Cheminformatics-aided discovery of small-molecule Protein-Protein Interaction (PPI) dual inhibitors of Tumor Necrosis Factor (TNF) and Receptor Activator of NF-κB Ligand (RANKL)

Abstract

We present an in silico drug discovery pipeline developed and applied for the identification and virtual screening of small-molecule Protein-Protein Interaction (PPI) compounds that act as dual inhibitors of TNF and RANKL through the trimerization interface. The cheminformatics part of the pipeline was developed by combining structure-based with ligand-based modeling using the largest available set of known TNF inhibitors in the literature (2481 small molecules). To facilitate virtual screening, the consensus predictive model was made freely available at: http://enalos.insilicotox.com/TNFPubChem/. We thus generated a priority list of nine small molecules as candidates for direct TNF function inhibition. In vitro evaluation of these compounds led to the selection of two small molecules that act as potent direct inhibitors of TNF function, with IC50 values comparable to those of a previously-described direct inhibitor (SPD304), but with significantly reduced toxicity. These molecules were also identified as RANKL inhibitors and validated in vitro with respect to this second functionality. Direct binding of the two compounds was confirmed both for TNF and RANKL, as well as their ability to inhibit the biologically-active trimer forms. Molecular dynamics calculations were also carried out for the two small molecules in each protein to offer additional insight into the interactions that govern TNF and RANKL complex formation. To our knowledge, these compounds, namely T8 and T23, constitute the second and third published examples of dual small-molecule direct function inhibitors of TNF and RANKL, and could serve as lead compounds for the development of novel treatments for inflammatory and autoimmune diseases.

Fig 1. Chemical structures of compounds SPD304 (published inhibitor), and T8, T23 (the two most potent inhibitors identified by our pipeline).

Fig 2. Strategy for the identification of new small-molecule PPI inhibitors for TNF and RANKL.

Fig 3. The sketching and structural modification facility for the prediction of new TNF inhibitors as provided by the Enalos cloud platform (screen shot).

Fig 4. Prediction and reliability results regarding TNF inhibition upon structure submission of T23, T8 and SPD304 (see SMILES Fig 3) to the Enalos cloud platform.

Fig 5. All-atom RMSD for compounds SPD304, T8 and T23 in complexes with TNF and RANKL.

Fig 6. Representative MD conformations of (a) SPD304, (b) T8 and (c) T23 in TNF and RANKL complexes.

Important residues for protein inhibition are displayed in orange; Gly122 and Gly278 are shown in red. Protein chains A and B are colored gray and magenta, respectively. For simplicity, interacting residues only on chains A are displayed and hydrogen atoms are not shown. Compounds are highlighted in green.

Fig 7. T8 and T23 obstruct the formation of active TNF and RANKL trimers.

Human TNF or RANKL was incubated with T8 or T23, chemically cross-linked, and subjected to SDS-PAGE. This was followed by western blotting to detect the various TNF and RANKL multimers. Both inhibitors were used at a molar ratio of 4:1 relative to TNF and 1:1 relative to RANKL. Numbers indicate molecular weights in kDa; NC = non cross-linked control (no inhibitor, no cross-linking); CC = cross-linked control (no inhibitor).