Structure-Aided Development of Small-Molecule Inhibitors of ENPP1, the Extracellular Phosphodiesterase of the Immunotransmitter cGAMP.

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 Ki < 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.

Chemical synthesis, purification, and characterization of 3'-5'-linked canonical cyclic dinucleotides (CDNs).

So far, four cyclic dinucleotides (CDNs) have been discovered as important second messengers in nature, where three canonical CDNs of c-di-GMP, c-di-AMP and c-AMP-GMP were found in bacteria containing two 3'-5' phosphodiester linkages and one non-canonical CDN 2'3'-c-GMP-AMP was identified in mammals containing mixed 2'-5' and 3'-5' phosphodiester linkages. The CDNs are produced by specific cyclases and degraded by phosphodiesterases (PDEs). All of the known CDNs could bind to the stimulator of interferon genes (STING) to induce type I interferon (IFN) responses and the three bacterial CDNs are sensed by specific riboswitches to regulate gene expression. The emerging physiological functions of bacterial CDNs lead the motivation to investigate other possible canonical CDNs. In recent years, many endeavors have been devoted to develop fast, convenient and cheap strategies for chemically synthesizing CDNs and their analogues. The phosphoramidite approach using commercial starting materials has attracted much attention. Herein, we describe an adapted one-pot strategy that enables fast synthesis of crude 3'-5'-linked canonical CDNs followed by purification of the obtained CDNs using reversed phase high-performance of liquid chromatography (HPLC). Furthermore, we report the full characterization of CDNs by mass spectrometry (MS) and nuclear magnetic resonance (NMR) techniques.

Purification of Cyclic GMP-AMP from Viruses and Measurement of Its Activity in Cell Culture.

Sensing of cytoplasmic DNA by cGAS is essential for the initiation of immune responses against several viruses. cGAS also plays important roles in some autoinflammatory and autoimmune diseases and may be involved in immune responses targeting cancer cells. Once activated, cGAS catalyzes the formation of the di-nucleotide 2'-3'-cyclic GMP-AMP (cGAMP), which propagates a signaling cascade leading to the production of type I interferons (IFNs). Interestingly, cGAMP is incorporated into enveloped viruses and is transferred to newly infected cells by virions. In this article, we describe a method to purify cGAMP from viral particles and a bioassay to measure its activity. This assay takes advantage of a reporter cell line that expresses the genes encoding green fluorescent protein (GFP) and firefly luciferase under the control of the IFNß promoter, allowing the testing of several samples in a single experiment taking not more than 3 days.

cGAMP Quantification in Virus-Infected Human Monocyte-Derived Cells by HPLC-Coupled Tandem Mass Spectrometry.

Upon virus infection, cells of the innate immune system such as dendritic cells and macrophages can mount type I interferon (IFN-I) responses that restrict viral dissemination. To inform host cells of virus infection, detection of cytosolic DNA is one important mechanism. Inappropriate sensing of endogenous DNA and subsequent induction of IFN-I responses can also cause autoimmunity, highlighting the need to tightly regulate DNA sensing. The cyclic GMP-AMP synthase (cGAS) was recently identified to be the major sensor of cytosolic DNA that triggers IFN-I expression. Upon DNA binding, cGAS synthesizes the second messenger cyclic guanosine-adenosine monophosphate (cGAMP) that induces IFN-I expression by the activation of the stimulator of interferon genes (STING). Notably, cGAMP does not only act in infected cells, but can also be relocated to noninfected bystander cells to there trigger IFN-I expression. Thus, direct quantification of cGAMP in cells of the innate immune system is an important approach to study where, when, and how DNA is sensed and IFN-I responses are induced. Here, we describe a method that allows specific quantification of cGAMP from extracts of virus-infected human myeloid cells by HPLC-coupled tandem mass spectrometry.

Quantification of cyclic dinucleotides by reversed-phase LC-MS/MS.

Cyclic dinucleotides such as bis-(3',5')-cyclic dimeric adenosine monophosphate (c-di-AMP) and bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP) represent important second messengers in bacteria. Although their synthesis has not been described in plants so far, they may be involved in the regulation of bacterial phytopathogen-plant interactions as well as rhizobium plant symbiosis. Here, we describe a sensitive and specific quantification method for c-di-AMP and c-di-GMP by HPLC-coupled tandem mass spectrometry. Additional linear dinucleotide metabolites and mononucleotides, as well as cyclic mononucleotides, can be simultaneously determined by this method.

The preparation of cyclic nucleotide-depleted regulatory subunits from type II adenosine 3':5'-monophosphate-dependent protein kinase by a nondenaturing method.

A nondenaturing method for the preparation of R subunits from type II cyclic AMP-dependent protein kinase is described. The procedure is based on the exchange of cyclic AMP, which is tightly bound to the R subunit, for more weakly bound cyclic GMP, which can be removed by washing and dialysis. Less than 5% of the available cyclic nucleotide-binding sites of R subunit prepared by this method contained cyclic AMP and less than 3% contained cyclic GMP. The C-subunit contamination (mol of C/mol of R monomer) was approximately 0.2%. These levels of contamination did not affect the properties of the R subunit as judged by (a) the ability of the R subunit to inhibit the activity of the C subunit and (b) the rate of exchange of cAMP into R2 . etheno-cAMP. The advantages of our method are that the protein is not subjected to denaturing conditions and that large amounts of material can be processed relatively rapidly.

A method for chromatographic separation and fluorometric quantification of 2',3'-cyclic nucleotide-3'-phosphodiesterase reaction products.

A sensitive method for separation and fluorometric quantification of 2',3'-cyclic nucleotide-3'-phosphodiesterase (EC 3.1.4.37) (CNPase) reaction products with 2',3'-cyclic adenosine monophosphate as substrate is presented. The 2'-AMP product was separated by cellulose thin-layer chromatography in 4 M MgSO4-0.5 M sodium acetate-2-propanol (80:18:2, v/v/v). After reaction with glyoxal dihydrate, the amount of reaction product was determined fluorometrically using excitation at 328 nm and emission at 382 nm. These results correlated well with those obtained using Kurihara and Tsukada's [(1967) J. Neurochem. 14: 1167-1174] paper chromatographic method (r = 0.96). With this fluorometric method, amounts as low as 0.20 nmol of 2'-AMP can be determined, and its sensitivity is comparable to that of the radiochemical method. The method is easy to use and sensitive enough for measuring CNPase activity in tissue with low enzyme activity.

Rapid separation and quantitation of 3',5'-cyclic nucleotides and 5'-nucleotides in phosphodiesterase reaction mixtures using high-performance liquid chromatography.

A simple, rapid high-performance liquid-chromatography system for the fractionation and direct quantitation of substrates and products in crude phosphodiesterase reaction mixtures is described. Phosphate buffers and a pellicular anion exchange resin are used at ambient temperature. The method is sensitive, measuring picomoles of products with ultraviolet detection and femtomoles with isotopic measurement, and offers several advantages over the more popular batch sorption and manual methods for measuring phosphodiesterase activity. The time required for analysis, less than 8 min for single substrate reaction mixtures, is a fraction of that required with other chromatographic systems, and precision is +/- 5%. Results of studies with an activatable form of phosphodiesterase demonstrate the accuracy, precision and utility of the procedure for biochemical analyses.

Reversed-phase liquid chromatographic separation of 3',5'-cyclic ribonucleotides.

A rapid, reversed-phase "high-performance" liquid-chromatographic separation of the five naturally occurring cyclic ribonucleotides is described. The separation, optimized for the measurement of these compounds in biological samples, is short (25 min), sensitive (50-100 pmol), and requires no sample pre-concentration steps. We also report an alternative isocratic elution mode, optimized for a rapid and selective analysis for adenosine 3',5'-cyclic phosphate. The identity of chromatographic peaks in biological extracts is confirmed by several methods: retention times, co-chromatography with the reference compounds, absorbance ratios, enzymatic peak-shift with cyclic nucleotide phosphodiesterase, and stopped-flow ultraviolet-scanning techniques.

A simple direct assay of 3',5'-cyclic nucleotide phosphodiesterase activity based on the use of polyacrylamide-bononate affinity gel chromatography.

A rapid, simple, and direct assay for 3',5'-cyclic nucleotide phospho-diesterase activity is based on the effective separation of cyclic AMP, cyclic GMP or cyclic CMP from their corresponding 5'-nucleotides and nucleosides by chromatography on a polyacrylamide-boronate gel. The affinity of the boronate residue for cis-diols results in the retention of 5'nucleotides and nucleosides while 3',5'-cyclic nucleotides are not retained. The coelution of all 5'-nucleotides and nucleosides allows for the accurate assessment of phosphodiesterase activity in preparations contaminated by other purine metabolizing enzymes such as 5'-nucleotidases and nucleotide and nucleoside deaminases. Phosphodiesterase activity assayed by this means yields linear reaction kinetics with respect to time and amount of enzyme protein. Low blank values obtained allow for detection of as little as 2-3% conversion of substrate to product.

Immunologic properties of mast cells from rats infected with Nippostrongylus brasiliensis.

The concentration of IgE in the serum of Sprague-Dawley rats increased after infection with Nippostrongylus brasiliensis (NB). The IgE concentration in normal rats was less than 1 mug/ml. After re-infection with NB, the concentration increased in 100 to 300 mug/ml. Mast cells were purified from peritoneal cells of both normal and NB-infected animals. Purified mast cells from the infected animals released histamine upon exposure to NB antigen. The antibody specific for IgE released histamine from purified mast cells of both normal and infected animals. Dose-reponse curves of histamine release suggested that mast cells from NB-infected animals bear more IgE molecules than normal mast cells. Binding of 125I-labeled rat E myeloma protein with normal mast cells was demonstrated by autoradiography. Under the same experimental conditions, mast cells of infected animals were not labeled with 125I-IgE. Mast cells from both normal and infected animals failed to combine 125I-labeled IgG. The number of IgE molecules bound per mast cell was determined by incubating 125I-labeled IgE with purified mast cells. When mast cells were incubated incubated in 0.6 to 2 mug/ml of IgE, the number of IgE molecules combined with the mast cells from infected animals was about 10% of that bound with normal mast cells. The results indicated that a large proportion of IgE receptors on mast cells of infected animals was occupied by their own IgE. No significant difference was observed between normal mast cells and those of infected animals with respect to histamine content and intracellular levels of cyclic nucleotides.

Quantitative separation of nucleotides on mercurated dextran.

Dextran gels of varying porosites (Sephadex G series) were chemically modified so as to contain covalently bound monofunctional mercury. Mercurated Sephadex of the porosity G-25 was then used to fractionate mixtures of mono- and dinucleotides into the constituent components. Separation is based on the affinity of the nitrogen binding sites of the purine and pyrimidine derivatives for organomercurial Hg+. Thus, a mixture composed of the four mononucleotides Cyd-5'-P, Ado-3'-P, Guo-2'(3')-P, dThd-5'-P and of the four dinucleotides Cyd-P-Cyd, Ado-P-Ado, Guo-P-Urd, and Urd-P-Urd could be separated into eight well-resolved fractions by using a combination Tris-bicarbonate/carbonate buffer system of increasing pH as an eluent. Complete separation was also achieved when a mixture of Ado 3:5'-P, Ado 5'-P, Ado-5'-PP, and Ado-5'-PPP was fractionated on mercurated Sephadex G-25. Again, Tris-bicarbonate/carbonate buffer served as an eluent. Lastly, fractionation can also be performed at a constant pH by offering other suitable ligands, for instance Cl-, that will compete with nucleotides for monofunctional Hg+. The fractionation behavior of mercurated Sephadex G-25 can be fully understood on the basis of the complexing properties of monofunctional Hg+. This has been shown by calculating the net retention volume ratios of several nucleotides with the help of the known interaction parameters of corresponding nucleosides with CH3 HgOH and by comparing the predicted ratios with the experimentally measured ones. Finally, the acid-base properties of mercurated Sephadex G-25 as well as its affinity for chloride and iodide ions have been determined. The data agree quite well with those known for CH3 HgOH.