Figure 5. DeMAND identifies the MoA of altretamine.
(A) GSH concentration following treatment of cells by negative control (DMSO, gray), sulfasalazine as a positive control (red), and altretamine (blue) show that sulfasalazine reduces active GSH levels compared to control, while altretamine results in active GSH levels indistinguishable from the control.
(B) The level of a GPX4-specific substrate (PC-OOH) is measured by mass spectrometry (a) without cell lysate (gray), (b) with untreated cell lysate (green), and (c) with cell lysate from altretamine treated cells (blue). PC-OOH levels in altretamine treated cells are similar to no-lysate, and markedly different from untreated lysate, indicating that altretamine inhibits GPX4 activity.
(C) Lipid reactive oxidative species (ROS) levels were measured by flow cytometry using DMSO treated cells (black curve, as control) and compound treated cells (red curve). Both altretamine and sulfasalazine significantly increases lipid-ROS levels, confirming the predicted similarity in their functional effect.
(D) Sulfasalazine is a known inhibitor of the System xc− cystine/glutamate antiporter. Its downstream effect on Glutathione (GSH) and GPX4 leads to accumulation of lipid ROS. DeMAND predicted significant similarity between sulfasalazine and altretamine and GPX4 but not GSH as altretamine specific MoA proteins, as experimentally confirmed panels (A–C).