S-1 subsite specificity of a recombinant cysteine proteinase, CPB, of Leishmania mexicana compared with cruzain, human cathepsin L and papain using substrates containing non-natural basic amino acids

We have explored the substrate specificity of a recombinant cysteine protei nase of Leishmania mexicana (CPB2.8 Delta CTE) in order to obtain data that will enable us to design specific inhibitors of the enzyme. Previously we have shown that the enzyme has high activity towards substrates with a basi c group at the P-1 position [Hilaire, P.M.S., Alves, L.C., Sanderson, S.J., Mottram, J.C., Juliano, M.A., Juliano, L., Coombs, G.H. & Meldal M. (2000) Chem. Biochem. 1, 115-122], but we have also observed high affinity for pe ptides with hydrophobic residues at this position. In order to have substra tes containing both features, we synthesized one series of internally quenc hed fluorogenic peptides derived from the sequence ortho-amino-benzoyl-FRSR Q-N-[2,4-dinitrophenyl]-ethylenediamine, and substituted the Arg at the P-1 position with the following non-natural basic amino acids: 4-aminomethyl-p henylalanine (Amf), 4-guanidine-phenylalanine (Gnf), 4-aminomethyl-N-isopro pyl-phenylalanine (Iaf), 3-pyridyl-alanine (Pya), 4-piperidinyl-alanine (Pp a), 4-aminomethyl-cyclohexyl-alanine (Ama), and 4-aminocyclohexyl-alanine ( Aca). For comparison, the series derived from ortho-amino-benzoyl-FRSRQ-N-[ 2,4-dinitrophenyl]-ethylenediamine was also assayed with cruzain (the major cysteine proteinase of Trypanosoma cruzi), human cathepsin L and papain. T he peptides ortho-amino-benzoyl-FAmfSRQ-N-[2,4-dinitrophenyl]-ethylenediami ne (k(cat)/K-m = 12 000 mm(-1).s(-1)) and ortho-amino-benzoyl-FIafSRQ-N-[2, 4-dinitrophenyl]-ethylenediamine (k(cat)/K-m = 27 000 mm(-1).s(-1)) were th e best substrates for CPB2.8 Delta CTE. In contrast, ortho-amino-benzoyl-FA maSRQ-N-[2,4-dinitrophenyl]-ethylenediamine and ortho-amino-benzoyl-FAcaSRQ -N-[2,4-dinitrophenyl]-ethylenediamine were very resistant and inhibited th is enzyme with K-i values of 23 nM and 30 nM, respectively. Cruzain hydroly zed quite well the substrates in this series with Amf, Ppa and Aca, whereas the peptide with Ama was resistant and inhibited cruzain with a K-i of 40 nM. Human cathepsin L presented an activity on these peptides very similar to that of CPB2.8 Delta CTE and papain hydrolyzed all the peptides with hig h efficiency. In conclusion, we have demonstrated that CPB2.8 Delta CTE has more restricted specificity at the S-1 subsite and it seems possible to de sign efficient inhibitors with amino acids such as Ama or Aca at the P-1 po sition.