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Small molecules activating RET for the treatment of Parkinson's disease

Final Report Summary - GDNF MIMETICS (Small molecules activating RET for the treatment of Parkinson's disease)

Small molecules activating RET for the treatment of Parkinson’s disease
Project acronym: GDNF mimetics, webpage:
INTRODUCTION. Parkinson’s disease (PD), caused by degeneration and progressive loss of midbrain dopamine (DA) neurons, is the second most common neurodegenerative disorder affecting 1-2 per 1000 of the population at any time. The disease is more common in elderly people and is diagnosed in 1% of the population above 60 years. No cure for this disease is currently available. The existing treatments are based mostly on DA replacement or stimulation of DA signaling. Although available drugs can alleviate the motor symptoms, they do not stop, prevent or reverse neurodegeneration. Glial cell line-derived neurotrophic factor (GDNF) protects and repairs DA neurons in vitro and in animal models of PD, but poor pharmacological properties of the GDNF protein complicate its clinical use. To exploit the therapeutic potential of GDNF and overcome the pharmacological problems of using of GDNF protein in clinical settings, we decided to develop small molecules with improved pharmacological properties and biological activity similar to GDNF. Initial set of hits with GDNF-like activity was developed by University of Helsinki and Molcode/GeneCode Ltd using a combination of high-throughput screening and rational drug design methods before the start of “GDNF mimetics” IAPP project. During the project life-span supported by IAPP funding, a number of derivatives of active hits were produced and their biological activity was analyzed in in vitro assays. The solubility and CaCo-2 permeability of novel derivatives of GDNF mimetics were also assessed and based on the biological activity and physicochemical properties, several leads were selected for further studies. Two GDNF activity mimicking compounds were also studied in animal model of PD and found to be able to alleviate motor symptoms of the disease. Several predictive QSAR models were also established to improve biological activity and physicochemical properties of small molecular weight GDNF mimetic. As alternative drug optimization approach, porous-silicone nanoparticles were used to improve the solubility of GDNF mimetics. Several potential GDNF mimetics with a different structure were also identified, but they still have to be studied further.
OBJECTIVES OF THE PROJECT. The major goal of the “Small molecules activating RET for the treatment of Parkinson’s disease” project was to strengthen the partnership between University of Helsinki (academic partner) and Molcode Ltd. (industrial partner) by seconding researchers from one partner to work in the premises of another partner, expanding their expertise to another field of knowledge and providing possibilities to conduct research in another environment. Three researchers from Molcode Ltd. and six researchers from University of Helsinki were seconded to partnering organization for 2 14 months during the project life span (01.11.2013 31.10.2017). Four experienced researchers were also recruited to the project to expand expertise of the existing team. All together recruited researchers worked for the project for 51 months and seconded researchers worked for hosting partners for 52.5 months. Project participants were trained in the areas of science mastered by the host organizations via formal training (courses, lectures, self-studies, discussions with specialists) and hands-on exercises required for the scientific progress of “GDNF mimetics” project. Participants also took part in other activities, for example dissemination or business development, improving their soft skills. In addition, technology from University of Helsinki was transferred to Molcode Ltd.
The scientific goal of the project was to develop drug-candidates efficiently protecting and supporting DA neurons in animal model of PD. Existing GDNF mimetics are selective to RET, but do not require the presence of GFRα co-receptors to elicit signaling. In the cells expressing GDNF receptors they slightly, but statistically significantly, reduce the binding of 125I-GDNF at 50 uM concentrations. It was also found that one of the derivatives of GDNF mimetics activated RET phosphorylation at Tyr1062 and Tyr905 residues similarly to GDNF. Reanalysis of HTS results (screening performed before the start of GDNF mimetics project) together with computational chemistry approaches enabled designing and purchasing of focused library of chemical compounds, where one potential novel GDNF novel mimetic was identified after screening in cells. A few compounds were also tested with known anti-PD activity. A compound promoting the survival of cultured DA neurons and restoring the density of DA innervation in mice model of PD was found. This compound failed to promote RET phosphorylation, but seemed to activate downstream signaling cascades MAPK and AKT. It was found that BT13 and BT44 are able to alleviated motor symptoms of PD (amphetamine-induced rotations) in rat 6-OHDA model of PD with comparable efficacy to GDNF. In the brains of experimental animals treated with BT13, a clear trend to increase in the density of DA innervation in the striatum was found. BT44 treated animals had the density of DA fibers comparable to that of GDNF-treated group, but statistically significant differences were not observed. What comes to the optimization of GFL mimetics, University of Helsinki’s goal in medicinal chemistry branch of the project was to improve the solubility of GDNF mimetics. To reach this purpose several approaches were used. A solvent (propylene glycol) was selected suitable for complete solubilization of GDNF mimetics and compatible with in vivo studies. Porous silicone nanoparticles were used to further improve the solubility of existing GDNF mimetics. It was shown that BT44 packed in nanoparticles retains biological activity and allows excluding dimethyl sulphoxide (that is not suitable for in vivo tests) from experimental settings without the loss in solubility and biological activity of BT44. Molcode/GeneCode Ltd concentrated on computational and medicinal chemistry approaches to improve biological activity and physicochemical properties of GDNF mimetics. 90 derivatives of BT13 compound were designed, synthesized and tested in HTS, solubility and CaCo-2 permeability assay. Based on these results, the most promising 6 compounds were shortlisted for further studies. Two of them promoted the survival of cultured DA neurons in 2 nM concentration (initial hit BT13 supports DA neurons in 100-1000 nM concentration). Based on the data, Molcode Ltd constructed fragmental QSAR model that guided the design and synthesis of 4th generation of GDNF mimetics consisting of 10 compounds. One of these derivatives (L9) was highly active in luciferase assay and its predicted solubility was expected to be better than that of GDNF mimetics from the 3rd generation. The ability of L9 to promote the survival of DA neurons survival is now being tested. Traditional SAR studies made upon recommendation of the project medicinal chemistry consultant Dr. Rod Porter resulted in a promising candidate for the development of novel scaffold of GDNF mimetics with better physicochemical characteristics.

Management of GDNF mimetics project and dissemination activities were performed according to the proposed plan. Audience attending dissemination events included students, school children, academic and industrial scientists, business partners, politicians, decision makers and health care professionals. A few peer review papers and one preprint was published during the project life-span. Several manuscripts have been prepared or considered to be prepared.
SUMMARY. the projected achieved most of the planned milestones (M1 - Selectivity to RET; M2 Activity in vivo; M3 – Protection of DA neurons in concentrations below 100 nM, M4 - selection of leads active in vivo) and produced majority of planned deliverables. We found compounds selective to RET that are able to support the survival of dopamine neurons in low nanomolar (<20 nM) concentrations and conducted a proof of concept studies in vivo to show that GDNF mimetics are able to alleviate symptoms in rat model of PD. Due to the limited funding we were not yet able to test all the selected leads in vivo. However, we made a good progress towards the fourth milestone. We plan to do PK studies at the beginning of 2018 to select the best compound(s) to be tested in animal model of PD in vivo. We have published 4 papers so far and although some papers were replaced by others, we reached the planned number of publication during this period. One paper was planned to be published within 6 months upon completion of the project and we are currently working on this manuscript. We also have several other manuscripts in preparation. We disseminated the information and the results of this project to broad audience. We also completed all planned secondments and recruitments, despite of changes in the secondment schedules and secondees’ selection.