Modeling of mechanistic effects of neurotrophin modulators

Neurotrophins (NTs) are growth factors that are expressed in the central and peripheral nervous systems. They are implicated in different phases of the development and maintenance of the nervous system and they can regulate neuronal survival, development, function, plasticity, as well as neuronal apoptosis. NTs can be used as therapeutics for the treatment of neurodegenerative disorders. However, their poor pharmacokinetic properties and their invasive administration to patients renders them inefficient for use as pharmaceuticals. A solution to this can be offered by small molecule NT mimetics, which can elicit NT mechanisms through binding to NT receptors, which are transmembrane (TM) glycoproteins. The mechanism of activation of NT receptors remains elusive and thus, in this thesis, I have investigated the mechanism of action of NT receptors and mimetics through molecular modeling and molecular dynamics (MD) simulations. I modeled and simulated the glycosylated state of the full extracellular (EC) domains of Tropomyosin receptor kinases A and B (TrkA, TrkB) NT receptors, which revealed that the glycans can shield the accessible surface area of the receptors and participate in the contact area between receptor and NT. Most importantly, glycosylation promoted the extended conformations of the EC domains, which might facilitate NT binding. Then, I performed coarse-grained MD simulations to study the possible arrangements of the TM helical homodimers of TrkA and TrkB receptors in micelles. The results revealed arrangements that could correspond to the active state of the receptors, while metadynamics simulations indicated a stronger binding for the TrkA helices by 10 kJ/mol compared to TrkB. Next, I modeled the full-length structures of the TrkA and TrkB receptors in their homodimeric, glycosylated state bound to their NTs. I embedded the receptors in a realistic model of a neuronal asymmetric membrane. I verified the proper behavior of the membrane and proteins with smaller systems comprising of the TM and intracellular monomers of the receptors. These test simulations revealed interactions between positively charged residues of the kinase domain of TrkA with negatively charged lipids of the inner leaflet of the membrane. These interactions were also formed in the full-length system, and they might stabilize the two kinase domains of the receptor dimer in an orientation that promotes activation, even though the kinase domains were not activated during the simulations. Also, in the full-length systems, the EC domains approached and lay down on the neuronal membrane, while interacting with membrane lipids, such as gangliosides, which are able to activate the NT receptors. Finally, I investigated the binding of small-molecule NT mimetics to the EC and TM domains of TrkA and TrkB receptors, with molecular docking and MD simulations. While plausible poses were 8 obtained, they were not able to explain the selectivity of the compounds for the receptors and the simulations showed that binding was weak. Due to the cholesterol core of the NT mimetics, I tested the ability of the compounds to enter the cell membrane with MD simulations. The compounds were able to spontaneously penetrate the membranes, indicating that their binding site could also lie in the TM region of the receptors. However, simulations with the compounds bound or close to the TM helices in the membrane environment, showed no specific binding. Further experimental exploration of the binding mechanism of these compounds is required. Overall, this thesis sheds light on the dynamic behavior of TrkA and TrkB NT receptors in membranes and the mechanistic insights provide a basis for future studies to develop NT mimetics.