Purpose

We have identified GDPD6 as a critical enzyme in the degradation of glycerophosphocholine (GPC) in choline metabolism, which can be detected using high-resolution (HR) ¹H magnetic resonance spectroscopy (MRS), and which is a potential therapeutic target in breast cancer. Using siRNA silencing, we have found a decrease in invasion and migration associated with the knockdown of GDPD6.¹ To date, no small molecule inhibitor is available to target GDPD6. Having a GDPD6 inhibitor available would be beneficial for studying the role of GDPD6 in the lab, as well as a potential therapeutic for use in the clinic. Using computational studies and ¹H HR MRS, we have identified dipyridamole, an FDA approved non-specific phosphodiesterase inhibitor, as a GDPD6 inhibitor.

Methods

The highly metastatic, triple-negative MDA-MB-231 breast cancer cell line was chosen to determine the effects of dipyridamole, as we have previously demonstrated that siRNA silencing of GDPD6 in MDA-MB-231 cells shows an increase in GPC by 1H HR MRS as well as a decrease in invasion and migration.¹ MDA-MB-231 cells were treated with 20 µM dipyridamole prior to dual-phase extraction and ¹H HR MRS of the water-soluble extract fractions. The choline metabolites including GPC, phosphocholine (PC), choline (Cho), and total choline-containing compounds (tCho, i.e. the sum of Cho, PC, and GPC) were quantified using previously established methods.² We have calculated the GPC/tCho ratio for several different time points of dipyridamole treatment in MDA-MB-231 cells as GDPD6 inhibition should lead to an increase in GPC levels relative to tCho. We have also identified a homology model for GDPD6 to determine the active site of GDPD6 and how dipyridamole acts as an inhibitor for GDPD6 computationally to guide further experiments in evaluating dipyridamole and related compounds as potential inhibitors for GDPD6. The human GDPD6 protein sequence (NP_062539.1) was retrieved from the NCBI database for 3D structure prediction. The 3D structure was predicted using the BioSerf server, which is a fully automated homology-modeling server.³ SwissDock⁴ was used to generate a series of potential conformations of dipyridamole binding.

Results

¹H HR MRS of choline metabolites, i.e. GPC, PC, Cho, and tCho (Figure 1A), from dipyridamole treated cells clearly shows an increased GPC/tCho ratio in MDA-MB-231 cells after 24, 48, 72, and 96 hours of treatment as compared to control cells treated with DMSO (Figure 1B). These data are in agreement with our previous findings showing an increase in GPC in MDA-MB-231 cells in which GDPD6 was silenced with siRNA.¹ Our new data indicate that dipyridamole inhibits GDPD6 activity. To understand the mechanism of this inhibition and potentially improve upon dipyridamole as an inhibitor, we have identified a homology model (Figure 2A), which is being used for docking experiments to determine how dipyridamole, or other potential inhibitor(s), binds to GDPD6. Some low energy models demonstrate competitive binding in the predicted active site, with potential interactions with active site residues (Figure 2B). The lower the energy of a computational binding model the higher is its likelihood of being a valid prediction for experimental results.

Discussion

We have previously identified GDPD6 as the primary enzyme responsible for the breakdown of GPC to Cho in choline phospholipid metabolism of breast cancer cells.1 GDPD6 silencing by siRNA in MDA-MB-231 breast cancer cells resulted in an increase in GPC levels as compared to control.¹ Our new results with dipyridamole are in agreement with these previous findings, as dipyridamole increased the GPC/tCho ratio in MDA-MB-231 cells. These data indicate that dipyridamole inhibits GDPD6 activity in MDA-MB-231 cells. A homology model was developed in order to allow us to access computational studies to understand the mechanism of inhibition of GDPD6, which will potentially allow us to improve a small molecule GDPD6 inhibitor based on dipyridamole, or identify other compounds that could potentially inhibit GDPD6.

Conclusions

GDPD6 has been identified in breast cancer cell lines as the enzyme primarily responsible for the breakdown of GPC to Cho. We have also previously demonstrated that siRNA silencing of GDPD6 leads to a decrease in invasion and migration of MDA-MB-231 cells, however, small molecule inhibitors are preferable for use in the lab and as potentially translatable therapeutics in the clinic. We have identified dipyridamole, a commercially available FDA-approved drug, as a potential non-specific GDPD6 inhibitor, and we are currently using computational methods to determine how dipyridamole acts as an inhibitor as well as identify other potential inhibitors.

Acknowledgements

We thank all members of the Division of Cancer Imaging Research in The Russell H. Morgan Department of Radiology and Radiological Science for their help and support.