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  • 67 7 We further analyzed selected hit compounds for their ab

    2024-04-12

    We further analyzed selected hit compounds for their ability to inhibit human AdK in intact cells. Human 1321N1 astrocytoma 67 7 were utilized which were found to express AdK. The whole cell assay was performed using a 96-well format. The standard AdK inhibitor 5-iodotubercidin was employed as a positive control whereas a sample without inhibitor served as a negative control. All tested hit compounds showed AdK inhibition in cultured cells which is an indication that the compounds were able to enter the cell to inhibit the cytosolic AdK (Fig. 4). As a next step, we investigated the potential interaction of the identified AdK inhibitors with adenosine receptors to investigate their selectivity. We performed radioligand binding studies using [3H]CCPA (A1) and [3H]MSX-2 (A2A), respectively. Except for compound 12 none of the identified AdK inhibitors showed affinity to the adenosine A1 receptor at the tested concentrations. Only compound 12 revealed relatively high affinity for the A1 adenosine receptor (Ki=130nM) whereas for the related compound 11 no binding could be observed at A1, A2A, A2B and A3 receptors. These results clearly indicate very different structure–activity relationships for the compounds at adenosine receptors as compared to adenosine kinase. In adenosine A2A binding studies only compound 5 displayed affinity revealing a Ki value of 3.02μM. The other identified AdK inhibitors showed no affinity for the adenosine A2A receptor at the tested concentrations. The radioligand binding data confirm that most of the newly identified non-nucleosidic AdK inhibitors exhibit no binding affinity for adenosine A1 and A2A receptors which suggests a low probability of inhibition of the compounds’ effects due to a blockade of adenosine receptors. Results of the binding studies for all tested hit compounds are shown in Table 3.
    Conclusion We established a high-throughput AdK assay using [3H]adenosine performed in a 96-well format. The new assay was shown to be robust, reproducible, and well suited for HTS (Z′=0.7). The newly established AdK assay represents a powerful tool for the discovery of novel AdK inhibitors. A first screening campaign of a purine receptor-targeted library of 189 compounds resulted in the discovery of 12 novel non-nucleoside AdK inhibitors (hit rate: 6%). The identified lead compounds inhibited human AdK both in vitro and in intact cells. The most potent AdK inhibitor of the present study was 10,11,12,13-tetrahydrobenzo[4,5]thieno[3,2-e]bis([1,2,4]triazolo)[4,3-a:4′,3′-c]pyrimidine-3,7-dithiol (1). 1 inhibited human AdK in a reversible manner with a Ki value of 0.184μM. The Ki values for the other 11 AdK inhibitors were in the range of 5.04–25.4μM. All hit compounds except for 5 and 12 showed selectivity versus adenosine A1 and A2A receptors. The 12 hits representing 6 novel non-nucleoside scaffolds are suitable for the design of potent, structurally novel AdK inhibitors.
    Acknowledgments We thank Julia Fischer, Saliha Harrach and Johannes Herzner for performing some of the AdK assays and Sonja Hinz and Nicole Florin for performing the A1 and A2A radioligand binding assays.
    Introduction Traumatic optic neuropathy (TON) is partial or complete loss of function of optic nerve (ON) due to either a direct injury or indirectly after head trauma sequelae, such as edema, hemorrhage, and concussion (Steinsapir and Goldberg, 2011). Unfortunately, there are currently no proven treatments that can prevent the damage associated with an acute TON. Optic nerve injury mediated loss of retinal ganglion cells (RGCs) through apoptosis has been hypothesized due to several underlying common mechanisms, including lack of neurotrophin support, increased extracellular glutamate levels, damage from free radicals, and disruption of cellular homeostasis (Pang et al., 2010). All these mechanisms cause activation of microglial cells and inflammatory responses such as release of free radicals, cytokines, and prostaglandins and complement molecules (Lucas et al., 2006). Therefore, counteracting inflammation may possess neuroprotective effect in TON.