The main protease of SARS‐CoV‐2 (M pro ), the causative agent of COVID‐19, constitutes a significant drug target. A new fluorogenic substrate was kinetically compared to an internally quenched fluorescent peptide and shown to be ideally suitable for high throughput screening with recombinantly expressed M pro . Two classes of protease inhibitors, azanitriles and pyridyl esters, were identified, optimized and subjected to in‐depth biochemical characterization. Tailored peptides equipped with the unique azanitrile warhead exhibited concomitant inhibition of M pro and cathepsin L, a protease relevant for viral cell entry. Pyridyl indole esters were analyzed by a positional scanning. Our focused approach towards M pro inhibitors proved to be superior to virtual screening. With two irreversible inhibitors, azanitrile 8 (k inac /K i =37 500 m −1 s −1 , K i =24.0 n m ) and pyridyl ester 17 (k inac /K i =29 100 m −1 s −1 , K i =10.0 n m ), promising drug candidates for further development have been discovered.

NAD kinase was purified 93-fold from Escherichia coli. The enzyme was found to have a pH optimum of 7.2 and an apparent Km for NAD+, ATP, and Mg2' of 1.9, 2.1, and 4.1 mM, respectively. Several compounds including quinolinic acid, nicotinic acid, nicotinamide, nicotinamide mononucleotide, AMP, ADP, and NADP+ did not affect NAD kinase activity. The enzyame was not affected by changes in the adenylate energy charge. In contrast, both NADH and NADPH were potent negative modulators of the enzyme, since their presence at micromolar concentrations resulted in a pronounced sigmoidal NAD+ saturation curve. In addition, the presence of a range of concentrations of the reduced nucleotides resulted in an increase of the Hill slope (nH) to 1.7 to 2.0 with NADH and to 1.8 to 2.1 with NADPH, suggesting that NAD kinase is an allosteric enzyme.These results indicate that NAD kinase activity is regulated by the availability of ATP, NAD+, and Mg2' and, more significantly, by changes in the NADP+/NADPH and NAD+/NADH ratios. Thus, NAD kinase probably plays a role in the regulation of NADP turnover and pool size in E. coli.Few reports have been devoted to the study of NAD kinase from bacteria. NAD kinase has been studied in a limited manner in crude, cell-free preparations from Escherichia coli (10). The enzyme has been purified up to 500-fold from Azotobacter vinelandii and has been found to have apparent Km values for NAD+ and ATP of 0.4 and 1 mM, respectively (7). NAD kinase has also been detected in immobilized Achromobacter aceris (25) and Brevibacterium ammoniagenes (9).Recently, we purified NAD kinase 180-fold from Bacillus licheniformis (31). Using this partially purified preparation, we have shown that this enzyme is subject to competitive inhibition by NADP+ and that it plays a key role in the regulation of NADP turnover in this organism (31). In addition, we have also purified NAD kinase from Bacillus subtilis and demonstrated that it is negatively modified by NADP+ and positively modified by NADPH (V. Lopez, E. Gomez, H. Kwong, and A. J. Andreoli, Fed. Proc. 44:2649, 1985).E. coli cells possess a functional NAD cycle that accounts for the turnover of NAD in this organism (2). In addition, E. coli cells also demonstrate NADP turnover (16; A. J. Andreoli, unpublished results). In the present investigation, we purified NAD kinase from E. coli to determine its role in the regulation of NADP turnover and pool size in this organism. Evidence is presented indicating that NADPH and NADH are potent negative modulators of this enzyme.

Nucleoside triphosphate diphosphohydrolase1 (NTPDase1, CD39) inhibitors have potential as novel drugs for the (immuno)therapy of cancer. They increase the extracellular concentration of immunostimulatory ATP and reduce the formation of AMP, which can be further hydrolyzed by ecto-5'-nucleotidase (CD73) to immunosuppressive, cancer-promoting adenosine. In the present study, we synthesized analogs and derivatives of the standard CD39 inhibitor ARL67156, a nucleotide analog which displays a competitive mechanism of inhibition. Structure-activity relationships were analyzed at the human enzyme with respect to substituents in the N 6-and C8-position of the adenine core, and modifications of the triphosph(on)ate chain. Capillary electrophoresis coupled to laserinduced fluorescence detection employing a fluorescent-labeled ATP derivative was employed to determine the compounds' potency. Selected inhibitors were additionally evaluated in an orthogonal, malachite green assay versus the natural substrate ATP. The most potent CD39 inhibitors of the present series were ARL67156 and its derivatives 31 and 33 with K i values of around 1 µM. Selectivity studies showed that all three nucleotide analogs additionally blocked CD73 acting as dual-target inhibitors. Docking studies provided plausible binding modes to both targets. The present study provides a full characterization of the frequently applied CD39 inhibitor ARL67156, presents structureactivity relationships, and provides a basis for future optimization towards selective CD39 and dual CD39/CD73 inhibitors.

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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.

Die Hauptprotease von SARS-CoV-2 (M pro ), dem Auslçser von COVID-19, ist ein wichtiges Arzneistoff-Target. Ein neues fluorogenes Substrat, das kinetischmit einem intern gequenchten fluoreszierenden Peptid verglichen wurde, erwies sich als ideal geeignet füre in Hochdurchsatz-Screening mit rekombinant exprimierter M pro .Z wei Klassen von Protease-Inhibitoren, Azanitrile und Pyridylester,w urden identifiziert, optimiert und biochemisch charakterisiert. Maßgeschneiderte Peptide mit einer reaktiven Azanitril-Kopfgruppe zeigten eine duale Inhibition von M pro und Cathepsin L, einer Protease, welche die virale Zellinvasion befçrdert. Zur Optimierung der Pyridylindolester wurde ein Positions-Scanning durchgeführt. Unser fokussierter Ansatz zur Entwicklung von M pro -Inhibitoren erwies sich dem virtuellen Screening als überlegen. Mit den beiden irreversiblen Inhibitoren Azanitril 8 (k inac /K i = 37 500 m À1 s À1 ,K i = 24.0 nm)u nd Pyridylester 17 (k inac /K i = 29 100 m À1 s À1 ,K i = 10.0 nm)w urden vielversprechende Kandidaten fürdie zukünftige Arzneistoffentwicklung entdeckt.

BackgroundAn important mechanism, by which cancer cells achieve immune escape, is the release of extracellular adenosine into their microenvironment. Adenosine activates adenosine A2A and A2B receptors on immune cells constituting one of the strongest immunosuppressive mediators. In addition, extracellular adenosine promotes angiogenesis, tumor cell proliferation, and metastasis. Cancer cells upregulate ectonucleotidases, most importantly CD39 and CD73, which catalyze the hydrolysis of extracellular ATP to AMP (CD39) and further to adenosine (CD73). Inhibition of CD39 is thus expected to be an effective strategy for the (immuno)therapy of cancer. However, suitable small molecule inhibitors for CD39 are not available. Our aim was to identify drug-like CD39 inhibitors and evaluate them in vitro.MethodsWe pursued a repurposing approach by screening a self-compiled collection of approved, mostly ATP-competitive protein kinase inhibitors, on human CD39. The best hit compound was further characterized and evaluated in various orthogonal assays and enzyme preparations, and on human immune and cancer cells.ResultsThe tyrosine kinase inhibitor ceritinib, a potent anticancer drug used for the treatment of anaplastic lymphoma kinase (ALK)-positive metastatic non-small cell lung cancer, was found to strongly inhibit CD39 showing selectivity versus other ectonucleotidases. The drug displays a non-competitive, allosteric mechanism of CD39 inhibition exhibiting potency in the low micromolar range, which is independent of substrate (ATP) concentration. We could show that ceritinib inhibits ATP dephosphorylation in peripheral blood mononuclear cells in a dose-dependent manner, resulting in a significant increase in ATP concentrations and preventing adenosine formation from ATP. Importantly, ceritinib (1–10 µM) substantially inhibited ATP hydrolysis in triple negative breast cancer and melanoma cells with high native expression of CD39.ConclusionsCD39 inhibition might contribute to the effects of the powerful anticancer drug ceritinib. Ceritinib is a novel CD39 inhibitor with high metabolic stability and optimized physicochemical properties; according to our knowledge, it is the first brain-permeant CD39 inhibitor. Our discovery will provide the basis (i) to develop more potent and balanced dual CD39/ALK inhibitors, and (ii) to optimize the ceritinib scaffold towards interaction with CD39 to obtain potent and selective drug-like CD39 inhibitors for future in vivo studies.