Gelatin degradation assay reveals MMP-9 inhibitors and function of O-glycosylated domain

Table 1 Set of used protease inhibitors in control experiments

Inhibitor (% inhibition)	Alternative name	MW (g/mol)	Target	Mechanism	Ref.
Aprotinin (5%)	Bovine pancreatic trypsin inhibitor	Approximately 6500	Serine proteases including plasmin, tissue plasminogen activator, kallikrein and thrombin	Nonspecific protease inhibitor	[33,34]
Batimastat (94%)	BB-94; [4-(N-hydroxyamino)-2R-isobutyl-3S-(thiopen-2-ylthiomethyl)succinyl]-L-phenylalanine-N-methylamide	478	MMP-1, -2, -3, -7 and -9	Peptide backbone similar to the cleavage site in collagen (= peptidomimetic inhibitor)	[6,9,35-37]
			TACE (MMP IC50 = 10-30 nmol/L)
Benzamidine (0%)	-	120	Trypsin, plasmin and thrombin	Competitive inhibitor	[36]
Bestatin (0%)	[(2S,3R)-3-amino-2-hydroxy-4-phenylbutanoyl]-L-leucine, Ubenimex	308	Aminopeptidase N	Slow-binding competitive inhibitor	[38-40]
Chymostatin (0%)	-	608	Proteinases including Serine, thiol, and carboxyl endopeptidases serine proteinases, chymotrypsin and Streptomyces griseus proteinase A, and several cysteine proteinases	Tetrapeptide analogue, formation of a hemiacetal or hemithioacetal adduct with the nucleophilic hydroxy or thiol group of the serine and cysteine proteinases	[41,42]
E-64d (0%)	Aloxistatin, EST, [2S,3S-trans-(Ethoxycarbonyloxirane-2-carbonyl)-L leucine-(3-methylbutyl) amide]	342	Specific thiol protease inhibitor such as papain and cathepsin B	Interaction with active thiol group	[43]
EGCG (33%)	Epigallocatechin-3-gallate	458	Multiple targets including MMP-2 and MMP-9 (MMP IC50 = 8-50 μmol/L)	Blocking the activation mechanism of MMP-2 induced by concanavalin A	[44-47]
				Other exact molecular targets remain unknown
Pefabloc (0%)	AEBSF; 4-(2-Aminoethyl) benzenesulfonyl fluoride hydrochloride	239	Serine protease inhibitor	Irreversible inhibition by covalent interaction with the active-site serine	[48,49]
Pepstatin (0%)	Isovaleryl-L-valyl-L-valyl-4-amino-3-hydroxy-6-methylheptanoyl-L- alanyl-4-amino-3-hydroxy-6-methylheptanoic acid	686	Pepsin and gastricsin (acid proteinase activity)	-	[50]
PMSF (0%)	Phenylmethylsulfonyl fluoride	174	Serine protease/carboxylesterase inhibitor	Covalent binding to the serine residue of the catalytic Ser-His-Asp triad	[51]
SB-3CT (91%)	-	306	MMP-2 and MMP-9 (MMP IC50 = 185-290 nmol/L)	Competitive, mechanism-based, thiirane-opening mechanism	[8,52]

The inhibition percentages are shown together with the compound names, as obtained in the initial screen with the compounds at 20 μmol/L. MMP: Matrix metalloproteinase.

Table 2 Michaelis-Menten parameters for different enzyme variants

	MMP-9 FL	MMP-9 ∆Hem	MMP-9 ∆OGHem	MMP-9 ∆OG	MMP-9 MutEC
Vmax (nmol/L per minute)	3.643	2.686	2.314	1.117	-
kcat/KM (nmol/L per minute)	0.097	0.086	0.088	0.058	-
Goodness of fit (R2)	0.9977	0.9900	0.9861	0.9470	0.7096
Difference from MMP-9 FL	-	P = 0.0207	P = 0.0049	P = 0.0020	P = 0.0010

The corresponding Michaelis-Menten curves are shown in Figure 3. The P-values were calculated with a Wilcoxon signed rank test. The V_max and KM values could not be determined for the matrix metalloproteinase (MMP)-9 MutEC.

Table 3 Results of the initial screening of 1612 compounds

	Fluorescence decrease
	1%-10%	11%-20%	> 20%
ChemDiv (555 compounds)	217	57	4 (Max = 33%)
InterBioScreen (360 compounds)	106	19	18 (Max = 100%)
ChemBridge (697 compounds)	134	50	15 (Max = 100%)