Specificity of S'1 and S'2 subsites of human tissue kallikrein using the reactive-centre loop of kallistatin: the importance of P'1 and P'2 positions in design of inhibitors

We have demonstrated that the S´₁ and S´₂ subsites of human tissue kallikrein (hK1) play determinant roles in the recognition and hydrolysis of substrates. The presence of serine at position P´₁ and arginine at P´₂ resulted in the best substrate, Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, which was derived from the kallistatin reactive-centre loop sequence and quencher groups o-aminobenzoic acid (Abz) and N-(2,4-dinitrophenyl)ethylenediamine (EDDnp). Serine and arginine are also the residues at positions P´₁ and P´₂ in human kininogen, from which hK1 releases Lys-bradykinin. Several peptide analogues of Abz-Ala-Ile-Lys-Phe-Phe-Ser-Arg-Gln-EDDnp, in which the Ser and Arg residues were substituted with various other amino acids, were synthesized and tested as substrates. Most of them were hydrolysed slowly, although they showed significant binding to hK1, as demonstrated by their competitive inhibition constants (K_i). Using this information, six peptides were designed, synthesized and assayed as inhibitors of hK1. Abz-Lys-Phe-Phe-Pro-Arg-Gln-EDDnp, Abz-Lys-Phe-Arg-Pro-Arg-Gln-EDDnp and acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH₂ inhibited hK1 in the range 20–30 nM (letters in italics denote the d-form of the amino acid). The peptide acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH₂ was a weak inhibitor for other serine proteases, as indicated by the higher K_i values compared with hK1, but this peptide was a potent inhibitor of human plasma kallikrein, which has a K_i value of 8 nM. This result was surprising, since this enzyme is known to be a restricted arginyl-hydrolase. In conclusion, acetyl-Lys-Phe-Phe-Pro-Leu-Glu-NH₂ can be used as a leader compound to design specific inhibitors for hK1, plasma kallikrein, or for both at same time, if the inhibition of kinin release is the main goal.