AdK as an important upstream regulator

AdK as an important upstream regulator of adenosine-based homeostasis represents a promising drug target since it leads to an increase in adenosine concentrations. Taking into account that the adenosine formation is only increased in affected tissues under stress with high adenosine levels, AdK inhibition might be particularly effective in these specific tissues and act in a site- and event-specific mode. One of the most intensively investigated diseases linked to AdK expression and function is epilepsy. Overexpression of AdK in astrocytes leads to an adenosine deficiency, consequently to a decreased inhibition of neuronal excitation and to seizure generation. In various in vivo mouse studies it could be demonstrated that the knockdown of the AdK gene can prevent seizures whereas transgenic overexpression of AdK triggers spontaneous seizures. Consequently, adenosine augmentation is considered as a potential therapeutic strategy for the treatment of epilepsy.4, 5, 6, 7 Recent findings showing an upregulation of AdK in human astrocytic entacapone tumors and in peritumoral tissues of glioma patients with epilepsy indicates that AdK might also play an important role in tumor-associated epilepsy. Moreover, it is well established that AdK inhibitors are highly effective as analgesic agents. The efficacy of AdK inhibitors in pain reduction could be demonstrated in several animal models of nociception.9, 10, 11 In addition to its roles in epilepsy and pain, it has become more and more evident that AdK inhibition could also serve as a potential therapeutic approach for the treatment of other neurological conditions, where adenosine homeostasis is disrupted, such as schizophrenia. It was demonstrated that the pharmacological inhibition of AdK using the AdK inhibitor ABT-702 produced an antipsycotic-like activity in mice indicating that AdK inhibitors might in fact be useful as therapeutic agents in schizophrenia.13, 14 Since adenosine is an endogenous anti-inflammatory agent, AdK inhibitors have also potential for the treatment of inflammatory diseases such as the chronic inflammatory bowel disease. Peripherally acting AdK inhibitors may be useful as site- and event-specific amplifier of endogenous adenosine. This therapeutic approach may allow a down-regulation of local inflammatory responses without exerting systemic side effects. Recently AdK was shown to act as a cell-autonomous negative regulator of β-cell replication suggesting potential of AdK inhibitors as therapeutic agents for diabetes. Moreover, AdK inhibitors may be effective in controlling epigenetic mechanisms. In addition to the above mentioned effects, adenosine exerts receptor-independent effects including epigenetic changes due to interference with DNA methylation.17, 18, 19 It is well-known that adenosine accumulation due to an adenosine kinase deficiency leads to increased levels of S-adenosylhomocysteine (SAH) which in turn causes an inhibition of DNA methyltransferases.20, 21 Therefore AdK inhibitors might act as indirect DNA methyltransferase inhibitors in cancer therapy. Besides, also parasitic AdK emerges as a promising drug target. In the past years, extensive research in the field of bacteriology and parasitology revealed differences between parasitic and human AdK opening up new possibilities to fight human pathogens such as Mycobacterium tuberculosis which is the causal agent of tuberculosis22, 23 or, Toxoplasma gondii that causes toxoplasmosis in humans, respectively, without affecting the host. Although considerable work has been performed to optimize AdK inhibitors in the past,9, 10, 25, 26, 27, 28 new structural classes leading to more specific AdK inhibitors are of great importance. Isoform-specific AdK inhibitors exerting tissue-specific effects, or non-brain-penetrating AdK inhibitors for peripheral indications, would be beneficial to overcome issues like toxicity limiting the therapeutic value of AdK inhibitors.
Material and methods Adenosine, magnesium chloride hexahydrate, adenosine 5′-triphosphate (ATP), DMSO, Tris (Trizma base), ABT-702 (4-amino-5-(3-bromophenyl)-7-(6-morpholino-pyridin-3-yl)pyrido[2,3-α]pyrimidine), 5 iodotubercidin (5-IT), lanthanum chloride (LaCl3) were purchased from Sigma (Taufkirchen, Germany). EDTA disodium salt and dipotassium hydrogenphosphate (K2HPO4) were obtained from Roth (Karlsruhe, Germany). [2,8-3H]Adenosine (33.1Ci/mmol) was obtained from PerkinElmer Life Sciences (Rodgau-Jügesheim, Germany). [3H]CCPA ([3H]2-chloro-N6-cyclopentyladenosine) was purchased from NEN Life Sciences (58Ci/mmol) and [3H]MSX-2 ([3H]3-(3-hydroxypropyl)-7-methyl-8-(m-methoxy-styryl)-1-propargyl-xanthine) from Amersham (84Ci/mmol, custom synthesis). The non-radioactive precursor of [3H]MSX-2 (MSX-1) was synthesized in our laboratory.29, 30 The human cDNA AdK clone (transcript variant AdK-short, accession number: NM_001123) was obtained from OriGene Technologies. The compounds tested for AdK inhibition were selected from our in-house compound library.