Drug discovery using chemical systems biology: weak inhibition of multiple kinases may contribute to the anti-cancer effect of nelfinavir

Abstract

Nelfinavir is a potent HIV-protease inhibitor with pleiotropic effects in cancer cells. Experimental studies connect its anti-cancer effects to the suppression of the Akt signaling pathway, but the actual molecular targets remain unknown. Using a structural proteome-wide off-target pipeline, which integrates molecular dynamics simulation and MM/GBSA free energy calculations with ligand binding site comparison and biological network analysis, we identified putative human off-targets of Nelfinavir and analyzed the impact on the associated biological processes. Our results suggest that Nelfinavir is able to inhibit multiple members of the protein kinase-like superfamily, which are involved in the regulation of cellular processes vital for carcinogenesis and metastasis. The computational predictions are supported by kinase activity assays and are consistent with existing experimental and clinical evidence. This finding provides a molecular basis to explain the broad-spectrum anti-cancer effect of Nelfinavir and presents opportunities to optimize the drug as a targeted polypharmacology agent.

Figure 1. The structural proteome-wide off-target pipeline integrating ligand binding site characterization and comparison, protein-ligand docking, MD simulation and MM/GBSA energy calculations, and biological network analysis.

Figure 2. Distribution of predicted off-targets on the human kinome tree.

Green represents off-targets with an SMAP p-value less than 1e-4. Yellow represents off-targets with an SMAP p-value less that than 1e-3 and greater than 1e-4.

Figure 3. Overlap between EGFR ATP binding sites and Nelfinavir binding sites predicted by SMAP.

The wheat cartoon represents the backbone structure of EGFR (PDB id: 2J6M). Green sticks represent the co-crystal ligand of EGFR (PDB ligand id: AEE). Magenta sticks represent superimposed Nelfinavir on EGFR according to SMAP alignment. Yellow sticks and balls represent the AEE binding site of EGFR. Orange sticks and balls represent the predicted Nelfinavir binding site for EGFR. Blue sticks and balls represent the overlap between the AEE binding site and the predicted Nelfinavir binding site for EGFR. The amino acid residues involved in the binding site are listed below the structure, colored accordingly.

Figure 4. Comparative Interactions between EGFR and Inhibitor (PDB ligand id AEE; A) and EGFR and Nelfinavir (PDB ligand id 1UN; B).

A: Cyan ribbon represents the backbone structure of EGFR bound to AEE (green). Cyan sticks represent the residues in contact with AEE. Black dash line represents hydrogen bonding interactions between AEE and EGFR. The distance between N and N is 3.22 Å. B: Yellow ribbon represents the backbone structure of EGFR bound to Nelfinavir. Yellow sticks represent the residues in contact with Nelfinavir. Black dash line represents hydrogen bonding interactions between Nelfinavir and EGFR. The distance between N and O is 3.16 Å.

Figure 5. Inhibition rates and calculated MM/GBSA binding free energies of Nelfinavir binding to EGFR, ErbB2, ErbB4, Akt1, Akt2 and Akt3.

Figure 6. Interactions between predicted off-targets and PI3K/Akt, MAPK, JNK, NF-κB, mTOR, Glucose uptake, and Glycogenolysis pathways.

Yellow circles represent predicted off-targets. Blue circle represents intermediate proteins. Green squares represent pathways. Pink squares represent cellular effects. Black lines represent activation. Red lines represent inhibition. Black dashed lines represent a dual effect (activation or inhibition).