Examples (sample input files and results included)
Example 1: Inverse screening of 6-bromo-indirubin-3'oxime (6BIO)  (sample ligand | result )

6-bromo-indirubin-3'oxime (6BIO) has been experimentally validated as an inhibitor of several kinds of protein kinases1. This example shows that not only the well-known kinase targets (CDK2, CDK5, GSK-3β) could be identified by idTarget, but also the kinase PDK11(PDB ID: 1OKY, 1UU7, 2BIY) and other proteins.

Job submission: First, upload the ligand coordinate file of 6BIO. AM1-BCC partial charges of this ligand has been assigned in the sample pdbqt file. Since the uploaded ligand has been determined protonated status beforehand, the option "Yes, no further adding polar hydrogens is needed" should be checked.

Fig 1. Job submission for Example 1

While a job is running but not yet finished, users can still check the job status at any time. For the privacy reason, only serial number and task name will be shown in the queue page.

Fig 2. Check the results of a submitted job

Fig 3. Using result page to monitor the progress of a running job

When the job is finished, the result page will show the final result and an email will be sent to the user if an valid email address is provided while submitting the job. If the users do not provide their email address, the Task ID should be kept so that the results can be retrieved later on. Two hierarchical tables will be shown. The two-layer table lists all docking results which are sorted by the docking energies from the robust AutoDock4 scoring functions 2. The PDB entries with the same target name would be merged, and one can click on to expand/collapse the next layer or click on to expand/collapse all results at once. The three-layer table groups the homologous proteins with a representative PDB shown in the first layer. The second and the third layers have the same relationship as the first and second layers of the table on the right.

Fig 4. Result of idTarget screening

Example 2: Dual inhibition of HMG-CoA and HDAC

Statins are used to lower cholesterol levels by inhibiting the enzyme HMG-CoA reductase. Previous study has shown that lovastatin possess a moderate inhibitory activity toward HDAC23. An example of off-target prediction for the dual inhibition of HMG-CoA and HDAC is shown here.

Off-target screening for 3max inhibitor (sample ligand | result)

First, download the x-ray crystallographic human HDAC2 complex from PDB (ID: 3max). The inhibitor is shown below:

Fig 1. N-(4-aminobiphenyl-3-yl)benzamide

Antechamber has been utilized to calculate AM1-BCC charges in the sample ligand pdbqt file. The MOCPAC 6 and "am1bcc" program have been embedded in the Antechamber package, and this can help us to obtain AM1-BCC charges easily. The Antechamber output was saved as Tripos mol2 file with AM1-BCC atomic charges. The pdbqt file of the ligand which remained structural information and AM1-BCC atomic charges can be prepared with ADT program (prepare_ligand4.py). The sample ligand pdbqt file can be downloaded here. In addition to HDAC complex (3max) and HMG-CoA complex (1hw9) proteins, another 8 receptors were chosen randomly from Protein Databank. Overall, there were 10 receptros in the protein set. The other settings of job submission can be referred in Fig 2.

Fig 2. Submit your job

The result can be retrieved by the Task ID: 1330668750gG.

Fig 3. The result of targets screening for 3max inhibitor

Off-target screening for 1hw9 inhibitor (sample ligand | result)

First, download the x-ray crystallographic complex of human HMG-CoA reductase with simvastatin(PDB ID: 1hw9). The 1hw9 inhibitor can be prepared in a similar way as described above, the sample pdbqt file with AM1-BCC charges can be downloaded here.

With the same settings as the target searching for 3max inhibitor, the results can be viewed and downloaded (Task ID: 1330857189rX). The ligand binding mode of 3max receptor visualized with Chimera can be seen in Fig 5.

Fig 4. The result of targets screening for 1hw9 inhibitor

Fig 5. The simvastatin (colored in magenta) of HMG-CoA reductase inhibitor also bind well to HDAC2 (3max) with the binding energy of -10.44 kcal/mol, and the interaction between a carboxylate group and zinc is observed. The original ligand of 3max complex is shown in cyan.

Example 3: acid proteases in SCOP  (sample ligand | PDB list | result)

This example will guide users to screen interested families or classes of protein targets by using the feature of "user-edited list". Protein Data Bank currently provides four well-known domain annotations for each PDB entry (SCOP, CATH, Pfam and GO). This is useful for users to select the protein sets of their interest. Take HIV-1 protease for example. PDB entry 2IEN is a crystal structure of HIV-1 protease with a potent inhibitor. The snapshot of the related annotations is shown below.

Fig 6. The snapshot of the annnotation page of PDB entry 2IEN.

We then select the superfamily "Acid proteases" in SCOP and obtain all the related PDB IDs.

Next, user can copy these PDB IDs and paste to the "user-edited list" in the job submit page to perform screening against these PDB entries. The sample results are shown here. In addition to Gag-Pol polyprotein (most of them are HIV-1 protease), beta-secretase 1 and plasmepsin-2 are on the top rank list as well. It has been known that beta-secretase 1 and plasmepsin-2 are the important targets for treatment of Alzheimer's disease and malaria, respectively. idTarget may be a useful tool for design of potent aspartic protease inhibitors to treat various diseases.

1. Zahler, S., et al., Inverse in silico screening for identification of kinase inhibitor targets. Chem Biol, 2007, 14, 1207-1214.
2. J.C. Wang, J.H. Lin, C.M. Chen, A.L. Perryman, A.J. Olson, Robust scoring functions for protein-ligand interactions with quantum chemical charge models, J. Chem. Inf. Model. 2011, 51, 2528-2537.
3. Lin, Y. C., Lin, J. H., Chou, C. W., Chang, Y. F., Yeh, S. H., and Chen, C. C., Statins increase p21 through inhibition of histone deacetylase activity and release of promoter-associated HDAC1/2. Cancer Res. 2008, 68, 2375-2383.

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