Discovery of a Potent Inhibitor Class with High Selectivity toward Clostridial Collagenases

Abstract

Secreted virulence factors like bacterial collagenases are conceptually attractive targets for fighting microbial infections. However, previous attempts to develop potent compounds against these metalloproteases failed to achieve selectivity against human matrix metalloproteinases (MMPs). Using a surface plasmon resonance-based screening complemented with enzyme inhibition assays, we discovered an N-aryl mercaptoacetamide-based inhibitor scaffold that showed sub-micromolar affinities toward collagenase H (ColH) from the human pathogen Clostridium histolyticum. Moreover, these inhibitors also efficiently blocked the homologous bacterial collagenases, ColG from C. histolyticum, ColT from C. tetani, and ColQ1 from the Bacillus cereus strain Q1, while showing negligible activity toward human MMPs-1, -2, -3, -7, -8, and -14. The most active compound displayed a more than 1000-fold selectivity over human MMPs. This selectivity can be rationalized by the crystal structure of ColH with this compound, revealing a distinct non-primed binding mode to the active site. The non-primed binding mode presented here paves the way for the development of selective broad-spectrum bacterial collagenase inhibitors with potential therapeutic application in humans.

Figure 1

Inhibition of selected MMPs and bacterial collagenases by N-aryl mercaptoacetamide compounds 3 and 7.

Figure 2

Peptidase domain of ColH in complex with the hydrolysis product of compound 3. Close-up view of the active site in ball-and-stick representation. The inhibitor (blue) is shown in sticks with the maximum likelihood weighted 2F_o – F_c electron density map contoured at 1σ. The catalytic zinc ion (dark gray), calcium ion (green), and water molecule (red) are shown as spheres. The S1′ site formed by Gly425 and Gly426 in the edge strand (shown in dark gray sticks) is indicated.

Figure 3

Conversion of thiocarbamates 7 and 12 into the respective corresponding free thiols: (a) compound 7 into 14 and (b) compound 12 into 15. Time course of hydrolysis in 10 mM Hepes, pH 7.4 (10% methanol), at 22.5 °C was monitored by LC-MS, showing conversion into corresponding thiol and to minor extent into another compound which is most likely the disulfide oxidation product.

Figure 4

Inhibition of selected MMPs and bacterial collagenases by thiol compounds 13 and 14.

Figure 5

Structure and inhibitory activity of the non-hydrolyzable carboxamide analogue 16.

Figure 6

Close-up on the superpositioned active sites of three MMPs (a) and of three clostridial collagenases (b) in the ligand-bound state. The ligands have been removed for better visualization. The HEXXH motif is shown in sticks, and the zinc ions (gray) and calcium ions (green) are shown as spheres. The edge strand on top of the catalytic zinc is highlighted in color.