Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis

Designing cysteine proteinase inhibitors as antitrichomonal drugs requires knowledge of which cysteine proteinases are essential to the parasite. In an attempt to obtain such information, the effects of a number of cysteine proteinase inhibitors on trichomonad growth in vitro and proteinase activity were investigated. The broad specificity inhibitor trans ‐epoxysuccinyl‐ l ‐leucylamido‐(4‐guanidino)butane (known as E‐64) had little effect on growth of Trichomonas vaginalis (27% inhibition at 280 μM, none at 28 μM) even though the addition of 2.8 μM E‐64 to growth medium resulted in inhibition of all but two (apparent molecular masses: 35 k and 49 k) of the parasite's proteinases detected by gelatin SDS‐PAGE. This shows that many of the parasite's cysteine proteinases are not essential for growth in axenic culture. In contrast, a peptidyl acyloxymethyl ketone, N ‐benzoyloxycarbonyl‐Phe‐Ala‐CH 2 OCO‐(2,6,‐(CF 3 ) 2 )Ph, at 16 μM killed T. vaginalis and severely inhibited growth of Tritrichomonas foetus . Exposure of Trichomonas vaginalis to 16 μM of this compound for 1 h resulted in both the 35 kDa and 49 kDa proteinases being inhibited, whereas some other proteinases were unaffected. Similar distinctions between the inhibitor sensitivity of the parasite's cysteine proteinases were apparent when a biotinylated peptidyl diazomethyl ketone was used to detect active proteinases. These data suggest that the growth inhibitory effects of the peptidyl acyloxymethyl ketone are through inhibition of cysteine proteinases that are not affected when the parasites are grown in the presence of E‐64. At least one of these enzymes, which include the 35 kDa and 49 kDa cysteine proteinases, must be essential and so a suitable target for chemotherapeutic attack.