Replacement of isobutyl by trifluoromethyl in pepstatin A selectively affects inhibition of aspartic proteinases

Two bis-trifluoromethyl pepstatin A analogues, carboxylic acid I and its methyl ester 2, have been synthesised in order to probe the properties and size of the trifluoromethyl (Tfm) group and compare it to the "bigger" isobutyl that is present in pepstatin A. The results demonstrate that Tfm can effectively replace the isobutyl chain as far as inhibitory activity against plasmepsin II (PMII), an aspartic proteinase from Plasmodium falciparum, is concerned. On the other hand, replacement of isobutyl by Tfm selectively affected activity against other asportic proteinases tested. Two lines of evidence led to these conclusions. Firstly, compounds 1 and 2 retained single-digit nanomolor inhibitory activity against PMII, but were markedly less active against PMIV cathepsin D and cathepsin E. Secondly, the X-ray crystal structures of the three complexes of PMII with 7, 2 and pepstatin A were obtained at 2.8, 2.4 and 1.7 A resolution, respectively. High overall similarity among the three complexes indicated that the central Tfm was well accommodated in the lipophilic S, pocket of PMII, where it was involved in tight hydrophobic contacts. The interaction of PMII with PheIII appeared to be crucial. Comparison of the crystal structures presented here, with X-ray structures or structural models of PMIV and cathepsin D, allowed an interpretation of the inhibition profiles of pepstatin A and its Tfm variants against these three enzymes. Interactions of the P, side chain with amino acids that point into the S, pocket appear to be critical for inhibitory activity. In summary, Tfm can be used to replace an isobutyl group and can affect the selectivity profile of a compound. These findings have implications for the design of novel bioactive molecules and synthetic mimics of natural compounds.