The data proves that a longer self-immolative linker of FMP improves the probe responsivity toward FAPα. To uncover the underlying mechanism, theoretical molecular docking simulation is further carried out to elucidate the different FAPα responsivity towards FMP and N-FMP by MolAICal 1.3. The X-ray crystal structure of FAPα from the Protein Data Bank (PDB code 1Z68) is used. 3D structures of FMP and N-FMP are obtained and energetically optimized by a ChemDraw 3D software. After molecular docking, FMP presents a strong hydrogen bond between the peptide substrate of the probe and FAPα residues at the site of Val540, Ser548, Gln547, Gly542, and Ser546 in the active pocket. By contrast, N-FMP shows relatively weak hydrogen bond interaction with only one site of FAPα at the Gln547 residue in the active pocket. The theoretical simulations indicate that the higher affinity of FAPα towards FMP and thus promotes the enzymatic cleavage efficiency relative to N-FMP, which is consistent well with the experimental results. Therefore, both theoretical simulations and experimental results confirm that FMP with the longer self-immolative linker has superior enzymatic kinetics for FAPα.

1.5 Docking Analysis. The X-ray structure of FAPα (PDB code 1Z68) is obtained from the Protein following Data Bank (http://www.rcsb.org/pdb). 3D structures of FMP and N-FMP are obtained and energetically optimized by ChemDraw 3D software. The MolAICal software package is used to process and save FAPα, FMP, and N-FMP in PDBQT molecular format. FAPα is the definition of a crystal ligand in the active pocket of molecular docking. Take x = 39.84 Å, y = 0.221 Å, and z = 59.383 Å as the center coordinate of the FAPα active pocket box, and the size of the grid box is set to 30 Å. The detained operations are as follows:

1.5.1 Software Requirement

1) MolAICal: https://molaical.github.io

2) UCSF Chimera: https://www.cgl.ucsf.edu/chimera

1.5.2 Prepare the Receptor Protein

The X-ray structure of FAPα (PDB code 1Z68) was obtained from the protein following data bank (http://www.rcsb.org/pdb). Save the file in 1Z68.pdb format.

1.5.3 Prepare the Ligand Molecule

3D structures of FMP and N-FMP are obtained and energetically optimized by ChemDraw 3D software. FMP and N-FMP are saved in the format of FMP.pdb and N-FMP.pdb. using the MolAICal software.

1.5.4 Convert the Receptor and Ligand to PDBQT Format

The CD command is used to switch to the folder where the receptor and ligand are located, following using the following command to get the PDBQT format of the receptor and ligand:

Receptor: #> MolAICal-xxx\molaical.exe -dock receptor -i 1Z68.pdb

Ligand: #> MolAICal-xxx\molaical.exe -dock ligand -i FMP (or N-FMP).pdb

MolAICal-xxx indicates the folder where the MolAICal software is stored.

1.5.5 Determine Box Size and Center Through UCSF Chimera

UCSF Chimera software is used to open the 1Z68.pdb and FMP (or N-FMP).pdb files.

Using UCSF Chimera Software Tools: Tools--> Surface/Binding Analysis--> Autodock Vina

In the dialog box, the receptor selects 1Z68.pdb and the ligand selects FMP (or N-FMP).pdb. Fill in "39.840, 0.221, 59.383" at the center coordinates, and fill in 30 for the box size.

1.5.6 Prepare Configuration Files

The final configuration file is set to:

out = all.pdbqt

cpu = 4

receptor = 1Z68.pdbqt

center_x = 39.840

center_y = 0.221

center_z = 59.383

size_x = 30

size_y = 30

size_z = 30

num_modes = 10

Save the file as conf.txt

1.5.7 Molecular Docking

MolAICalD from MolAICal package is used for molecular docking of receptor and ligand:

#> MolAICal-xxx\molaicald --config conf.txt --ligand FMP (or N-FMP).pdbqt

1.5.8 Result Processing

At the end of the simulation, the results are split into individual molecules:

#> MolAICal-xxx\molaical.exe -tool pdbqt -i all.pdbqt -o ./

Individual molecules are named 1.pdbqt, 2.pdbqt, or 3.pdbqt, etc.

1.pdbqt, 2.pdbqt or 3.pdbqt can be checked directly through Pymol or UCSF Chimera software.

The above operations are carried out in the window system using MolAICal and UCSF Chimera software.

 Software requirement

1) MolAICal: https://molaical.github.io

2) UCSF Chimera: https://www.cgl.ucsf.edu/chimera