Targeting GDNF receptors with small molecules for the treatment of Parkinson’s disease

Motor symptoms of Parkinson’s disease (PD) are caused by the degeneration of dopamine (DA) neurons. Currently, no disease-modifying therapies against PD are available. Therefore, the major challenge is developing a drug to protect and restore DA neurons. Glial cell line-derived neurotrophic factor (GDNF) has been identified and characterized for its ability to promote the survival of cultured nigrostriatal dopamine neurons and protect and repair them in animal models of PD. However, the outcomes of the clinical trials with GDNF in PD patients remain inconclusive. GDNF is a promising protein that halts neurodegeneration. GDNF does not cross through the blood-brain barrier (BBB) and has poor diffusion properties, complicating clinical use in PD patients. This thesis aimed to characterize the biological activity of GDNF family ligand (GFL) receptor agonists in both in vitro and in vivo preclinical models of PD. We have developed small molecular weight compounds acting similarly to GDNF in the dopamine system (GFL receptor agonist) with improved pharmacological characteristics. The first-generation GFL receptor agonist, a compound named BT13, potently activated the GDNF receptor REarranged during transfection (RET) and its downstream targets. It protected cultured dopamine neurons from neurotoxin-induced cell death and stimulated dopamine release in the mouse striatum. We further optimized BT13 and developed the second-generation GFL receptor agonist BT44 with ten-fold improved potency. BT44 activated RET, supported cultured DA neuron's survival and protected them from neurotoxin lesions, induced functional recovery, and protected striatal dopaminergic fibers in rats with experimentally induced PD. We also studied the selectivity of both BT13 and BT44 using different functional assays. Our results demonstrate that both BT13 and BT44 are selective towards RET. The compounds promote survival and protect the cultured dopamine neurons only in the presence of RET. BT44 is a promising lead compound that could be potentially developed as a drug for PD treatment. However, BT44 suffers from intrinsic poor aqueous solubility like many other hydrophobic drugs, making its preclinical development and clinical translation difficult. Therefore, to improve BT44 solubility, we developed nanoformulation of BT44 with Pox-based ABA triblock copolymers. We found that nanoformulated BT44 retains its biological activity and has improved absorption and blood-brain barrier (BBB) penetration compared to the pristine BT44. We also demonstrate that nanoformulated BT44 binds to the GFL receptors and competes with GDNF for binding to the RET receptor. The receptor binding studies were not possible with pristine BT44 due to its poor solubility. Taken together, our results indicate that both BT13 and BT44 can activate the RET receptor and its downstream signaling cascade necessary for neuronal survival and protection from neurotoxin insults. Further, our results show that in vitro BT44 is active in 10 nM concentrations, which is a realistic target concentration for actual drugs. Moreover, we developed a nanoformulation of BT44 to improve the solubility of the pristine compound and showed that this nanoformulation retains biological activity. With further optimization and detailed studies, BT44 could be developed as a drug-like candidate for disease-modifying treatment for PD.Tiivistelmä ei saatavilla