TopII has become an enzyme of major interest because it is targeted by anticancer drugs that are routinely used in the clinic, such as etoposide and doxorubicin [12
]. Most clinically active drugs that target TopII generate protein-DNA covalent complexes [13
]. The generation of TopII–DNA complexes has profound effects on cell physiology, such as blocking transcription and replication. Following treatment with TopII poisons, DNA strand breaks are rapidly detected, and most are covalently attached to proteins [16
]. Cells subsequently undergo apoptosis [18
The repair mechanism of TopII-DNA complexes is not very clear, although it is likely that blocking the repair of the TopII-DNA complex can increase the efficacy of drugs like etoposide and doxorubicin. Recently, it was shown that TDP2 can cleave TopII-DNA adducts [9
]. It was proposed that TDP2 functions in conjunction with components of the non-homologous end-joining machinery. Ku and DNA ligase IV probably control events at the double strand breaks (DSBs) before and/or after TDP2 activity because Lig4
−/− and Ku70
−/− DT40 cells have exhibited high levels of sensitivity to etoposide [19
Like TDP1, it is likely that effective inhibition of TDP2 can be targeted to improve the efficacy of TopII poisons [10
]. DT40 cells with a targeted deletion of TDP2 are hypersensitive towards etoposide but not to methyl methane sulfonate (MMS) or the TopI poison camptothecin. Thus,
TDP2 inhibitors may have therapeutic utility in treating cancers that are refractory to TopII-poison treatment. To facilitate inhibitor screening in a high throughput manner, we developed an efficient assay system and studied the kinetic properties of TDP2 using chromogenic T5PNP as a substrate in a 96-well format. We also compared those properties with the established TDP2 substrates.
TDP2 displayed a fast reaction rate for cleavage of the 5′-phosphotyrosyl adduct, although the overall efficiency of TDP2 towards 5-sub was much higher than towards T5PNP. Our study indicates the 3′-phosphotyrosyl phosphodiesterase activity of TDP2 is not very efficient. A recent study showed that DT40 cells lacking TDP2 were not hyper-sensitive to camptothecin but that they were to etoposide. This result indicates that the 3′ enzymatic activity of TDP2 is not very prominent in the presence of TDP1 in vivo
]. The fast 5′-phosphotyrosyl phosphodiesterase activity of TDP2 is quite unique among human repair enzymes. To our knowledge, the only known comparable human repair enzymes are APE1 and TDP1. APE1 requires such high reactivity for cell survival because the spontaneous production of abasic sites is lethal [20
]. It has been shown that deficiency in the TDP1-mediated repair pathway in humans causes spinocerebellar ataxia with axonal neuropathy (SCAN1) by affecting large, terminally differentiated, non-dividing neuronal cells [22
]. TDP2 is another fast enzyme in the repair field. The biological need for such a highly efficient enzyme to remove 5′-phosphotyrosyl adducts will obviously be interesting to study in the future. The Km
value of TDP2 towards T5PNP was determined to be 54 mM (). This value is much higher than the Km
for 5-sub used in the gel-based assay, which offered a more extensive surface for binding although the turnover actually is higher for T5PNP compare to 5-sub (). Salt and pH effects were similar towards all 3 substrates. Increasing the salt concentration actually decreased TDP2 activity, possibly indicating the presence of more charged amino acids in the catalytic pocket. The exact nature of the macromolecular substrate, i.e., the TopII–DNA covalent complex, required by TDP2 for efficient catalysis remains unknown. Sodium orthovanadate is known to inhibit TDP2, shown in gel based assay (10
). Our result showed sodium orthovanadate also inhibits product formation from T5PNP (IC50
=40 mM). TDP1 and APE1, the closest relatives of TDP2, were not able to form product, so T5PNP is not a nonspecific substrate for any phosdiesterase.
In this study, the synthetic chromogenic substrate T5PNP was used to determine the kinetic parameters of TDP2, which hydrolyzes the phosphodiester bond linking the TopII enzyme and its DNA substrate in the presence of TopII poisons, such as etoposide. Inhibitors of TDP2 might potentiate or synergize the cytotoxic effect of TopII poisons used as cancer therapeutic agents. The 96-well format developed for this assay should facilitate drug screening in a HT manner, and we obtained a signal to noise ratio of 5 when we used only 90 nM protein. However, one probable limitation of this assay may be the spectrophotometric detection of p-nitrophenol because of p-nitrophenol’s relatively poor extinction coefficient (15,000 M−1cm−1). TDP2 reaction kinetics are rapid, which explains why it needs a minimum protein concentration of 90 nM to obtain the signal in only 30 min. Also, because this assay does not require any washing steps, the signal to noise ratio is high. Although fluorescence-based assays may be more sensitive, the background signal is typical of fluorescent methods. Furthermore, auto-fluorescence of some compounds may increase this background and limit the utility of this assay. Overall, our assay offers a quick, simple, and straightforward method that can be used in a HT manner for inhibitor screening.