A composite docking approach for the identification and characterization of ectosteric inhibitors of cathepsin K

<div><p>Cathepsin K (CatK) is a cysteine protease that plays an important role in mammalian intra- and extracellular protein turnover and is known for its unique and potent collagenase activity. Through studies on the mechanism of its collagenase activity, selective ectosteric sites were identified that are remote from the active site. Inhibitors targeting these ectosteric sites are collagenase selective and do not interfere with other proteolytic activities of the enzyme. Potential ectosteric inhibitors were identified using a computational approach to screen the druggable subset of and the entire 281,987 compounds comprising Chemical Repository library of the National Cancer Institute-Developmental Therapeutics Program (NCI-DTP). Compounds were scored based on their affinity for the ectosteric site. Here we compared the scores of three individual molecular docking methods with that of a composite score of all three methods together. The composite docking method was up to five-fold more effective at identifying potent collagenase inhibitors (IC₅₀ < 20 μM) than the individual methods. Of 160 top compounds tested in enzymatic assays, 28 compounds revealed blocking of the collagenase activity of CatK at 100 μM. Two compounds exhibited IC₅₀ values below 5 μM corresponding to a molar protease:inhibitor concentration of <1:12. Both compounds were subsequently tested in osteoclast bone resorption assays where the most potent inhibitor, 10-[2-[bis(2-hydroxyethyl)amino]ethyl]-7,8-diethylbenzo[g]pteridine-2,4-dione, (NSC-374902), displayed an inhibition of bone resorption with an IC₅₀-value of approximately 300 nM and no cell toxicity effects.</p></div

Chemical similarity mapping of hits identified through molecular docking.

<p>The composite method identified 25 compounds that could be grouped into four different scaffolds. Group 4 contained the highest number of compounds with 13 identified putative inhibitors. A complete list of compounds can be found in the Supplementary Information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186869#pone.0186869.s001" target="_blank">S1 Table</a>).</p

Top binding poses of compounds 1 and 3 from composite docking.

<p>Top binding poses of the most potent collagenase inhibitors, <b>1 (A)</b> and <b>3 (B)</b>, as docked using the three docking methods. The poses are depicted using sticks and colored orange (Glide), green (Surflex), and yellow (GOLD). Ligplot diagrams depicting the predicted binding of compounds <b>1 (C)</b> and <b>3 (D)</b> into ectosteric site 1 using the best binding pose calculated from Glide. Hydrogen bonds with the binding site residues are highlighted in green with the respective distances and hydrophobic interactions are shown with red dashes.</p

Screening and evaluation workflow for the identification of ectosteric site 1 inhibitors of CatK.

<p>The screening procedure used to identify potential collagenase inhibitors of CatK from the NCI/DTP Repository. A composite virtual screening method involving GOLD, Glide, and Surflex was used to screen and identify potential hits and a total of 160 compounds were tested in <i>in vitro</i> assays. Eight compounds were identified as potent collagenase inhibitors with IC₅₀ values below 20 μM and two compounds were tested in osteoclast-based bone degradation assays with both inhibitors displaying bone resorption inhibition.</p

Collagenase inhibitory activity of compounds 1 and 3.

<p>The two most potent compounds, <b>1</b> (A) and <b>3</b> (B), identified through molecular docking are shown with their respective structures. Collagenase inhibitory activities with representative collagenase degradation gels are depicted with the corresponding IC₅₀ curves determined from three separate experiments (n = 3). The IC₅₀ values for the inhibition of collagenase activity of CatK were 4.7 ± 0.4 μM and 4.9 ± 0.3 μM for compounds <b>1</b> and <b>3</b>, respectively. * represents the α1 type I collagen peptide used to quantify the collagenase activity of CatK.</p

Summary of collagenase inhibitors identified through composite docking from the NCI-DTP repository.

<p>Summary of collagenase inhibitors identified through composite docking from the NCI-DTP repository.</p