Materials and Reagents

ViewPlate-96 black assay plates (PerkinElmer, catalog number: 6005225 )
Recombinant human hepsin protein (R&D Systems, catalog number: 4776-SE-010 )
DMSO (Sigma-Aldrich, catalog number: D8418 )
Fluorogenic peptide substrate Boc-Gln-Arg-Arg-AMC (Bachem, catalog number: I-1655.0025 )
Small-molecule compounds were purchased either from Asinex Corporation (Winston-Salem, NC, http://www.asinex.com) or MolPort (Riga, Latvia, https://www.molport.com/shop/index)
WX-UK1 was a kind gift from Dr. Ramachandra (Aurigene Discovery Technologies Limited, Bangalore, India), however, it is also commercially available (for example, AURUM Pharmatech, catalog number: Z-3200 )
Trizma base (Tris) (Sigma-Aldrich catalog number: T1503 )
Calcium chloride (CaCl₂) (Sigma-Aldrich, catalog number: 499609 )
Brij-35 (Brij L23 30% solution in H₂O) (Sigma-Aldrich, catalog number: B4184 )
Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 31434-M )
Activation buffer for recombinant human hepsin protein (see Recipes)
Assay buffer for recombinant human hepsin protein (see Recipes)

Equipment

ELISA plate reader FLUOstar Omega (BMG LABTECH, model: FLUOstar Omega )
Computer hardware provided by CSC - IT Center for Science Ltd (Espoo, Finland)

HP Proliant SL230s (Hewlett Packard Development Company, model: SL230s )
Intel Xeon E5-2670 2.6GHz processors (100 cores with 16 GB memory per core) (Intel Corporation, model: E5-2670 )

Software

Modeling software
Schrödinger Suite (Schrödinger, LLC, New York, NY) with Protein Preparation Wizard (Sastry et al., 2013), LigPrep, MacroModel and Glide (Friesner et al., 2004 and 2006; Halgren et al., 2004) modules, and a protocol version 2016-2 (https://www.schrodinger.com/suites/small-molecule-drug-discovery-suite)
Databases

Screening and validation: ZINC database (downloadable via http://zinc15.docking.org/). ZINC database includes over 100 million purchasable compounds in 3D formats (Sterling and Irwin, 2015)
Protein structure database (http://www.rcsb.org/pdb/home/home.do)

Procedure

Preparation of the target protein structure for virtual screening
The hepsin protein structure (PDB id 1O5E, from protein structure database, www.rcsb.org, see ‘Online tools’) can be automatically downloaded with Protein Preparation Wizard of Schrödinger suite using ‘biological unit’-option. With other types of softwares one should use the manual downloading and read in the structure by software preferred method. The pre-processing is carried out with default methods except keeping all the water molecules. H-bond refinement should be carried out with default pH value 7. All water molecules with at least 3 H-bonds with non-water atoms are saved and the whole structure is minimized with default settings with heavy atoms converged at RMSD 0.3Å. The Glide Grid file is created using the above mentioned structure and the center of the Grid was at Tyr94 (Figure 2). The center of the GRID is referring to the center of molecular field calculation during the GRID creation. Hydroxyl groups of Tyr94, Tyr146, Ser195 and Tyr228 are allowed to rotate for Grid generation. Addition of free rotatable bonds doubles the Glide calculation time and should be limited to keep the computational time at reasonable level. Also, it is strongly recommended that the Glide Grid file will be constructed so that OPLS3 force field is used, if possible. If another software is used, one should use the corresponding protein preparation method. The critical point is to make sure that the structural quality of the protein is checked, proper ionization status are fixed for polar sidechains and right rotamers for (pseudo)symetric residues are used.

Figure 2. Definition of GRID region for docking. The center of the GRID is based on the location of residues TYR94, TYR146 and SER195 (residues marked with labels) and the GRIF region is shown with magenta box.
Preparation of the virtual screening compound library
The virtual screening docking library must be prepared using the methods and settings recommended by the authors of the virtual screening software. When choosing the virtual compound library, one should take into account the number of compounds intended for screening, possibilities for own synthesis and if the resulting hits are intended to be used as such or further modified in subsequent medicinal chemistry programs. We use the drug-like subset of ZINC library, downloaded as a SDF-file from the ZINC web page. SDF-file format is supported by all the major software brands. The SDF-file is prepared by Schrödinger LigPrep module with default settings (Figure 3). In short the preparation includes checking of 2D structure quality, fast 2D-3D transformation, analysis and modification of protonation state, modification of all reachable tautomers and further minimization of resulting molecules. In a typical case each molecule will be represented with 2-4 different ionization/protomer/tautomer states. For other types of software other than the Schrödinger software, we recommend ligand setup via LigPrep. In our own experience, the benefit of this method is that it yields high quality predictions for both ionization status (i.e., pKa) and also for tautomers. The prediction of tautomers option is not included in most of the other software packages.

Figure 3. LigPrep window under Schrödinger Suite. The library for virtual screening is prepared according to the settings selected; the shown setting are the default ones in our protocol.
Virtual screening and analysis of the results
In our study (Tervonen et al., 2016) virtual screening was carried out as follows:
1. SP-docking is performed using the above-defined Grid file and the best 10% of compounds are further redocked with XP settings.
2. Clustering compounds was initially performed by visually studying the resulting docking poses.
3. Compounds were then clustered by using the Interaction Fingerprint script available within Schrödinger.
4. As a result 24 clusters were created.
5. In each cluster the most representative compounds are located in the center of the cluster. Those compounds are selected for in vitro assays.
Validation of screen results by in vitro cell-free peptide cleavage assay for hepsin enzymatic activity protocols
1. Recombinant human hepsin (rhHepsin) is diluted to 100 µg/ml in activation buffer.
2. rhHepsin is activated by incubating the solution at 37 °C for 24 h.
3. rhHepsin is diluted into 0.1 nM solution with assay buffer just before use.
4. Small-molecule compounds are dissolved in DMSO to give 10 mM stock solution.
5. The assay is performed in assay buffer in a 96-well plate.
6. 0.1 nM final concentration of rhHepsin and 10 µM final concentration of small-molecule compound (and equal volume of DMSO control) in 100 µl reaction volume are incubated for 30 min at room temperature. Assay buffer is used as blank control.
7. The reaction is started by adding a final concentration of 30 µM peptide substrate BOC-Gln-Arg-Arg-AMC.
8. The plate is analyzed with ELISA plate reader by 350 nm excitation and 450 nm emission capture at room temperature.
9. The inhibition % is determined by using the following formula:

Data analysis

The described virtual screening protocol resulted in hundreds of potential hits. Using the protocols described above and in Tervonen et al. (2016), we performed an initial in vitro cell-free validation. More than 30% reduction in peptide cleavage relative to DMSO control with three technical replicates and two repeats was considered as a cut-off for hits. We ordered and tested 24 compounds identified by virtual screening as potential hits (Figure 1B) and few compounds made it close to the cut-off value (see Tervonen et al. [2016]). These compounds were used as skeletons for the next round of virtual screening, which is a reiterative process. In the study Tervonen et al. (2016) we chose to use WX-UK1, which was identified in parallel screen, because its binding pose against hepsin and superb inhibition efficiency in comparison to other tested molecules.

Notes

Computer hardware
In addition to listed hardware above, we note that any high-end PC computer with any of the major operating system (Windows, Mac or Linux) and with either Intel or AMD processors is suitable for the virtual screening job but the process will then require more time.
Small-molecule compounds
We note that while the virtual screening stage of the project was low-cost and non-time consuming, one bottleneck in the screening was the price of the compounds. The typical price for commercially available compounds ranges from 50 € to 200 € per mg. Thus, screening of hundreds of compounds in cell-free assays would be the most expensive stage of the project. Also, the fact that most of the compounds are sold with minimum 1 mg pack size (synthesis limit) and that the initial cell-free screening only requires fraction of that amount makes purchasing of vast amount of compounds unreasonable.
Feasibility of virtual screening In our experience
Virtual screening as a method for identification of molecular tool compounds performed well in finding a small set of skeleton compounds even though only relatively small number of compounds identified as hits by molecular modeling were eventually tested in vitro (cell free assays with recombinant hepsin). These results indicate that virtual screening is in principle a feasible method for identification of transmembrane serine protease inhibitors but it would be advisable to use virtual screening in combination with other approaches. For example, starting with skeleton compounds with previously established inhibitory action against the transmembrane serine protease of interest or against closely related serine protease. The virtual screening strategy described here would be greatly facilitated by a possibility to purchase just a small microgram quantity of the candidate molecule instead of a milligram. There would be a significant market for any contract research laboratory able to miniaturize compound synthesis for purpose of testing significant number of virtual screening hits with low-cost.

Recipes

Activation buffer for recombinant human hepsin (pH 8.0)
0.1 M Tris
10 mM CaCl₂
0.15 M NaCl
0.05% Brij-35
Assay buffer for recombinant human hepsin (pH 9.0)
50 mM Tris

Acknowledgments

Protocol is adapted from Tervonen et al. (2016). This study was funded by the Academy of Finland, Sigrid Jusélius Foundation, the Finnish Cancer Society, the Research Funds of the Helsinki University Central Hospital, Jane and Aatos Erkko Foundation, and Helsinki Graduate Program in Biotechnology and Molecular Biology and Innovative Medicines Initiative Joint Undertaking under grant agreement No.115188.

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