Chemical Insights in the Concept of Hybrid Drugs:  The Antitumor Effect of Nitric Oxide-Donating Aspirin Involves A Quinone Methide but Not Nitric Oxide nor Aspirin

Abstract

Hybrid drug 1 (NO-ASA) continues to attract intense research from chemists and biologists alike. It consists of ASA and a -ONO2 group connected through a spacer and is in preclinical development as an antitumor drug. We report that, contrary to current beliefs, neither ASA nor NO contributes to this antitumor effect. Rather, an unsubstituted QM was identified as the sole cytotoxic agent. QM forms from 1 after carboxylic ester hydrolysis and, in accordance with the HSAB theory, selectively reacts with cellular GSH, which in turn triggers cell death. Remarkably, a derivative lacking ASA and the -ONO2 group is 10 times more effective than 1. Thus, our data provide a conclusive molecular mechanism for the antitumor activity of 1. Equally importantly, we show for the first time that a "presumed invisible" linker in a hybrid drug is not so invisible after all and is in fact solely responsible for the biological effect.

Amplification of oxidative stress by a dual stimuli-responsive hybrid drug enhances cancer cell death

“…We first performed liquid chromatography-mass spectrometry (LC-MS/MS) analysis to confirm the generation of QM and cinnamladehyde in cells. The generation of QM from QCA was indirectly confirmed by measuring the level of cellular GSH, because it is impossible to detect QM that is an intermediate and rapidly reacts with nucleophilic GSH 7,20 . As shown in Supplementary Fig.…”

“…Cells were treated with various concentrations of QCA or Q 1 for 1.5 h and then the level of GSH was quantitatively measured using Ellman's reagent as previously reported 7 . As shown in Fig.…”

“…Accumulating evidence has suggested that abnormal cancer cells are under oxidative stress associated with an increased production of ROS due to disrupted ROS homeostasis and cancer cells have also adapted to oxidative stress by activating antioxidant systems often through the upregulation of glutathione (GSH) 2,[7][8][9][10] . Ironically, cancer cells are also known to utilize ROS to drive proliferation and other events required for tumour development 11,12 .…”

“…QM was reported to rapidly alkylate GSH, one of the key antioxidant systems in cells and trigger apoptotic cell death 20 . Hulsman et al 7 reported that the potent anticancer activity of nitric oxide-donating aspirin is attributed to QM, but not nitric oxide. Combined use of an ROSgenerating agent with an inhibitor of ROS scavenging systems has been also reported to amplify oxidative stress and be effective in the treatment of advanced solid tumours in terms of both efficacy and selectivity 8,19 .…”

NO-donating NSAIDs and cancer: An overview with a note on whether NO is required for their action

“…Recently, our group [57]and Hulsman et al [58] raised the possibility that, at least in the case of NO-ASA, the NO releasing moiety is not required for its anti-cancer effect. Both sets of data demonstrate that carboxylic ester hydrolysis of NO-ASA leads to the formation of quinone methide which reacts at least with cellular GSH.…”