Next-Generation TNF Inhibitors from Natural Products
Building on Previous TNF Discoveries
Tumor necrosis factor (TNF) is a critical inflammatory cytokine implicated in autoimmune and inflammatory diseases. Previous work identified Ampelopsin H as a natural product TNF inhibitor capable of disrupting TNF trimers. The challenge was to find new, more potent analogs and demonstrate the power of a fully automated drug discovery pipeline. Using the upgraded Enalos Asclepios KNIME nodes, NovaMechanics sought structural analogs in the vast PubChem chemical database to identify superior TNF inhibitors. The goal extended beyond mere discovery — to showcase the comprehensive capability of the Enalos Asclepios workflow spanning from similarity searches through docking to extended 1000ns molecular dynamics simulations and binding free energy calculations.
Our Approach
A fully automated drug discovery pipeline from PubChem search to TNF-TNFR1 binding validation
Similarity Search & Compound Selection
Searched PubChem for structural analogs of Ampelopsin H, a known TNF trimer disruptor. Identified 113 compounds through similarity search, refined through docking to 53 promising candidates, of which 9 were commercially available for immediate testing.
Molecular Docking with Enalos Asclepios
Used the advanced Enalos Asclepios KNIME nodes for automated preparation and docking of all candidates into the TNF active site. Ampelopsin H scored −45.140 as the reference benchmark, with candidates ranked by docking score and binding energetics.
Extended Molecular Dynamics (1000 ns)
Ran 1000ns molecular dynamics simulations using the fully automated Enalos Asclepios pipeline. This provided deep insight into binding stability, protein-ligand interaction dynamics, and conformational equilibration.
Binding Free Energy Calculations
Computed MM-GBSA and YANK binding affinity calculations from the extended MD trajectories. Nepalensinol B showed the lowest binding free energy (~−35 kcal/mol) and the highest number of hydrogen bonds with TNF, establishing it as the most promising candidate.
Phenotypic Pharmacological Testing
Tested 9 commercially available compounds in L929 cytotoxicity assays and primary murine joint synovial fibroblasts. Two compounds — Nepalensinol B and Miyabenol A — efficiently reduced TNF-induced cytotoxicity and chemokine production.
TNF-TNFR1 Binding Disruption
Nepalensinol B completely abolished TNF-TNFR1 binding at non-toxic concentrations, with 100% blockade of the interaction at concentrations exceeding 50 μM. This makes Nepalensinol B a validated lead compound for TNF inhibitor drug development.