Epoxysuccinate containing peptidomimetic inhibitors of several cysteine-dependent proteins, including calpain and cathepsins, are candidate therapeutic agents, in particular for anti-neurodegenerative therapy. Epoxysuccinates have been administered as ethyl ester prodrugs to circumvent their poor pharmacokinetic properties; for example, E64d, a therapeutic candidate for Alzheimer's disease (AD) that has been recently entered clinical trials. The stability and reactivity of epoxysuccinate salts and their putative ethyl ester prodrugs was measured using LC-MS/MS analysis. The parent carboxylates were stable in the presence of excess thiols: reactivity with GSH was negligible after 24 h at physiological pH and temperature.  In contrast, the ester prodrugs were reactive towards thiols. Importantly, formation of the desired parent drug by hydrolysis of the ester prodrug was observed to be a secondary reaction pathway. Activity directed probes bearing epoxysuccinate carboxylate and ester moieties were designed and studied in incubation with recombinant protein, and cells and their lysates. Covalently modified proteins were visualized in gels using clicking techniques and/or by LC-MS/MS. These methods confirmed that epoxysuccinate ester prodrugs react with cysteine residues of multiple off-target proteins. Assigning the in vivo efficacy of epoxysuccinate ester prodrugs to on-target cysteine protease inhibition is therefore problematic. This previously unappreciated reactivity of epoxysuccinate esters creates challenges in drug design. In order to improve on the pharmacokinetic properties of epoxysuccinates while avoiding promiscuous reactivity, cysteine protease inhibitors with novel warheads were explored. A thiol-reactive functional group was developed that may effectively substitute for epoxysuccinate.

Epoxysuccinyl prodrugs: Discovery of an overlooked prodrug breakdown pathway and its implications in drug development

248th National Meeting (2014)

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