Covalent Enzyme Inhibitors in Drug Design

Epoxysuccinates have shown promising activity as irreversible inhibitors of calpain and cathepsin in indications from cancer to Alzheimer’s disease (AD). Their poor pharmacokinetic properties have been addressed by use of simple epoxysuccinate ester prodrugs in preclinical and clinical trials. A prodrug strategy requires the prodrug to be converted efficiently to the drug molecule and for the prodrug itself not to engage off-target biomolecules. We observe that the epoxysuccinate esters are both more reactive and provide an alternative reaction pathway for covalent modification of biomolecules. Reactivity was studied towards GSH, glutathione-S-transferase (GST-P1), and in plasma. The parent epoxysuccinates are stable, whereas ester prodrugs undergo ready base-catalyzed reaction. The enhanced thiophilic reactivity of the neutral epoxide forces a dominant reaction pathway that does not yield the parent drug. This unappreciated reactivity of epoxysuccinyl prodrugs creates challenges in drug design and must be taken into account in interpretation of preclinical and clinical results, since these prodrugs will engage targets other than the cysteine proteases that the parent drug is designed selectively to inhibit. These findings make it obvious that epoxysuccinate esters are unsuitable as clinical candidates due to off-target reactivity. To obtain clinical leads maintaining the desirable pro-cognitive effects of the compounds but lacking the general chemical reactivity, several routes can be taken. Firstly, the electrophilic warhead can be modified in order to solve the problem of high off-target reactivity. A tight-binding reversible inhibitor would be superior in many respects to an irreversible inhibitor. Since a peptidomimetic recognition group is available that confers potent binding to the target enzymes, an alternative electrophile has the potential of solving the problems associated with the epoxysuccinate reactivity. A library of molecular probes was synthesized with the goal of maintaining potent inhibition of target cysteine proteases with attenuated reactivity towards general nucleophiles. An increased membrane permeability compared to the epoxysuccinates through the absence of negative charge was also desired. Several novel electrophilic warheads as well as groups known to react with active cysteines were explored with the intent to identify inhibitors of the target enzymes, calpain 1 and cathepsin B