Posters
Presentation Type
Poster
Discipline Track
Other
Abstract Type
Research/Clinical
Abstract
Background: Neurodegenerative disease is a debilitating and incurable condition that affects millions of people around the world. The loss of functions or malfunctions of neural cells causes mortality. A proteasome inhibitor, MG132, is well known to cause neurodegeneration in vitro when model neuronal-derived cell lines are exposed to it. Niclosamide, an anthelmintic drug, which has been used for more than 50 years, has recently attracted renewed attention in drug repurposing because it has been found as a good candidate against various diseases in screenings. We recently found that all markers of MG132-induced neuronal cell toxicity, including the accumulation of ubiquitinated proteins, were prevented by niclosamide. In addition, niclosamide was shown to enhance autophagy induced by MG132. Therefore, our results suggest that niclosamide could be a potential neuroprotective agent. In the present study, niclosamide derivatives were synthesized by changing substituents, and their structure-activity relationship (SAR) of the protein ubiquitination induced by MG132 and cell survival signaling pathways for neuroprotective function were studied.
Methods: The 12 niclosamide derivatives were synthesized; mostly, they were prepared from the corresponding benzoic acid and aniline derivatives in the presence of PCl3 in dry xylene under reflux conditions. Niclosamide and the 12 derivatives were dissolved in DMSO. The SH-SY5Y cells were cultured at 5% CO2 at 37 °C in EMEM: Ham’s F-12K medium (1:1), 5% horse serum with penicillin (100 units/mL), and streptomycin (100 μg/mL). The cells were sub-cultured weekly in 60 mm or 100 mm cell culture dishes and used for experiments at 85-90% confluence of the cell monolayer. SH-SY5Y cells were treated with niclosamide or derivatives and 5 μM MG132 for 24 h. The cell lysates were prepared for Western blot assays using anti-ubiquitin antibodies, including ubiquitin, PARP, p-JNK, CHOP, cyclin D, and p53.
Results: Our results indicate that when phenol OH was present, the compounds demonstrated neuroprotective activity, while the presence or absence of Cl (5- or 2’-Cl) showed almost the same neuroprotective effect. 4’-NO2 can be replaced by N3 or CF3 to have neuroprotective activity, whereas NH2 significantly decreased activity. Yet, when there is no substituent at the 4’- position, there is no significant activity. All the bioassays showed that niclosamide and certain derivatives showed a neuroprotective function. While there is no evidence for the direct bindings of niclosamide and their derivatives to any specific proteins, the results indicate that the phenol OH plays an important role, and chloride at 2’-Cl or 5-Cl, does not affect the neuroprotective activity. 4’-NO2 can be replaced with N3 or CF3. However, 4’-NH2 and 4’-H significantly decreased the neuroprotective function. This suggested the substituent at 4’ position plays some role in bindings. The inhibition of p53 expression by these compounds may have a different mechanism of action, and further investigation will be required in the future.
Conclusions: Based on the results of the present study, niclosamide, and its derivatives can be new target molecules for the prevention of Parkinson’s disease (PD) and other neurodegenerative diseases. Also, these findings provide valuable information for the development of the next generation of niclosamide analogs.
Academic/Professional Position
Faculty
Mentor/PI Department
Medical Education
Recommended Citation
Mito, Shizue; Cheng, Benxu; Garcia, B. A.; Ooi, Xin Yee; Gonzalez, D.; Ruiz, T. C.; Elisarraras, Francisco Xavier; and Tsin, Andrew, "SAR Study of Niclosamide Derivatives for Neuroprotective Function in SH-SY5Y Neuroblastoma" (2024). Research Symposium. 92.
https://scholarworks.utrgv.edu/somrs/2023/posters/92
Included in
SAR Study of Niclosamide Derivatives for Neuroprotective Function in SH-SY5Y Neuroblastoma
Background: Neurodegenerative disease is a debilitating and incurable condition that affects millions of people around the world. The loss of functions or malfunctions of neural cells causes mortality. A proteasome inhibitor, MG132, is well known to cause neurodegeneration in vitro when model neuronal-derived cell lines are exposed to it. Niclosamide, an anthelmintic drug, which has been used for more than 50 years, has recently attracted renewed attention in drug repurposing because it has been found as a good candidate against various diseases in screenings. We recently found that all markers of MG132-induced neuronal cell toxicity, including the accumulation of ubiquitinated proteins, were prevented by niclosamide. In addition, niclosamide was shown to enhance autophagy induced by MG132. Therefore, our results suggest that niclosamide could be a potential neuroprotective agent. In the present study, niclosamide derivatives were synthesized by changing substituents, and their structure-activity relationship (SAR) of the protein ubiquitination induced by MG132 and cell survival signaling pathways for neuroprotective function were studied.
Methods: The 12 niclosamide derivatives were synthesized; mostly, they were prepared from the corresponding benzoic acid and aniline derivatives in the presence of PCl3 in dry xylene under reflux conditions. Niclosamide and the 12 derivatives were dissolved in DMSO. The SH-SY5Y cells were cultured at 5% CO2 at 37 °C in EMEM: Ham’s F-12K medium (1:1), 5% horse serum with penicillin (100 units/mL), and streptomycin (100 μg/mL). The cells were sub-cultured weekly in 60 mm or 100 mm cell culture dishes and used for experiments at 85-90% confluence of the cell monolayer. SH-SY5Y cells were treated with niclosamide or derivatives and 5 μM MG132 for 24 h. The cell lysates were prepared for Western blot assays using anti-ubiquitin antibodies, including ubiquitin, PARP, p-JNK, CHOP, cyclin D, and p53.
Results: Our results indicate that when phenol OH was present, the compounds demonstrated neuroprotective activity, while the presence or absence of Cl (5- or 2’-Cl) showed almost the same neuroprotective effect. 4’-NO2 can be replaced by N3 or CF3 to have neuroprotective activity, whereas NH2 significantly decreased activity. Yet, when there is no substituent at the 4’- position, there is no significant activity. All the bioassays showed that niclosamide and certain derivatives showed a neuroprotective function. While there is no evidence for the direct bindings of niclosamide and their derivatives to any specific proteins, the results indicate that the phenol OH plays an important role, and chloride at 2’-Cl or 5-Cl, does not affect the neuroprotective activity. 4’-NO2 can be replaced with N3 or CF3. However, 4’-NH2 and 4’-H significantly decreased the neuroprotective function. This suggested the substituent at 4’ position plays some role in bindings. The inhibition of p53 expression by these compounds may have a different mechanism of action, and further investigation will be required in the future.
Conclusions: Based on the results of the present study, niclosamide, and its derivatives can be new target molecules for the prevention of Parkinson’s disease (PD) and other neurodegenerative diseases. Also, these findings provide valuable information for the development of the next generation of niclosamide analogs.