Nucleotide Analog ARL67156 as a Lead Structure for the Development of CD39 and Dual CD39/CD73 Ectonucleotidase Inhibitors

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 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.

Receptor-specific Ca2+ oscillation patterns mediated by differential regulation of P2Y purinergic receptors in rat hepatocytes

“…To exclude the possibility that the differences in [Ca 2+ ] c oscillation patterns seen with ADP and UTP might reflect the generation of other components by ectonucleotidases, we evaluated the [Ca 2+ ] c responses evoked by both agonists in the presence of ARL 67156, an inhibitor of nucleoside triphosphate diphosphohydrolase-1 (CD39) and ecto-5′-nucleotidase (CD73) ( Schakel et al., 2020 ). Preincubation for 5 min with ARL 67156 (100 μM), did not change the spike with of the typical narrow ADP-induced [Ca 2+ ] c oscillations (22.8 ± 1.3 s vs 21.3 ± 1.0 s; ADP and ARL 67156 + ADP, respectively).…”

Sulfated Polysaccharides from Macroalgae Are Potent Dual Inhibitors of Human ATP-Hydrolyzing Ectonucleotidases NPP1 and CD39

“…So far, only moderately potent and/or non-selective NPP1 and CD39 inhibitors have been described, which can be divided into nucleotides and non-nucleotides. N 6 -Diethyl-β,γ-dibromomethylene-ATP (ARL 67156, 1 ) is a weak dual CD39/CD73 inhibitor with low metabolic stability [ 16 , 17 ]. Several other negatively charged compound classes including (poly)sulfonates such as suramin ( 2 ) and sulfoanthraquinones [ 18 , 19 , 20 ], and polyoxometalates (POMs), e.g., [TiW 11 CoO 40 ] 8− ( 3 ) and [Co 4 (H 2 O) 2 (PW 9 O 34 ) 2 ] 10− ( 4 ) [ 21 , 22 ], were found to inhibit CD39 and/or NPP1.…”

“…So far, only moderately potent and/or non-selective NPP1 and CD39 inhibitors have been described, which can be divided into nucleotides and non-nucleotides. N 6 -Diethylβ,γ-dibromomethylene-ATP (ARL 67156, 1) is a weak dual CD39/CD73 inhibitor with low metabolic stability [16,17]. Several other negatively charged compound classes including Recently, there has been an enormous interest in identifying and developing ectonucleotidase inhibitors, antibodies and small molecules, as novel cancer immunotherapeutics [4,6,7].…”

2‐Substituted thienotetrahydropyridine derivatives: Allosteric ectonucleotidase inhibitors

“…Only a few small molecule inhibitors of CD39 have been described so far, all of which show limited potency and/or lack of metabolic stability. [26][27][28][29][30][31][32] Nucleotide derivatives, analogs of the natural substrates, have been investigated, for example, ARL 67156 (I) and its derivatives (e.g., II). [27,33,34] Nonnucleotide inhibitors comprise polyoxometalates (POMs, e.g., III), anthraquinones, and various other heterocyclic compounds including the clinically approved drugs ticlopidine (IV) and clopidogrel (V), and indole derivatives such as VI.…”