Inhibition of bacterial and human zinc-metalloproteases by bisphosphonate- and catechol-containing compounds

Figures & data

Figure 1. Inhibitory effect of 100 μM of the catechol containing compounds on the activity of the human metalloproteases, MMP-9 and MMP-14, and the bacterial metalloproteases TLN, PLN and ALN. The inhibition experiments were performed by using a fixed concentration of 4 μM of both the MMP-9 and MMP-14 substrate McaPLGL(Dpa)AR-NH₂ and the ALN, PLN and TLN substrate McaRPPGFSAFK(Dnp)-OH. The v_i/v₀ (mean ± sd) were based on 4–6 experiments.

Table 1. K_i values of the catechol containing compounds for TLN, PLN, ALN, MMP-14, and MMP-9(T).

Compound	Ki ± sd (μM)
	McaRPPGFSAFK(Dnp)-OH			McaPLGL(Dpa)AR-NH2
	TLN	PLN	ALN	MMP-14	MMP-9(T)
ML32	N.D.	38 ± 8	N.D.	19 ± 0.8	51 ± 17
BF471	13 ± 2	9 ± 3	49 ± 5	6.6 ± 0. 6	13 ± 2
BF489	14 ± 5	16 ± 3	N.D.	8.3 ± 0.6	12.6 ± 0.6
MT336	N.D.	N.D.	N.D.	12 ± 1	13 ± 1

K_i values determined for both bacterial and human metalloproteases using two different fluorescence quenched peptide substrates, McaRPPGFSAFK(Dnp)-OH (for TLN, PLN, and ALN) and McaPLGL(Dpa)AR-NH₂ (for MMP-9(T) and MMP-14). The concentration of the substrates used was 4 µM and the highest concentration of tested inhibitors was 100 µM. Compounds included are only those where 100 µM of the compound reduced the enzymatic activity by 60% or more as described in the text. The K_i values are based on 4–6 experiments.

N.D.: not determined.

Figure 2. Dose response plot of BF482 for MMP-9(A). The enzyme with and without inhibitor was pre-incubated for 30 min at room temperature and the reaction was started by adding McaPLGL(Dpa)AR-NH₂ (4 μM in assay). The reaction was allowed to proceed for 4 h at 37 °C and stopped by the addition of EDTA (10 mM end concentration). The relative fluorescence was determined with the Clariostar plate reader as described in the Experimental Section, where v_i and v₀ are the reaction rates in the presence and absence of BF482, respectively. Each point on the curve shows the mean ± sd (N = 5 for all points except for two points where N = 4). The regression coefficient r² is 0.97, with a determined IC₅₀ value of 38.8 ± 0.9 μM and a K_i value of 19.4 ± 0.4 μM.

Figure 3. BF471 docked into PLN, TLN, ALN and MMP-9, and ML32 docked into ALN and MMP-9. Zinc is shown in dark grey. The side chain of zinc coordinating amino acids are shown in blue. The side chains of some amino acids of importance for ligand binding are displayed with the following colour coding of atoms: oxygen: red; nitrogen: blue, carbon: yellow, hydrogen: grey. Colour coding of ligand atoms: oxygen: red; sulphur: green; nitrogen: blue; hydrogen; grey; carbon (BF471): pink; carbon (ML32): orange.

Table 2. Functionally important amino and amino acids in the S_1-, S₁′- and S₂′-subpockets of TLN, PLN, ALN, MMP-9, and MMP-14 contributing to the binding of catechol or bisphosphonate containing compounds tested in the present study.

	TLN	PLN	ALN	MMP-9	MMP-14
S1-subpocket	W115, Y157	W115, Y155	W117, Q152, N167	H190, P193	F204
S1′-subpocket	L133, V139, H142, I188	L132, V137, H140, I190	V137, H144, I186, V189	L397, V398, H401, L418, Y420, P421, Y423, R424, T426	L199, E219, W221, N231, H239
S2′-subpocket	N111, F130, Y193, L202	E111, F129, L197	N114, F132, L199	G186, L187, Y218	L235, V236, Y261, Q262
Catalytic Glu	E143	E141	E145	E402	E240
Known inhibitor binding residues	N112, A113, F114, R203, H231	E111, N112, A113, R198, H223	N112, Y114, R200, H228	A189, H190, A191	A200, H201
Zn^2+ ligated	H142, H146, E166	H140, H144, E164	H144, H148, E168	H401, H405, H411	H239, H243, H248

The subpocket amino acids of MMP-9 in the table are based on the X-ray structure of the inactive MMP-9 mutant E402A lacking the pro, FnII, hinge and HPX domains bound to a chromogenic substrate (PDB id: 4JIJ)⁷¹, while the numbering is as in the PDB id: 1l6j, which includes the three FnII-like repeats in the catalytic site⁷². The subpocket amino acids of TLN are based on Krimmer et al.⁷³. Subpocket amino acids of the other enzymes are based on structural superimposition with TLN (PLN and ALN) and MMP-9 (MMP-14).

Figure 4. Inhibitory effect of 100 μM bisphosphonate containing compounds on the activity of the human metalloproteases, MMP-9 and MMP-14, and the bacterial metalloproteases TLN, PLN, and ALN. The inhibition experiments were performed by using a fixed concentration of 4 μM of both the MMP-9 and MMP-14 substrate McaPLGL(Dpa)AR-NH₂ and the ALN, PLN and TLN substrate McaRPPGFSAFK(Dnp)-OH. The v_i/v₀ (mean ± sd) were based on 4–6 experiments.

Table 3. The obtained K_i values of the various bisphosphonate containing compounds for TLN, PLN, ALN, MMP-14 and MMP-9.

Compounds	Ki ± sd (µM)
	McaRPPGFSAFK(Dnp)			McaPLGL(Dpa)AR-NH2
	TLN	PLN	ALN	MMP-14	MMP-9(T)
LS4	N.D.	58 ± 4	N.D.	N.D.	N.D.
RC2	16.2 ± 0.4	22 ± 3	N.D.	N.D.	N.D.
ML45	N.D.	37 ± 6	N.D.	17 ± 1	N.D.
MT363	7 ± 1	12 ± 4	N.D.	7.2 ± 0.6	6.6 ± 0.4

The K_i values for both bacterial and human metalloproteases were measured with two different fluorescence quenched substrates, McaPLGL(Dpa)AR-NH₂ (for MMP-9 and MMP-14) and McaRPPGFSAFK(Dnp)-OH (for TLN, PLN and ALN). The concentration of the substrates used was 4 µM and the highest concentration of the inhibitor compounds tested was 100 µM. Compounds tested were only those where 100 µM of the compound reduced the enzymatic activity by 60% or more. The K_i values are based on 4–6 experiments.

N.D.: not determined.

Figure 5. MT363 and RC2 docked into PLN, TLN MMP-9 (MT363) and MMP-14 (RC2). TLN, PLN and MMP-9. Zinc is shown in dark grey. The side chain of zinc coordinating amino acids are shown in blue. The side chains of some amino acids of importance for ligand binding are displayed with the following colour coding of atoms: oxygen: red; nitrogen: blue; carbon: yellow; hydrogen: grey. Colour coding of ligand atoms: oxygen: red; sulphur: green; nitrogen: blue; phosphate: orange; hydrogen: grey; carbon – MT363: purple; carbon – ML32: orange.

Tranchant I, Vera L, Czarny B, et al. Halogen bonding controls selectivity of FRET substrate probes for MMP-9. Chem Biol 2014;21:408–13.

 PubMedGoogle Scholar

Elkins PA, Ho YS, Smith WW, et al. Structure of the C-terminally truncated human ProMMP9, a gelatin-binding matrix metalloproteinase. Acta Crystallogr D Biol Crystallogr 2002;58:1182–92.

 PubMedGoogle Scholar

Krimmer SG, Cramer J, Betz M, et al. Rational design of thermodynamic and kinetic binding profiles by optimizing surface water networks coating protein-bound ligands. J Med Chem 2016;59:10530–48.

 PubMed Web of Science ®Google Scholar