Background

Controlled proteolytic activity plays a fundamental role in cellular processes and signaling, as evidenced by the presence of proteases in all organisms, including viruses, prokaryotes and eukaryotes. Not surprisingly, aberrantly regulated protease activity is causal to wide variety of human pathologies such as cardiovascular and inflammatory diseases, osteoporosis, neurological disorders and cancer (Turk, 2006; Bachovchin and Cravatt, 2012). In particular, development of primary cancer and metastatic capacity has been linked to several different classes of proteases including matrix metalloproteinases (MMPs), cysteine proteases (cathepsins) and membrane-associated serine proteases (Lopez-Otin and Matrisian, 2007). Recent clinical, genetic and functional data, suggesting a critical role for membrane-associated serine proteases in solid cancers, including cancer of prostate, ovarian and breast, have prompted new interest in the development of small molecule serine protease inhibitors for the treatment of cancer. Hepsin is a type II transmembrane serine protease and an attractive target for serine protease drug development due to frequent hepsin overexpression in common solid cancers, such as prostate and breast cancer, confinement of its overexpression on the membranes of cancer cells and due to positioning of the catalytic domain to the extracellular, i.e., more reachable, side of the cells (Antalis et al., 2010).

We describe here protocols for structure-based virtual screening of serine protease inhibitors using the catalytic site of hepsin for docking with drug-like subset of ZINC database. On the basis of virtual screening results, altogether 24 candidate compounds were purchased for further biochemical validation. Cell-free ELISA-based fluorogenic enzymatic assay using recombinant hepsin and fluorogenic peptide substrate Boc-Gln-Arg-Arg-AMC (BACHEM) was used for experimental validation and 30% inhibition of peptidolytic activity was set as threshold. Cut-off value was based on a notion that with the initial high micromolar concentration of compounds more than 30% inhibition would be required to determine reasonable IC50 value (Goswami et al., 2015; Tervonen et al., 2016) (Figures 1A and 1B). With these criteria, 3 out of 24 tested compounds showed inhibition potency (Tervonen et al., 2016). In conclusion, even though structure-based virtual screening is often considered as a complementary drug screening approach, the protocols reported here allowed us to demonstrate the feasibility and provided meaningful structural scaffolds for further development of specific serine protease inhibitors. However, it is important to stress that virtual screening hits typically do not demonstrate high potency, which is also true to the present screen. High-affinity ligands typically become available only after skillful medicinal chemistry optimization of selected hit structures.


Figure 1. Schematic figure illustrating the workflow. A. Virtual screening (compound) library is prepared and the target protein structure analyzed and prepared. The virtual screening is carried out by docking (Glide SP/XP) and the putative binders are validated by in vitro biochemical assays. B. Examples of candidate ligands (a-d).

The virtual screening protocol presented here is a modified version from the original protocols described in our recent research paper (Tervonen et al., 2016). The modifications make the protocol fully compatible with the most updated versions of ZINC database and Schrödinger software. While the current virtual screening protocol is tailored for users of Schrödinger software and ZINC database, the protocol is versatile and also compatible with other molecular modeling packages. However, implementation of the present virtual screening protocol with other modeling packages would require a careful platform-specific validation of the docking settings. As a guidance, we provide a general overview of the validation procedures used in the present screen.