The Design, Synthesis and in Vitro Evaluation of a Novel Pro-Oxidant Anticancer Prodrug Substrate Targeted to Acylamino-Acid-Releasing Enzyme

Document Type

Book Contribution

Publication Date

1-1-2014

Description

Cancer cells often exhibit a high level of intrinsic oxidative stress due to an increased formation of reactive oxygen species and a decreased expression of enzymatic antioxidants. Prodrugs inducing additional oxidative stress can selectively induce apoptosis in cancer cells already having a high level of intrinsic oxidative stress. This study focused on the rational design and in vitro evaluation of a novel prodrug ester, (4- [(nitrooxy)methyl]phenyl-N-acetyl-L-alaninate or NPAA) activated by acylamino-acidreleasing enzyme (AARE, EC 3.4.19.1) to yield a quinone methide (QM) intermediate capable of depleting glutathione (GSH), a key intracellular antioxidant. NPAA shares structural features with both nitric oxide donating aspirin (NO-ASA), a wellcharacterized QM releasing anticancer prodrug, and N-acetyl-L-alanine-4-nitroanailide (AANA), a known specific substrate for AARE. AARE is a serine peptidase that is overexpressed in some tumors and cancer cell lines. The overall approach was to first predict the 3-dimensional structure of both rat (rAARE) and human AARE (hAARE) and then use the resulting low-resolution models to determine if NPAA was a plausible prodrug by estimating its affinity to hAARE and rAARE in comparison to AANA. The AARE models were constructed using a bioinformatic-based protein structure prediction webserver (I-TASSER) followed by energy minimization and refinement. The resulting models were subjected to a variety of structural quality assessments. The optimal models of hAARE and rAARE were found to have similar three-dimensional structures with a ß- propeller domain and an a/ß-hydrolase domain containing an exopeptidase catalytic site with active site residue distances typically found in serine peptidases. Protein-ligand docking studies showed that both AANA and NPAA could bind to the exopeptidase catalytic site of the hAARE and rAARE models with reasonable affinities and in a region with a highly druggable pocket. In order to validate the in silico results, NPAA was synthesized, purified, physically characterized and evaluated for its in vitro ability to deplete GSH in the presence of rAARE. As anticipated, NPAA was found to deplete GSH and this effect was completely blocked by diisopropylfluorophosphate (DFP), an irreversible inhibitor of serine proteases, including rAARE. These studies support further efforts to optimize the design of QM releasing anticancer prodrugs targeted to AARE. Moreover, the molecular models presented here could be useful for the rational design of AARE inhibitors, which could also be exploited as potential anticancer agents.

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