In Silico Investigation of the Binding of MCoTI-II Plant Defense Knottin to the γ-NGFserine Protease of the 7S Nerve Growth Factor Complex and Biological Activity of Its NGF Mimetic Properties.

Nerve Growth Factor (NGF) induces neurotrophic effects primarily through the tropomyosin-related kinase receptor TrkA NGF. While trophic proteins hold promise for the treatment of neuronal injury and disease, use of NGF is limited by its large molecular weight, lack of permeability through the blood brain barrier and peripheral side effects. High throughput screening on > 1000 natural plant based substances using a PC-12 neurite outgrowth model found only a single NGF mimetic hit, the seed pit of bitter gourd Momordica cochinchinensis. Bioactivity guided fractioning of the M. cochinchinensis seed extract confirmed the active component to be a protein of approximately 17kD MW, and not of chemical nature. By a theoretical understanding, it has been shown that in vivo, NGF exists as an inactive high molecular weight complex known as 7S NGF, which comprises pairs of two serine proteinases α-NGF and γ-NGF, and the trypsin inhibitor β-NGF. Active NGF is comprised of the free β-NGF dimer. Here we use computational modeling, which demonstrates that MCoTI-II can bind stably to the serine protease γ-NGF within the 7S NGF complex. This interaction may inhibit 7S NGF complex formation, potentially freeing the active NFG protein, and may explain the neuritogenic NGF mimetic activity of M. cochinchinensis seed extract. Recombinant purified MCoTI-II tested in rat PC-12 cells grown on collagen failed to initiate outgrowth relative to the control. While theoretical dockings studies show a plausible hypothetical explanation for trophic effects, biological studies do not support a role in neurotrophic activity of the pure MCoTI-II protein.