The coronavirus main protease, M(pro), is considered to be a major target for drugs suitable for combating coronavirus infections including severe acute respiratory syndrome (SARS). An HPLC-based screening of electrophilic compounds that was performed to identify potential M(pro) inhibitors revealed etacrynic acid tert-butylamide (6a) as an effective nonpeptidic inhibitor. Docking studies suggested a binding mode in which the phenyl ring acts as a spacer bridging the inhibitor's activated double bond and its hydrophobic tert-butyl moiety. The latter is supposed to fit into the S4 pocket of the target protease. Furthermore, these studies revealed etacrynic acid amide (6b) as a promising lead for nonpeptidic active-site-directed M(pro) inhibitors. In a fluorimetric enzyme assay using a novel fluorescence resonance energy transfer (FRET) pair labeled substrate, compound 6b showed a K(i) value of 35.3 muM. Since the novel lead compound does not target the S1', S1, and S2 subsites of the enzyme's substrate-binding pockets, there is room for improvement that underlines the lead character of compound 6b.

Cysteine proteases are connected to various viral and parasitic infections, as well as to other severe diseases like arthritis, stroke and cancer. Due to its alpha,beta-unsaturated carbonyl moiety etacrynic acid, a well known diuretic, can inhibit cysteine proteases in a Michael-type reaction by reaction with the nucleophilic cysteine residue of the active site. For first structure-activity-relationship studies modifications at various positions of the etacrynic acid structure have been investigated concerning inhibition potency against the CAC1 protease papain: length of the side chain, substitution pattern of the aromatic ring as well as influence and necessity of acidic groups, esters or amides. Additionally, the effect of the aromatic ring was evaluated by replacement with a cyclohexyl moiety.

In comparison to the huge number of peptidic and peptidomimetic inhibitors of cysteine proteases which have been developed during the last twenty years the number of non-peptidic compounds with cysteine protease inhibiting properties is restricted to a few substance classes. In contrast to peptidic and peptidomimetic inhibitors the non-peptidic lead structures have mainly been discovered by computational or enzymatic industrial screenings and not by a rational approach. But, the growing number of resolved X-ray structures of the target enzymes as well as molecular modeling methods have supported the further development of potent inhibitors beginning from these lead structures. In this review we will focus on new non-peptidic cysteine protease inhibitors which have been developed during the last years. Discovery, structure-activity-relationship and inhibition mechanisms will be discussed.

A series of etacrynic acid derivatives was synthesized and screened for their in vitro activity against Plasmodium falciparum, as well as their activity against recombinantly expressed falcipain-2 and -3. The two most active compounds of the series displayed IC50 values of 9.0 and 18.8 μM against Plasmodia.