Abstract
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 laser-induced 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 Ki 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 structure-activity relationships, and provides a basis for future optimization towards selective CD39 and dual CD39/CD73 inhibitors.
Highlights
Nucleoside triphosphate diphosphohydrolase1 (NTPDase1, nucleoside triphosphate diphosphohydrolase1 (CD39), EC 3.6.1.5) catalyzes the hydrolysis of extracellular nucleoside tri- and diphosphates producing the corresponding monophosphates (Zimmermann et al, 2012)
The ATP analog ARL67156 (I), which is known as a standard inhibitor of CD39, was selected as a lead structure, and different substitutions of the adenine base and modifications of the phosphate chain were performed
Since 8-BuS-AMP (II), 8-BuS-ADP and 8-Bu-ATP were described as CD39 inhibitors (Lecka et al, 2013), we introduced an 8-butyl substituent to study its effect on the ATP analogs as well
Summary
Nucleoside triphosphate diphosphohydrolase (NTPDase, CD39, EC 3.6.1.5) catalyzes the hydrolysis of extracellular nucleoside tri- and diphosphates producing the corresponding monophosphates (Zimmermann et al, 2012). Many tumor cells overexpress ectonucleotidases (De Marchi et al, 2019; Horenstein et al, 2019) which metabolize proinflammatory ATP to immunosuppressive, angiogenic, prometastatic, and tumor growth-promoting adenosine (Vitiello et al, 2012). Due to its pathophysiological role, CD39 represents a promising potential drug target that requires, further validation. For this purpose, potent, selective, and metabolically stable inhibitors need to be identified. Besides selective CD39 inhibitors, dual inhibition of CD39 and CD73 is of interest and may be synergistic since the substrate of CD73, extracellular AMP, may be formed by alternative ectonucleotidases, such as nucleotide pyrophosphatase/ phosphodiesterase (NPP1) (Lee and Müller, 2017; Lee et al, 2017a)
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