that dCTPP1 plays a role in regulation of Wnt/β-catenin signaling most likely through a direct interaction with USP7., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Antibiotic resistance remains a pressing medical challenge for which novel antibacterial agents are urgently needed. The phenylthiazole scaffold represents a promising platform to develop novel antibacterial agents for drug-resistant infections. However, enhancing the physicochemical profile of this class of compounds remains a challenging endeavor to address to successfully translate these molecules into novel antibacterial agents in the clinic. We extended our understanding of the SAR of the phenylthiazoles' lipophilic moiety by exploring its ability to accommodate a hydrophilic group or a smaller sized hetero-ring with the objective of enhancing the physicochemical properties of this class of novel antimicrobials. Overall, the 2-thienyl derivative 20 and the hydroxyl-containing derivative 31 emerged as the most promising antibacterial agents inhibiting growth of drug-resistant Staphylococcus aureus at a concentration as low as 1 μg/mL. Remarkably, compound 20 suppressed bacterial undecaprenyl pyrophosphatase (UppP), the molecular target of the phenylthiazole compounds, in a sub nano-molar concentration range (almost 20,000 times more potent than the lead compounds 1a and 1b). Compound 31 possessed the most balanced antibacterial and physicochemical profile. The compound exhibited rapid bactericidal activity against S. aureus, and successfully cleared intracellular S. aureus within infected macrophages. Furthermore, insertion of the hydroxyl group enhanced the aqueous solubility of 31 by more than 50-fold relative to the first-generation lead 1c., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

With the aim to discover novel, efficient and selective inhibitors of human alkaline phosphatase and nucleotide pyrophosphatase enzymes, two new series of pyrazolyl pyrimidinetriones (PPTs) (6a-g) and thioxopyrimidinediones (PTPs) (6h-n) were synthesized in good chemical yields using Knoevenagel condensation reaction between pyrazole carbaldehydes (4a-g) and pharmacologically active N-alkylated pyrimidinetrione (5a) and thioxopyrimidinedione (5b). The inhibition potential of the synthesized hybrid compounds was evaluated against human alkaline phosphatase (h-TNAP and h-IAP) and ectonucleotidase (h-NPP1 and h-NPP3) enzymes. Most of the tested analogs were highly potent with a variable degree of inhibition depending on the functionalized hybrid structure. The detailed structure-activity relationship (SAR) of PPT and PTP derivatives suggested that the compound with unsubstituted phenyl ring from PPT series led to selective and potent inhibition (6a; IC 50  = 0.33 ± 0.02 µM) of h-TNAP, whereas compound 6c selectively inhibited h-IAP isozyme with IC 50 value of 0.86 ± 0.04 µM. Similarly, compounds 6b and 6h were identified as the lead scaffolds against h-NPP1 and h-NPP3, respectively. The probable binding modes for the most potent inhibitors were elucidated through molecular docking analysis. Structure-activity relationships, mechanism of action, cytotoxic effects and druglikeness properties are also discussed., (Copyright © 2019. Published by Elsevier Inc.)

Ectonucleoside Triphosphate Diphosphohydrolase 5 (ENTPD5) has been shown to be important in maintaining cellular function in cancer, and its expression is upregulated through multiple, unique pathways in certain cancers, including laryngeal, glioblastoma multiforme, breast, testicular, and prostate. ENTPD5 supports cancer growth by promoting the import of UDP-glucose, a metabolite used for protein glycosylation and hence proper glycoprotein folding, into the ER by providing the counter molecule, UMP, to the ER antiporter. Despite its cancer-supporting function, no small molecule inhibitors of ENTPD5 are commercially available, and few studies have been performed in tissue culture to understand the effects of chemical inhibition of ENTPD5. We performed a high-throughput screen (HTS) of 21,120 compounds to identify small molecule inhibitors of ENPTD5 activity. Two hits were identified, and we performed a structure activity relationship (SAR) screen around these hits. Further validation of these probes were done in an orthogonal assay and then assayed in cell culture to assess their effect on prostate cancer cell lines. Notably, treatment with the novel ENTPD5 inhibitor reduced the amount of glycoprotein produced in treated cells, consistent with the hypothesis that ENTPD5 is important for glycoprotein folding. This work serves as an important step in designing new molecular probes for ENTPD5 as well as further probing the utility of targeting ENTPD5 to combat cancer cell proliferation., Competing Interests: The authors have declared that no competing interests exist.

Protein inhibitors of key DNA repair enzymes play an important role in deciphering physiological pathways responsible for genome integrity, and may also be exploited in biomedical research. The staphylococcal repressor StlSaPIbov1 protein was described to be an efficient inhibitor of dUTPase homologues showing a certain degree of species-specificity. In order to provide insight into the inhibition mechanism, in the present study we investigated the interaction of StlSaPIbov1 and Escherichia coli dUTPase. Although we observed a strong interaction of these proteins, unexpectedly the E. coli dUTPase was not inhibited. Seeking a structural explanation for this phenomenon, we identified a key amino acid position where specific mutations sensitized E. coli dUTPase to StlSaPIbov1 inhibition. We solved the three-dimensional (3D) crystal structure of such a mutant in complex with the substrate analogue dUPNPP and surprisingly found that the C-terminal arm of the enzyme, containing the P-loop-like motif was ordered in the structure. This segment was never localized before in any other E. coli dUTPase crystal structures. The 3D structure in agreement with solution phase experiments suggested that ordering of the flexible C-terminal segment upon substrate binding is a major factor in defining the sensitivity of E. coli dUTPase for StlSaPIbov1 inhibition., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Overproduction of extracellular diphosphate due to hydrolysis of ATP by NPP1 leads to pathological calcium diphosphate (pyrophosphate) dihydrate deposition (CPPD) in cartilage, resulting in a degenerative joint disease that today lacks a cure. Here, we targeted the identification of novel NPP1 inhibitors as potential therapeutic agents for CPPD deposition disease. Specifically, we synthesized novel analogs of AMP (NPP1 reaction product) and ADP (NPP1 inhibitor). These derivatives incorporate several chemical modifications of the natural nucleotides including (1) a methylene group replacing the P α,β -bridging oxygen atom to provide metabolic resistance, (2) sulfonate group(s) replacing phosphonate(s) to improve binding to NPP1's catalytic zinc ions, (3) an acyclic nucleotide analog to allow flexible binding in the NPP1 catalytic site, and (4) a benzimidazole base replacing adenine. Among the investigated compounds, adenine-N9-(methoxy)ethyl-β-bisphosphonate, 10, was identified as an NPP1 inhibitor (K i 16.3 μM vs. the artificial substrate p-nitrophenyl thymidine-5'-monophosphate (p-Nph-5'-TMP), and 9.60 μM vs. the natural substrate, ATP). Compound 10 was selective for NPP1 vs. human NPP3, human CD39, and tissue non-specific alkaline phosphatase (TNAP), but also inhibited human CD73 (K i 12.6 μM). Thus, 10 is a dual NPP1/CD73 inhibitor, which could not only be of interest for treating CPPD deposition disease and calcific aortic valve disease but may also be considered for the immunotherapy of cancer. Compound 10 proved to be a promising inhibitor, which almost completely reduces NPPase activity in human osteoarthritic chondrocytes at a concentration of 100 μM.

Background This first-in-human phase 1 study assessed the safety of TAS-114, a novel deoxyuridine triphosphatase inhibitor, combined with S-1 to determine its maximum tolerated dose (MTD) and recommended dose (RD). Methods In this dose-escalation study with a 3 + 3 design, TAS-114 and S-1 were concurrently administered orally under fasting conditions at 5-240 mg/m 2 and 30-36 mg/m 2 , respectively, in patients with advanced solid tumors. Safety, efficacy, and pharmacokinetics (PK) were evaluated. Results Seventy-six patients were enrolled. The MTD and RD were TAS-114 200 mg/m 2 plus S-1 36 mg/m 2 and TAS-114 240 mg/m 2 plus S-1 30 mg/m 2 , respectively. Common treatment-related adverse events were anemia, lymphocytopenia, leukopenia, neutropenia, decreased appetite, rash, nausea, and pigmentation disorder. Partial response (PR) was observed in 10 patients (non-small cell lung cancer [NSCLC], n = 5; pancreatic neuroendocrine tumor, n = 2; gastric cancer, n = 2; gallbladder cancer, n = 1). Of these, four patients achieved PR despite prior treatment history with S-1. Patients administered TAS-114 exhibited linear PK and CYP3A4 induction, with no effect on the PK of S-1. Conclusion TAS-114 plus S-1 showed tolerable, safe, and potentially effective results. To confirm safety and efficacy, two phase 2 studies are ongoing in NSCLC and gastric cancer patients. Clinical trial registration ClinicalTrials.gov ( NCT01610479 ) .

Membrane-bound pyrophosphatases are homodimeric integral membrane proteins that hydrolyze pyrophosphate into orthophosphates, coupled to the active transport of protons or sodium ions across membranes. They are important in the life cycle of bacteria, archaea, plants, and parasitic protists, but no homologous proteins exist in vertebrates, making them a promising drug target. Here, we report the first nonphosphorus allosteric inhibitor of the thermophilic bacterium Thermotoga maritima membrane-bound pyrophosphatase and its bound structure together with the substrate analog imidodiphosphate. The unit cell contains two protein homodimers, each binding a single inhibitor dimer near the exit channel, creating a hydrophobic clamp that inhibits the movement of β-strand 1-2 during pumping, and thus prevents the hydrophobic gate from opening. This asymmetry of inhibitor binding with respect to each homodimer provides the first clear structural demonstration of asymmetry in the catalytic cycle of membrane-bound pyrophosphatases.

The UDP-2,3-diacylglucosamine pyrophosphatase LpxH in the Raetz pathway of lipid A biosynthesis is an essential enzyme in the vast majority of Gram-negative pathogens and an excellent novel antibiotic target. The 32 P-radioautographic thin-layer chromatography assay has been widely used for analysis of LpxH activity, but it is inconvenient for evaluation of a large number of LpxH inhibitors over an extended time period. Here, we report a coupled, nonradioactive LpxH assay that utilizes the recently discovered Aquifex aeolicus lipid A 1-phosphatase LpxE for quantitative removal of the 1-phosphate from lipid X, the product of the LpxH catalysis; the released inorganic phosphate is subsequently quantified by the colorimetric malachite green assay, allowing the monitoring of the LpxH catalysis. Using such a coupled enzymatic assay, we report the biochemical characterization of a series of sulfonyl piperazine LpxH inhibitors. Our analysis establishes a preliminary structure-activity relationship for this class of compounds and reveals a pharmacophore of two aromatic rings, two hydrophobic groups, and one hydrogen-bond acceptor. We expect that our findings will facilitate the development of more effective LpxH inhibitors as potential antibacterial agents.

Due to the widespread prevalence, deoxyuridine triphosphatase (UTPase) is considered by modern biochemists and physicians as a promising target for the development of drugs with a wide range of activities. The therapeutic effect of these drugs will be due to suppression of DNA biosynthesis in various viruses, bacteria and protozoa. In order to rationalize the search for new dUTPase inhibitors, domestic and foreign researchers are actively using the QSAR methodology at the selection stage of hit compounds. However, the practical application of this methodology is impossible without existence of valid QSAR models. With the use of the GUSAR 2013 program, a quantitative analysis of the relationship between the structure and efficacy of 135 dUTPase inhibitors based on uracil derivatives was performed in the IC50 range of 30¸185000 nmol/L. Six statistically significant valid consensus models, characterized by high descriptive ability and moderate prognostic ability on the structures of training and test samples, are constructed. To build valid QSAR models for dUTPase inhibitors can use QNA or MNA descriptors and their combinations in a consensus approach.

Pseudoxanthoma elasticum (PXE), a prototype of heritable ectopic mineralization disorders, is caused by mutations in the ABCC6 gene encoding a putative efflux transporter ABCC6. It was recently shown that the absence of ABCC6-mediated adenosine triphosphate release from the liver and, consequently, reduced inorganic pyrophosphate levels underlie the pathogenesis of PXE. Given that tissue-nonspecific alkaline phosphatase (TNAP), encoded by ALPL, is the enzyme responsible for degrading inorganic pyrophosphate, we hypothesized that reducing TNAP levels either by genetic or pharmacological means would lead to amelioration of the ectopic mineralization phenotype in the Abcc6 -/- mouse model of PXE. Thus, we bred Abcc6 -/- mice to heterozygous Alpl +/- mice that display approximately 50% plasma TNAP activity. The Abcc6 -/- Alpl +/- double-mutant mice showed 52% reduction of mineralization in the muzzle skin compared with the Abcc6 -/- Alpl +/+ mice. Subsequently, oral administration of SBI-425, a small molecule inhibitor of TNAP, resulted in 61% reduction of plasma TNAP activity and 58% reduction of mineralization in the muzzle skin of Abcc6 -/- mice. By contrast, SBI-425 treatment of Enpp1 mutant mice, another model of ectopic mineralization associated with reduced inorganic pyrophosphate, failed to reduce muzzle skin mineralization. These results suggest that inhibition of TNAP might provide a promising treatment strategy for PXE, a currently intractable disease., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene ( ITPA ) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies. IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase., (Copyright © 2018 Nyström et al.)

In the present work we report the synthesis of new aryl pyrazole derivatives using 1,3-dicarbonyl motifs. The reaction was proceeded by the cyclization of pentane-2,4-dione (1a), 3-chloropentane-2,4-dione (1b) or ethyl 3-oxobutanoate (1c) with different aryl hydrazines. The products, which can be regarded as 1H-pyrazol-1-yl-one analogues (3a-f, 3g-o, 4a-c, 5a-b) and represent drug like molecules along with well-developed structure-activity relationships, were obtained in good to excellent yield. The structures of synthesized compounds were charcterized on the basis of FT-IR, 1 H NMR, 13 C NMR and mass spectroscopic data. Considering alkaline phosphatases (APs), nucleotide pyrophosphatases/phosphodiesterases (NPPs) and nucleoside triphosphate diphosphohydrolase as the molecular targets, the effects of these synthesized compounds were investigated on different isozymes of APs, NPPs and NTPDases. The data revealed that the synthesized compounds inhibited both enzymes but most of them inhibited tissue non-specific alkaline phosphatase (TNAP) more selectively. The antitumor activity results indicated that the synthesized derivatives have strong inhibitory effects on the growth of selected cell lines from different tissues such as breast, bone marrow and cervix (MCF-7, K-562 and Hela) but with varying intensities. Moreover the binding mode of interactions were explained on the basis of molecular docking and in-silico studies., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

5-Fluorouracil (5-FU) is an antimetabolite and exerts antitumor activity via intracellularly and physiologically complicated metabolic pathways. In this study, we designed a novel small molecule inhibitor, TAS-114, which targets the intercellular metabolism of 5-FU to enhance antitumor activity and modulates catabolic pathway to improve the systemic availability of 5-FU. TAS-114 strongly and competitively inhibited deoxyuridine 5'-triphosphate nucleotidohydrolase (dUTPase), a gatekeeper protein preventing aberrant base incorporation into DNA, and enhanced the cytotoxicity of fluoropyrimidines in cancer cells; however, it had little intrinsic activity. In addition, TAS-114 had moderate and reversible inhibitory activity on dihydropyrimidine dehydrogenase (DPD), a catabolizing enzyme of 5-FU. Thus, TAS-114 increased the bioavailability of 5-FU when coadministered with capecitabine in mice, and it significantly improved the therapeutic efficacy of capecitabine by reducing the required dose of the prodrug by dual enzyme inhibition. Enhancement of antitumor efficacy caused by the addition of TAS-114 was retained in the presence of a potent DPD inhibitor containing oral fluoropyrimidine (S-1), indicating that dUTPase inhibition plays a major role in enhancing the antitumor efficacy of fluoropyrimidine-based therapy. In conclusion, TAS-114, a dual dUTPase/DPD inhibitor, demonstrated the potential to improve the therapeutic efficacy of fluoropyrimidine. Dual inhibition of dUTPase and DPD is a novel strategy for the advancement of oral fluoropyrimidine-based chemotherapy for cancer treatment. Mol Cancer Ther; 17(8); 1683-93. ©2018 AACR ., (©2018 American Association for Cancer Research.)