Major current targets for cancer therapeutic drugs include growth signaling pathways, the cellular division machinery, and cellular DNA itself. Although cytotoxic DNA-damaging agents such as IR have been used in cancer treatment for many years, there have been few attempts to target the DNA repair machinery. As the molecular mechanisms for cellular DNA damage responses have become more clearly understood, there has been increasing interest in enhancing the effects of chemotherapy by disrupting these systems.3
This approach maybe particularly important in cancers that have lost some capacity to repair DNA rendering them hypersensitive to additional loss of ordinarily redundant mechanisms. Several molecules that target the DNA damage response network have been identified, with some of them, such as PARP1 inhibitors that block the repair of DNA single-strand breaks and Chk1 and Chk2 kinase inhibitors that inhibit cell cycle progression,26,27
currently being evaluated in clinical trials.
The hypersensitivity of Lig4-defective cells to IR and DNA-damaging drugs suggests that this enzyme is a good target for the development of inhibitors that potentiate the cytotoxic effects of DNA damage. Although several small molecules inhibitors of human DNA ligases have been identified using structure-based computational design,28
none of the inhibitors were specific for Lig4. And unlike Lig1 and Lig3,23,29,30
there is no available structural information for the Lig4 catalytic domain. Chen and coworkers described a Lig4 assay with potential use in screening large chemical libraries. The assay makes use of a dual-labeled DNA strand with a donor fluorophore (D) and a quencher fluorophore (Q) within proximity to each other and separated by the ligation site. To distinguish between the ligated and unligated DNA oligonucleotides, as both exhibit 100% fluorescence intensities, a thermal denaturation step is performed using a thermocycler in which the DNA duplex is denatured to ssDNA oligonucleotides upon exposure at 95°C for 5min. Upon cooling of the reaction, the ligated ssDNA oligonucleotides adopt secondary structures bringing Q in close proximity to D resulting in a loss of fluorescence intensity.31
The described denaturation step could potentially result in many false positives as compounds would bind nonspecifically to the ssDNA oligonucleotides as the temperature is cooling down to RT at a rate of 2°C per minute for a total of 92
min. Other ligation assays using fluorescently labeled DNA substrates have also been reported for bacterial DNA ligases,32,33
and have been shown to be amenable to miniaturization and used in screening large chemical libraries; however, they do require a denaturation step using 8M urea to separate the fully ligated DNA strand harboring the fluorophore from the other strand template harboring the quencher.32,33
The SPA is a radioisotopic assay technique that has been widely used in screening chemical libraries of various therapeutic targets;34,35
it is homogeneous in nature and does not require washing steps. Like fluorescence-based assay technologies, SPA does also suffer from sensitivity to various colored compounds yielding higher hit rates due to false hits scored during screening campaigns. However, the recent introduction of the PS imaging beads results in “red-shifted” assay readouts that appear to be relatively insensitive to colored compounds, especially those absorbing in the yellow, red, and blue ranges of the light spectrum.35
Other inherent limitations of SPA are as follows: (i) the requirements for specific infrastructure to handle radioactive material usage and disposal, which is typically expensive and (ii) the NPE due to the excitation of the fluorophore in the beads by the nonspecific radiation from the tracer when using higher energy radioisotopes such as [32
P], or [125
I]; this NPE effect limits the specific activity of the reporter radioisotope to be used in the assay and in many instances renders the use of SPA inadequate. The ultimate advantage of SPA is the direct capture of the reaction product harboring a radioactive tracer35
; this attribute was an important consideration in designing our Lig4 assay because we were monitoring the loss of an incorporated radioactive tracer in the DNA substrate upon complete ligation. Further, the preformed Lig4-[α-33
P]-AMP complex offers the additional advantage for the SPA approach as it would exclude nucleotide analogs from scoring as hits in this format, but not in the fluorescence-based assay formats described previously. DNA interchelators, on the other hand, are another class of nonspecific inhibitors to which the SPA would be insensitive, but the secondary assays in place would eliminate these hits from further consideration.
For this purpose, we have successfully developed a scintillation proximity-based assay to screen for inhibitors of the enzymatic activity of Lig4 where our strategy was to block the deadenylation step of the Lig4-AMP complex and potentially resulting in an extra selectivity for actives in chemical libraries. A chemical screen was completed against a library of 5,280 compounds, containing known bioactives and FDA-approved drugs, resulting in four confirmed hits (rabeprazole, cytochalasin A, U73122, and NSC95397) with IC50
values ranging from 1 to 30
μM (); though not particularly potent, these compounds have proven useful nonetheless in follow-up mechanistic studies and in their potential use as tool compounds.
The Lig4 inhibitors found in this study are known bioactives. Rabeprazole is a PPI. The PPIs suppress gastric acid secretion by inhibiting the final transport of hydrogen ions into the gastric lumen in the gastric H+
The PPIs become active forms upon entering the lower pH environment (e.g.
, inside parietal cells), and then they can bind to H+
-ATPase and inhibit ATPase function. Because the pH of our designed assay is 8.0, rabeprazole remains in its inactive form. Two other PPIs were included in the screening library and were initially selected as outliers in the screen and not pursued (); this suggests that this chemical scaffold maybe important for binding to the Lig4 complex. It is unlikely that the inhibitory mechanism of Lig4 by rabeprazole is the same as the inhibition of the H+
-ATPases. The U73122 is a phospholipase C inhibitor.38,39
Phospholipase C cleaves phosphatidylinositol 4,5-bisphosphate into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3
). The IP3
works to increase concentration of cytosolic calcium, and DAG and calcium work together to activate protein kinase C that triggers the signal transduction pathway. The U73122 also shows inhibitory effects on a variety of receptor-mediated signal transductions.40,41
The mechanism of inhibition of Lig4 by U73122 remains unclear.
Chemical Structures of Rabeprazole Derivatives in the Screening Collection
Rabeprazole, cytochalasin A, U73122, and NSC95397 were found to inhibit the AMP transfer step of the ligation process by Lig4, though at a high compound concentration of 100
μM, supporting the argument that such Lig4 inhibitors could be found through screening against larger chemical libraries. Both rabeprazole and U73122, when tested in an in vitro
DNA joining assay, were found to inhibit the formation of ligation products by up to 35% (), whereas cytochalasin A and NSC95397 had little or no effect on the DNA joining reaction. Close examination of the inhibitory effects revealed that rabeprazole and U73122 observed effects were not mediated by blocking dsDNA substrate to the enzyme thus ruling out their effects as nonspecific DNA intercalators (). Although the three human DNA ligases share a related catalytic domain and a common catalytic mechanism, the two most active Lig4 inhibitors, rabeprazole and U73122, identified in the screen had no detectable inhibitory activity against both Lig1 and Lig3. It is conceivable that these inhibitors interact with the enzyme–substrate complex and that there are significant conformational constraints between the enzyme–substrate complexes formed by the three human DNA ligases. Thus, it may also be possible to identify specific inhibitors of Lig1 and Lig3 by adapting our scintillation proximity-based assay platform to screen for inhibitors of Lig1 and Lig3.
Although rabeprazole and U73122 only partially inhibited specifically the Lig4 function in vitro, they exhibited intended cellular activity by slowing down the repair of gamma irradiation–induced DNA breaks in HeLa cells. This is a good indication for their potential use in combination therapy with DNA-damaging agents and IR to treat cancer. While rabeprazole and U73122 are not potent enough for use effectively in cancer xenograft models, as a starting point followed conceptually by human clinical trials, our experiments can provide the basis for a proof-of-concept and rationale to target Lig4 as a druggable entity, identify inhibitors and develop them as drugs to treat cancer in combination therapies, or for additional screens against larger chemical libraries to identify more potent and novel Lig4 inhibitors.