Heparins are potent inhibitors of ectonucleotide pyrophosphatase/phospho-diesterase-1 (NPP1) - a promising target for the immunotherapy of cancer.

Introduction: Heparins, naturally occurring glycosaminoglycans, are widely used for thrombosis prevention. Upon application as anticoagulants in cancer patients, heparins were found to possess additional antitumor activities. Ectonucleotidases have recently been proposed as novel targets for cancer immunotherapy. Methods and results: In the present study, we discovered that heparin and its derivatives act as potent, selective, allosteric inhibitors of the poorly investigated ectonucleotidase NPP1 (nucleotide pyrophosphatase/phosphodiesterase-1, CD203a). Structure-activity relationships indicated that NPP1 inhibition could be separated from the compounds' antithrombotic effect. Moreover, unfractionated heparin (UFH) and different low molecular weight heparins (LMWHs) inhibited extracellular adenosine production by the NPP1-expressing glioma cell line U87 at therapeutically relevant concentrations. As a consequence, heparins inhibited the ability of U87 cell supernatants to induce CD4+ T cell differentiation into immunosuppressive Treg cells. Discussion: NPP1 inhibition likely contributes to the anti-cancer effects of heparins, and their specific optimization may lead to improved therapeutics for the immunotherapy of cancer.

Fluorescent Probes for Ecto-5'-nucleotidase (CD73).

Ecto-5'-nucleotidase (CD73) catalyzes the hydrolysis of AMP to anti-inflammatory, immunosuppressive adenosine. It is expressed on vascular endothelial, epithelial, and also numerous cancer cells where it strongly contributes to an immunosuppressive microenvironment. In the present study we designed and synthesized fluorescent-labeled CD73 inhibitors with low nanomolar affinity and high selectivity based on N 6 -benzyl-α,ß-methylene-ADP (PSB-12379) as a lead structure. Fluorescein was attached to the benzyl residue via different linkers resulting in PSB-19416 (14b, K i 12.6 nM) and PSB-18332 (14a, K i 2.98 nM) as fluorescent high-affinity probes for CD73. These compounds are anticipated to become useful tools for biological studies, drug screening, and diagnostic applications.

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

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.