ecto-5'-Nucleotidase (eN, CD73) catalyzes the hydrolysis of extracellular AMP to adenosine. eN inhibitors have potential for use as cancer therapeutics. The eN inhibitor α,β-methylene-ADP (AOPCP, adenosine-5'-O-[(phosphonomethyl)phosphonic acid]) was used as a lead structure, and derivatives modified in various positions were prepared. Products were tested at rat recombinant eN. 6-(Ar)alkylamino substitution led to the largest improvement in potency. N(6)-Monosubstitution was superior to symmetrical N(6),N(6)-disubstitution. The most potent inhibitors were N(6)-(4-chlorobenzyl)- (10l, PSB-12441, Ki 7.23 nM), N(6)-phenylethyl- (10h, PSB-12425, Ki 8.04 nM), and N(6)-benzyl-adenosine-5'-O-[(phosphonomethyl)phosphonic acid] (10g, PSB-12379, Ki 9.03 nM). Replacement of the 6-NH group in 10g by O (10q, PSB-12431) or S (10r, PSB-12553) yielded equally potent inhibitors (10q, 9.20 nM; 10r, 9.50 nM). Selected compounds investigated at the human enzyme did not show species differences; they displayed high selectivity versus other ecto-nucleotidases and ADP-activated P2Y receptors. Moreover, high metabolic stability was observed. These compounds represent the most potent eN inhibitors described to date.

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Ecto‐5′‐nucleotidase (CD73, EC 3.1.3.5) catalyzes the extracellular hydrolysis of AMP yielding adenosine, which induces immunosuppression, angiogenesis, metastasis, and proliferation of cancer cells. CD73 inhibition is therefore proposed as a novel strategy for cancer (immuno)therapy, and CD73 antibodies are currently undergoing clinical trials. Despite considerable efforts, the development of small molecule CD73 inhibitors has met with limited success. To develop a suitable drug candidate, a high resolution (2.05 Å) co‐crystal structure of the CD73 inhibitor PSB‐12379, a nucleotide analogue, in complex with human CD73 is determined. This allows the rational design and development of a novel inhibitor (PSB‐12489) with subnanomolar inhibitory potency toward human and rat CD73, high selectivity, as well as high metabolic stability. A co‐crystal structure of PSB‐12489 with CD73 (1.85 Å) reveals the interactions responsible for increased potency. PSB‐12489 is the most potent CD73 inhibitor to date representing a powerful tool compound and novel lead structure.

Fluorescent ligands represent powerful tools for biological studies and are considered attractive alternatives to radioligands. In this study, we developed fluorescent antagonists for A adenosine receptors (AARs), which are targeted by antiasthmatic xanthines and were proposed as novel targets in immuno-oncology. Our approach was to merge a small borondipyrromethene (BODIPY) derivative with the pharmacophore of 8-substituted xanthine derivatives. On the basis of the design, synthesis, and evaluation of model compounds, several fluorescent ligands were synthesized. Compound 29 (PSB-12105), which displayed high affinity for human, rat, and mouse AARs ( K = 0.2-2 nM) and high selectivity for this AR subtype, was selected for further studies. A homology model of the human AAR was generated, and docking studies were performed. Moreover, 29 allowed us to establish a homogeneous receptor-ligand binding assay using flow cytometry. These compounds constitute the first potent, selective fluorescent AAR ligands and are anticipated to be useful for a variety of applications.

The following members of the ecto-nucleoside triphosphate diphosphohydrolase family, NTPDase1 (CD39), NTPDase-2, -3, and -8, play an important role in purinergic signal transduction by regulating extracellular nucleotide levels. Potent and selective NTPDase inhibitors are required as pharmacological tools and have potential as novel drugs, e.g. for anti-cancer and anti-bacterial therapy. We have developed fast and sensitive NTPDase fluorescence polarization (FP) immunoassays using the natural substrates (ATP or ADP). During the NTPDase1-catalyzed reaction, the substrate is dephosphorylated to ADP which is further dephosphorylated yielding AMP as the final product (by NTPDase1). NTPDase3 and -8 yield AMP and ADP, while NTPDase2 results mainly in the formation of ADP. Direct quantification of the respective product, AMP or ADP, is achieved by displacement of an appropriate fluorescent tracer nucleotide from a specific antibody leading to a change in fluorescence polarization. The assays are highly sensitive and can be performed with low substrate concentrations (20 μM ATP or 10 μM ADP) below the KM values of NTPDases, which simplifies the identification of novel competitive inhibitors. Optimized antibody and enzyme concentrations allow the reproducible detection of 2 μM ADP and 1 μM AMP (at 10% substrate conversion). Validation of the assays yielded excellent Z'-factors greater than 0.70 for all investigated NTPDase subtypes indicating high robustness of the analytical method. Furthermore, we tested a standard inhibitor and performed a first exemplary screening campaign with a library consisting of >400 compounds (Z'-factor: 0.87, hit rate 0.5%). Thereby we demonstrated the suitability of the FP assay for IC50 value determination and high-throughput screening in a 384-well format. The new FP assays were shown to be superior to current standard assays.