Definition of peptide inhibitors from a synthetic peptide library by targeting gelatinase B/matrix metalloproteinase-9 (MMP-9) and TNF-α converting enzyme (TACE/ADAM-17)

Figures & data

Table 1.  Design of library B.

P3	P2	P1	P1′	P2′	P3′	P4′
Pro	D-Pyr	D-Cys	Bip	Cys,D-Cys,Arg,D-Arg or Lys	Gly	Glu
Pro	Arg	D-Cys	Bip	Cys,D-Cys,Arg,D-Arg or Lys	Gly	Glu
Pro	Pyr	D-Cys	D-Bip	Cys,D-Cys,Arg,D-Arg or Lys	Gly	Glu
Pro	D-Arg	D-Cys	Val	Cys,D-Cys,Arg,D-Arg or Lys	Gly	Glu
Pro	Arg	Cys	D-Bip	Cys,D-Cys,Arg,D-Arg or Lys	Gly	Glu
Pro	Pyr	D-Cys	D-Bip	Cys,D-Cys,Arg,D-Arg or Lys	Gly	Glu

Figure 1.  Design of library A and inhibitor screen of the samples of library A with MMP-9 and TACE. (A) Amino acid candidates selected for different positions (from P₂ to P₂′) for library A. (B) and (C) Inhibitory effects of samples from library A on MMP-9 (B) and TACE (C) activity. Each bar represents the inhibitory effect (Z-axis) of a mixture of five peptides. The X-axis indicates the amino acids selected for P₂ position. The Y-axis indicates the amino acids selected for P₁′ position. The histograms on the left indicate the inhibitory effects of samples with cysteine at the P₁ position, whereas the right side figure indicates the inhibitory effects of samples with D-form cysteine at the P₁ position. Pyr indicates pyridylalanine, Bip indicates biphenylalanine.

Table 2.  Percentage inhibition of MMP-9 activity by peptides from library B.

P2 to P1′	P2′
	Cys	D-Cys	Arg	D-Arg	Lys
D-Pyr-D-Cys-Bip	63	0	61	12	64
Arg-D-Cys-Bip	55	6	47	0	44
Pyr-D-Cys-D-Bip	15	17	22	62	44
D-Arg-D-Cys-Val	0	15	1	4	6
Arg-Cys-D-Bip	0	0	38	62	29
Pyr-Cys-Val	0	11	0	7	4

Table 3.  Percentage inhibition of TACE activity by peptides from library B.

P2 to P1′	P2′
	Cys	D-Cys	Arg	D-Arg	Lys
D-Pyr-D-Cys-Bip	15	30	30	20	30
Arg-D-Cys-Bip	27	24	36	12	15
Pyr-D-Cys-D-Bip	17	27	18	10	19
D-Arg-D-Cys-Val	8	26	16	9	9
Arg-Cys-D-Bip	13	19	15	9	4
Pyr-Cys-Val	15	23	12	21	4

Table 4.  IC₅₀ values of the selected peptide inhibitors on the target enzymes.

Sequence	Enzymes
	MMP-9/gelatinase B	MMP-8/collagenase 2	ADAM-17/TACE	MMP-3/stromelysin 1
Arg-Cys-(D-Bip)-(D-Arg)	0.75	2.3	20	19.5
Arg-(D-Cys)-Bip-Arg	0.9	0.9	2.5	22.5
(D-Pyr)-(D-Cys)-Bip-(D-Cys)	8.65	0.73	0.6	28
(D-Pyr)-(D-Cys)-Bip-Arg	2.75	0.8	5.5	4.75
(D-Pyr)-(D-Cys)-Bip-Lys	3.5	0.65	8	30

The IC₅₀ values were based on the reaction systems indicated in the section of Materials and Methods in which commercially available fluorogenic peptides were used as substrates. D-Bip indicated D-form biphenylalanine; D-Pyr indicated D-form pyridylalanine.

Table 5.  K_I or K_I′ values of the selected peptide inhibitors on MMP-9.

Sequence (P2 to P2′)	KI or KI′ (µM)
	Inhibitor concentration (μM)	KI	KI′
Arg-Cys-(D-Bip)-(D-Arg)	1.95	1.95	0.43
Arg-(D-Cys)-Bip-Arg	1.95	0.87	0.57
(D-Pyr)-(D-Cys)-Bip-(D-Cys)	7.82	2.41	1.72
(D-Pyr)-(D-Cys)-Bip-Arg	1.95	0.87	0.43
(D-Pyr)-(D-Cys)-Bip-Lys	7.82	3.91	1.38

The K_I or K_I′ values were based on the reaction systems indicated in the section of Materials and Methods.

Figure 2.  Lineweaver-Burk plots of inhibition of MMP-9 by the selected peptide inhibitors. Enzyme cleavage of the fluorogenic peptide substrate was performed with the substrate concentration of 4, 2, 1.33, 1 and 0.8 uM in the presence or absence of a peptide inhibitor. Both intercepts and the slope changed in the presence of each inhibitor, which indicated the mode of mixed non-competitive inhibition.