Identification and pharmacological characterization of nucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) and -4 (NPP4) inhibitors

Abstract

Ectonucleotidases are enzymes involved in different physiological functions and mainly in the regulation of purinergic signaling; in fact, they regulate the duration of purinergic receptor activation by catalyzing the hydrolysis of nucleotides (mainly ATP)producing the nucleoside adenosine. As a result, they are involved in a broad range of physiological process, and have emerged as promising drug targets. <br /> Ectonucleotidases are allocated into four subfamilies: ecto-nucleoside triphosphates diphosphohydrolases (NTPDases), alkaline phosphatases (APs), ecto-5′-nucleotidase (CD73), and ecto-nucleotide pyrophosphatases/phosphodiesterases (NPPs). The NPP family consists of seven structurally related subtypes, NPP1-7, and despite their enormous therapeutic potential, the availability of powerful and selective modulators remains still limited. As a matter of fact, molecules acting as activators or inhibitors of their enzymatic activity are useful tool for pharmacological investigations to advance the basic research of these enzymes, their physiological functions as well as their participation in diseases. Additionally, tool compounds may be used as lead structures for drug development. The objective of this project was to advance the field of drug development for two NPP isoenzymes, human NPP1 and NPP4. The results were published in three original research articles presented in Sections 2-4. Sections 5 and 6 describe experimental results that have not been published yet. <br /> Section 1 provides an overview of hNPP1 and -4, with an emphasis on the current level of knowledge concerning enzyme structures and kinetics, as well as the state of the art regarding modulators and their activity, in particular inhibitors including tool compounds, and therapeutic candidates. In order to identify new, potent, and selective hNPP1 inhibitors, we studied the potential inhibitor activity of sulfated polysaccharides derived from sea algae, which were reported to have anti-cancer effects, and to be nontoxic and well tolerated by humans (Section 2). We identified and comprehensively characterized them in vitro with respect to potency, selectivity versus related enzymes and mechanism of inhibition. These sulfated polysaccharides, natural constituent of some marine algae, are the most potent NPP1 inhibitors known to date, with nano- to sub-nanomolar potencies. A sulfated polysaccharide isolated from Saccharina latissima was identified as a dual inhibitor of NPP1 and CD39. Due to their involvement in ATP-hydrolysis, this dual activity may be beneficial in cancer immunotherapy. Further advancement in the identification of new hNPP1 inhibitors is described in Section 3. Heparin and its derivatives including marked antithrombotic as well as newly synthetized analogs, were found to negatively modulate hNPP1 activity and thus inhibit the breakdown of ATP. Moreover, using heparins we observed reduction of adenosine formation in a cellbased assay using tumor cells with native NPP1 expression. Adenosine is an immunosuppressive and tumor-promoting compound and, although still speculative,the reported hNPP1 inhibition by heparin and its derivatives may be crucial to explain the clinical evidence of heparinized cancer patients showing improved survival rates. In order to discover novel enzyme inhibitors, we developed reliable analytical methods for the monitoring of NPP4 enzymatic reactions. Chapter 4 describes the development of an HTS (High-throughput screening) assay, using Ap4A (diadenosine tetraphosphate) as substrate and bio-luminescence as detection method. The assay was fully optimized and validated following FDA (Food and Drug Administration) and ICH (International Council for Harmonisation) guidelines for bio-analytical assays. It was subsequentially used for the screening of a compound library from which two new hNPP4 inhibitors were discovered, further characterized, and validated with a second analytical method based on capillary electrophoresis (CE) with Diode-Array Detection (DAD). The compounds PSB-POM145 and PSB-POM146 are the first hNPP4 inhibitors reported in the literature showing IC50 values of 0.298 and 3.36 μM, respectively. These compounds represent essential tools for further studies on NPP4. In order to discover new chemical scaffold, with potential as lead compounds, for drug development, additional screening of compound libraries was performed. The obtained data are presented in Chapter 5. We identified new hit molecules with (moderate) inhibitory potency and selectivity for hNPP4, which may serve as lead structures for future optimization. <br /> Section 6 describes an extensive study on NPP4 enzyme kinetics for a wide range of (potential) substrates. Novel natural substrates of hNPP4 were discovered and characterized. <br /> In conclusion, this work advances the field of ligand discovery for a therapeutically important class of ecto-enzymes. The discovered and reported powerful NPP1 and NPP4 inhibitors will be useful tools for in vitro and in vivo studies aiming to elucidate the enzymes’ roles in physiology and pathology. The new tool compounds may contribute to the validation of NPP1 and NPP4 as novel therapeutic targets, e.g., in cancer (immune)therapy and antithrombotic therapy, respectively.