Discovery of Novel CCR5 Ligands as Anticolorectal Cancer Agents by Sequential Virtual Screening

Abstract

C-C chemokine receptor type 5 (CCR5) is a member of the G protein-coupled receptor. CCR5 and its interaction with chemokine ligands have been crucial for understanding and tackling human immunodeficiency virus (HIV)-1 entry into target cells. In recent years, the change in CCR5 expression has been related to the progression of different cancer types. Patients treated with the CCR5 ligand, maraviroc (MVC), showed a deceleration in tumor development especially for metastatic colorectal cancer. Based on the crystal structure of CCR5, we herein describe a multistage virtual screening protocol including pharmacophore screening, molecular docking, and protein-ligand interaction fingerprint (PLIF) postdocking filtration for discovery of novel CCR5 ligands. The applied virtual screening protocol led to the identification of four hits with binding modes showing access to the major and minor pockets of the MVC binding site. Compounds 2-4 showed a decrease in cellular proliferation upon testing on the metastatic colorectal cancer cell line, SW620, displaying 12, 16, and 4 times higher potency compared to MVC, respectively. Compound 3 induced apoptosis by arresting cells in the G0/G1 phase of the cell cycle similar to MVC. Further in vitro assays showed compound 3 drastically decreasing the CCR5 expression and cellular migration 48 h post treatment, indicating its ability to inhibit metastatic activity in SW620 cells. The discovered hits represent potential leads for the development of novel classes of anticolorectal cancer agents targeting CCR5.

Figure 1

Two-dimensional (2D) chemical structures of CCR5 ligands used as training set compounds (T1–16) and their activity values (half-maximal inhibitory concentration (IC₅₀)).

Figure 2

Ligand-based pharmacophore model (PH9) for CCR5 inhibitors: (A) three-dimensional (3D) spatial arrangement and distance constraints between the chemical features of the pharmacophore model represented by blue (cationic center, Cat), orange (aromatic center, Aro), and green (hydrophobic, Hyd) spheres. (B) Overlay of the crystal coordinates of MVC on the pharmacophore model (PH9).

Figure 3

Chemical structures of hit compounds showing the basic nitrogen (blue) and functional groups fitting in TMS1 (green) and the hydrophobic subpocket of TMS2 (red). Interactions between the ligands and specific residues derived from the CCR5 X-ray structure (PDB code: 4MBS) are depicted by dotted lines. Structure of MVC is added for comparison.

Figure 4

Graphical representation of the MVC binding pocket of CCR5 (PDB code: 4MBS) with the docked pose of compounds (A) 1, (B) 2, (C) 3, and (D) 4 (green sticks) showing residues from TMS1 and TMS2 (gray sticks). Only side-chain atoms are shown for clarity.

Figure 5

In vitro effect of compound 1 on the viability of SW620 CRC cells showing an approximate EC₅₀ value of 300 μm.

Figure 6

In vitro cytotoxic effects induced by MVC, compounds 2–4 in SW620 CRC cells 48 hrs post treatment. IC₅₀ values with 95% confidence limits are given below the respective curves.

Figure 7

Impact of compound 3 IC₅₀ on the cell cycle of SW620 CRC cells at 48 h post treatment in comparison to the control samples C-1 (cells treated with compound 3 solvent) and C-2 (cells only).

Figure 8

Impact of compound 3 on migration of SW620 CRC cells at 24, 48, and 72 h post treatment in comparison to MVC, C-1 (cells treated with solvent of compound 3), C-2 (cells treated with MVC solvent), and C-3 (cells only).

Figure 9

Impact of compound 3 IC₅₀ on the CCR5 protein expression level in SW620 CRC cells at 12 and 48 h post treatment in comparison to controls: C-1 (cells only), C-2 (cells treated with MVC solvent), and C-3 (cells treated with compound 3 solvent).