Alternative mechanisms of AG126

Tyrphostins as a novel class of PTK inhibitors were originally designed as chemotherapeutic agents to fight cancer. PTK’s have a plethora of regulatory functions in signaling cascades of metabolism, cell proliferation, angiogenesis and immune system of multicellular organisms. PTK malfunction can lead to diseases with their abnormal activity affecting whole signaling pathways (Levitzki and Mishani, 2006). During the developmental process, tyrphostins were synthesized and tested for selective inhibitory capacity on epidermal growth factor receptor (EGFR) kinase, leading for example to AG18 and AG213, also known as RG50864 and RG50810 (Yaish et al., 1988; Gazit et al., 1989; Lyall et al., 1989). For other tyrphostins, additional specific PTK’s targets could be assigned, such as for AG879 to nerve growth factor pp140^c−trk(Ohmichi et al., 1993), AG490 to Jak2 (Meydan et al., 1996) and AG1295 to the platelet derived growth factor receptor (Kovalenko et al., 1994). However, most if not all the specific inhibitory effects of these tyrphostins were determined in special assay systems.

Over the years, additional PTK-independent functions could be observed, especially in cellular systems. Antioxidative features, decoupling functions on oxidative phosphorylation and binding capacities to hormone receptors could be shown for certain tyrphostins.

We thus raised the question whether PTK-independent modes of action could also help ex-plaining the biological profile of AG126. Antioxidative properties for AG126 as shown for the JAK2 inhibitor AG490/B42 (Gorina et al., 2007) or AG18 and AG82 (here named as A23 and A25, Sagara et al., 2002 were not tested because AG126 does not exhibit quinone-like structures.

Of course, a chemical degradation or metabolic modification of AG126 in an aquous environment or by enzymatical conversion in a cell cannot be ruled out fully to also lead to a compound with antioxidative features. Yet reactions towards such a structure are not obvious and we, therefore, rendered antioxidant mechanisms unlikely.

5.4.1. AG126 does not act as a decoupler

Oxidative phosphorylation guarantees the synthesis of ATP by ATPase in mitochondria. The reactions of electron transport, providing energy for ATPase activity, and phosphorylation of ADP to ATP by ATPase are coupled. Decoupling substances have the ability to separate these two processes and to thereby suppress the ATP synthesis (Fig. 2.1). Tyrphostin AG17

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(A9 or SF6847) can decouple the respiratory chain (Terada, 1990). Tyrphostin AG10 and AG18 are also able to act as mitochondrial uncouplers (besides inhibitory functions on EGFR autophosphorylation and antioxidative features, Sagara et al., 2002; Soltoff, 2004). Because of these published effects, we examined such potential functions for AG126.

Using JC-1 for a membrane potential-depending fluorescence staining of mitochondria in TLR-activated or control microglia with and without AG126 treatment, we could not detect a clear effect. For a potent uncoupler a dissociable group and a hydrophobic moiety (given by BZ residue) as well as a strong electron withdrawing group are required (given by the MN residue, Terada, 1990. Yet for AG126, no decoupling property seemed to play role (Fig. 4.15).

In contradiction to our results, Sagara et al. (2002) concluded from there experiments that AG126 acts as a mitochondrial decoupler. They induced cytotoxcicity in the hippocampal neuronal cell line HT-22 by glutamate treatment, causing cell death as well as membrane dis-ruption, with a low JC-1 monomer/aggregate ratio. Tyrphostins A9 and AG126 were able to increase the JC-1 monomer/aggregate ratio as the known decoupler FCCP (cyanide p-trifluoromethoxyphenylhydrazone) did. Different to our experimental setup, they dissolved the tyrphostins in Me2SO instead of DMSO. Furthermore, Sagara et al. tested cells in a state of oxidative stress, caused by glutamate and following excessive production of ROS in mitochondria (Davis and Maher, 1994). ROS accumulation is caused by insufficient ROS elimination. Our microglia, however, were in a ‘endangered’ and activated state, but not in a cytotoxic environ-ment. Nevertheless, AG126 may affect mitochondrial functions, even though not like a classical decoupler. Regulation Ca²⁺ influx (Tan et al., 1998) and [Ca²⁺]i could be an AG126-relevant feature. In this regard, our earlier observations on [Ca²⁺]i may require some re-consideration (Hoffmann et al., 2003; Kann et al., 2004).

5.4.2. AG126 does not signal via the GR

For some ‘master template’ tyrphostins, such as the genistein (inhibits EGFR pp60^v−src and pp110^{gag−f es}, Akiyama et al., 1987), PTK-independent functions are known. The compound was originally known as a phytoestrogen ligand to the estrogen receptor (Martin et al., 1978).

Later on, Young et al. (1993) published genistein to have inhibitory effects on the fatty acid synthesis, lactate transport, mitochondrial oxidative phosphorylation as well as on aldehyde dehydrogenase.

We considered AG126 to probably use the steroid receptor, the GR in microglia, for in-tracellular signaling. Indeed, GR expression in microglia and GR interaction with TLR’s in macrophages were previously shown (Sierra et al., 2008; Ogawa et al., 2005). Protective func-tions of GRs during innate immune responses, also in the brain, have been described (Glezer and Rivest, 2004)

GRs are ligand-induced transcription factors that are localized in a non-active state in the cy-toplasm, in association with some heat shock proteins (hsp) 90, 70 and 56 (Pratt, 1993). Ligand binding leads to a conformational change, dissociation from the hsp complex and translocation

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to the nucleus (Picard and Yamamoto, 1987). GR then influence gene transcription by binding to the GC response element or by the interaction with transcription factors (Nancy Ing and O’Malley, 1995).

In agreement with a AG126 effect, GR modulation is regulated by the TLR adapter protein MyD88. LPS activation of IRF3-dependent genes, that were dexametasone (dex)- sensitive inwt macrophages became dex-resistant in MyD88ko cells (Ogawa et al., 2005). This study revealed that dex was able to repress the release of TNFα and KC in TLR1-2- and TLR6-2-activated cells, quite similar as AG126 did. In some contrast to AG126, dex also repressed the release activities of TLR4-activated microglia (Fig. 4.18).

Moreover, the aziridine precursor from the African shrubSalsola tuberculatiformis Botschantzev, CpdA, is able to bind to the GR, although it has no steroid structure, but rather a tyrosine-related one. As a GR ligand, this compound is able to mediate gene-inhibitory effects on TC10 endothelial cells (De et al., 2005). In the TLR-activated microglia, CpdA revealed then a sim-ilar release profile as described for AG126. Interestingly, while CpdA repressed the release in Pam₃CSK₄- and MALP-activated microglia, it could not repress the release of KC and TNFα in LPS-activated cells — another striking parallel to AG126. High concentrations of CpdA were toxic to microglia, as it decomposes into aziridine intermediates known to act as alkylating pro-apoptotic agents (Fig. 4.18; Wüst et al., 2009).

Despite similar impacts on the release profiles obtained with CpdA and AG126 and the tyrosine relatedness of both compounds, GRko-based studies excluded a potential action of AG126 via the GR signaling pathway (Fig. 4.19).

5.4.3. AG126 does not act via adrenergic receptors

Noradrenaline (NA) is a stress hormone as well as a neurotransmitter and is known — along with adrenaline — for its impact on the ‘fight or flight’ response of the autonomic nervous system.

NA is primarily produced by the Locus caeruleus in the CNS, and by the adrenal gland in the periphery, and signalsvia the adrenergic receptors. The amino acid tyrosine is the starting product for its biosynthesis. Regarding its tyrosine-related structure, the question arose whether AG126 may signal — similar to NA — through the adrenergic receptors. Many aspects support the idea that AG126 may act in a comparable fashion.

In the periphery, NA is known to modulate innate immune function, e.g. phagocytosis and TNFαresponse of macrophages (Gosain et al., 2006; García et al., 2003; Hu et al., 1991; Spengler et al., 1994). As a neurotransmitter, NA has excitatory and modulating functions on neurons (Benarroch, 2009). NA has also modulating functions for inflammatory gene expression in the brain and, thereby, influences CNS diseases, including also MS, AD or PD. It reduces cytokine expression in microglial, astroglial and brain endothelial cells in vitro (Feinstein et al., 2002).

Influences of NA on microglia have been documented several times (Colton and Chernyshev, 1996; Färber et al., 2005; Prinz et al., 2001). The expression of the respective adrenergic receptors (α1,α2,β1andα2receptors) for (nor)adrenalin on microglia was previously shown (Mori et al.,

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2002; Tanaka et al., 2002). We confirmed such patterns for our own microglial preparations (van Rossum, unpublished).

In accordance with reports of Prinz et al. (2001); Feinstein et al. (2002); Heneka et al. (2010), to which our group contributed, the present study could show, that NA is able to reduce the release of diverse pro-inflammatory cytokines in a TLR-activated microglia (Fig. 4.16). We thus assumed that AG126, like NA, could act as a adrenergic agonist. However, experiments involving antagonists could not confirm such AG126 activity. The studies were based on Pam3CSK4 -activated microglia, but ruled out the α1/α2 or β1/β2 adrenergic receptors to mediate the suppressive influence of the tyrphostin (Fig. 4.17).