SAR study of niclosamide derivatives for neuroprotective function in SH-SY5Y neuroblastoma

Abstract

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 are the causes of mortality. A proteosome 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 to treat tapeworm infections for more than 50 years, has recently attracted renewed attention in drug repurposing because it has been found to be a good candidate in many drug development screenings. We recently found that all markers of MG132-induced neuronal cell toxicity, including the accumulation of ubiquitinated proteins, were prevented by the presence of niclosamide. In addition, niclosamide was shown to enhance autophagy induced by MG132. There results suggested that niclosamide could act as a neuroprotective agent. In the present study, niclosamide derivatives were synthesized, and the structure–activity relationship (SAR) were determined with respect to protein ubiquitination induced by MG132 and effect on cell survival signaling pathways for neuroprotective function. Our results indicate that phenol OH plays a significant role in neuroprotective activity while the niclosamide derivatives without Cl (5- or 2′-Cl) showed almost the same neuroprotective effect. 4′-NO₂ can be replaced by N₃ or CF₃ whereas NH₂ significantly decreased activity. These findings provide guidance for the development of new niclosamide analogues against neurodegenerative diseases including Parkinson’s disease.

Parkinson’s disease (PD) is the second-most common neurodegenerative disease after Alzheimer’s disease. Nearly one million people in the U.S. lives with PD, and about nine million individuals worldwide.¹ Globally, disability and death due to PD are increasing faster than for any other neurological disorder.² PD is defined pathologically by a loss of melanized dopaminergic neurons in the substantia nigra pars compacta (SNc) coupled with cytoplasmic inclusions known as Lewy bodies.³

Lewy bodies contain multiple ubiquitin-proteasome system (UPS) components, such as ubiquitin and proteasome subunits.⁴ UPS is a non-lysosomal proteolytic pathway involved in the regulation of the normal functions of the nervous system, including degradation of damaged and regulatory intracellular proteins. The presence of ubiquitin-positive aggregates has been involved in many neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and Huntington’s disease (HD).^5,6 In the case of PD, Lewy bodies also consist of highly ubiquitinated protein aggregates in degenerated dopaminergic neurons.⁷ This indicates that PD may be related to a defect in the capacity of the UPS to clear unwanted proteins, resulting in protein accumulation, aggregation, and cytotoxicity.

In addition, research on the possible association of PD with neurodegeneration indicated that PD could regulate the aggresome-autophagy pathway.⁸ Most neurodegenerative diseases are marked not only by the presence of ubiquitin-positive protein aggregates, but also by the presence of autophagy abnormalities.⁹ Since aggresome formation is essential for activation of protein degradation via autophagy, it is thought that this process could be an effective therapeutic target for neurodegenerative diseases.

Niclosamide (trade name Niclocide, Table 1, compound 1) is an FDA-approved anthelmintic drug that has been used in humans for more than 50 years.¹⁰ In the past decade, this drug has renewed attention in drug repurposing because it has had a number of hits in drug development screenings programs.^11-15 In our previous research, niclosamide induced cell toxicity in the human glioblastoma U87-MG cell line, showing an increase in protein ubiquitination, ER stress, and autophagy.¹⁶ We also investigated the structure–activity relationship (SAR) of niclosamide derivatives and found the key determinants for anti-glioblastoma activity.¹⁷ It has been reported that niclosamide can block protein ubiquitination caused by proteasome inhibition in human SH-SY5Y cells.¹⁸ Recently, we reported that niclosamide significantly reduced protein ubiquitination induced by MG132 treatment in human neuroblastoma SH-SY5Y and rat PC12 neural cells in a dose-dependent manner. This suggests that niclosamide may be effective in protecting neuronal cells against proteasome dysfunction triggered by the aggregation of ubiquitinated proteins. Furthermore, niclosamide also suppressed MG132-induced apoptosis by inhibiting the upregulation of p53 expression and ER stress in both cell lines.¹⁹ Overall, these results indicate that niclosamide may offer a new treatment of neurodegenerative diseases such as PD. Thus, we hypothesized that the niclosamide derivatives may also have similar effects. Herein, we tested 12 niclosamide derivatives with the replacement of substituents for neuroprotective activity in the presence of a proteosome inhibitor, MG132.

Table 1.

Structures of Niclosamide (1) and Derivatives

Twelve niclosamide derivatives were synthesized (Table 1, 2–13) and their bioactivities were compared with niclosamide, (1). Compounds 2, 3, 7–9, 12, 13 were prepared from the corresponding benzoic acid and aniline derivatives in the presence of PCl₃ in dry xylene under reflux conditions (Scheme 1).

Scheme 1.

Synthesis of niclosamide derivatives by PC1₃ coupling.

Compound 5 was prepared by reduction of niclosamide (Scheme 2), and 10 was obtained by reduction and dichlorination of 2 using H₂/Pd-C in MeOH (Scheme 3).^20,21 Compounds 4 and 11 were obtained by acetylation of 5 and niclosamide 1, respectively. Compound 6 was synthesized from 4 via the diazonium salt.²² All compounds were purified by silica gel column chromatography or recrystallization. The experimental details are provided in the Supporting Information. The structures were confirmed by NMR spectroscopy and their purity was confirmed by HPLC.

Scheme 2.

Synthesis of niclosamide derivatives via reduction, acetylation, and azidation from niclosamide.

Scheme 3.

Synthesis of 10 from 2 via reduction and dechlorination.

Niclosamide 1 and the derivatives 2 – 13 were dissolved in DMSO and stored at −20 °C. 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 subcultured weekly in 60 mm or 100 mm cell culture dishes and used for experiments at 85–90 % confluence of the cell monolayer. For Hoechst 33342 nucleic acid staining, cells were grown in 4-well chamber slides.

SH-SY5Y cells were treated with niclosamide 1 in the presence or absence of 5 μM MG132 for 24 h (Fig. 1A, lanes 2 and 4). The cell lysates were prepared for Western blot assays using anti-ubiquitin antibody. As we reported previously,¹⁹ Western blot analysis confirmed MG132 induces a large increase in ubiquitinated protein levels, especially at higher molecular weights (lane 3). On the other hand, the protein ubiquitination in MG132 treated cells was clearly suppressed with the addition of niclosamide 1 (lane 4). For the SAR investigation, SH-SY5Y cells were treated with niclosamide derivatives 2–7 in the presence of 5 μM MG132 for 24 h. Compounds 2, 6, 7 showed the suppression of protein ubiquitination associated with MG132 (lanes 5, 9, and 10, Fig. 1A). To verify whether these compounds also inhibit ubiquitination-induced apoptosis, in the present study, SH-SY5Y cells were treated with 5 μM niclosamide derivatives in the presence of MG132 for 24 h, then Western blotting was performed with PARP antibody. As shown in the Fig. 1A, cleaved PARP expression is concomitant with the compounds which inhibited protein ubiquitination induced by MG 132, indicating that niclosamide 1 and the compound 2, 6, 7 treatment reduced apoptosis by decreasing protein ubiquitination. From these results, it can be speculated that the OH in the phenol group at R¹ is important for the effect. 2 does not have Cl at C’-2 (R³) but still demonstrated similar activity to niclosamide. This indicates that Cl at R³ is not a pharmacophore. The substituent at C’-4 (R⁴) does not need to be NO₂, and N₃ (6), CF₃ (7), also showed bioactivity similar to niclosamide (1). Compound 5 showed a lower activity with NH₂ at R⁴. The lower activity can be attributed to the contribution of additional hydrogen bonding along with OH at R¹; the increased hydrogen bonding may induce a structural change in the target protein.

Fig. 1.

Effect of niclosamide and derivatives on protein ubiquitination, apoptosis, and ER Stress in SH-SY5Y cells under conditions of proteasome inhibition. SH-SY5Y cells were treated with 5 μM niclosamide 1 and the niclosamide derivatives 2–13 in the presence or absence of 5 μM MG132 for 24 h. Western blot was performed with antibodies specific for ubiquitin, PARP and cleaved PARP, P-JNK, p53, CHOP, cyclin D and β-catenin.

Based on these observations, the niclosamide derivatives 8–13 (Table 1) were also tested (Fig. 1 B). Compounds 11 and 13 were the most active among the set of these 6 compounds. 13 does not have Cl at C-5 (R²), but still demonstrated similar neuroprotective activity to niclosamide. This indicates that Cl at R² is also not a pharmacophore. It means the drug structure can be simplified without Cl, which will be less expensive. Compound 8, without OH at R¹ did not suppress ubiquitination significantly. A comparison between 8 and 13 indicates that phenol OH plays an important role in the mechanism of action. Compound 10, with OH at R¹ and NH₂ is at R^4, does not show the activity, similar to 5. Without a substituent at R⁴ (9 and 12), the activity of the derivatives is diminished. This suggested that some substituent is required at R⁴. Compound 11 showed inhibition of ubiquitination while 4 did not, although both compounds have an acetylated phenol OH group at R¹. This may be attributed to the acetyl group undergoing hydrolysis, and the OAc at R¹ of 11 is converted to OH, forming niclosamide (1). The difference in activity between 4 and 11 may be due to the NHAc at R⁴ of 11 which causes different interactions with the target including additional hydrogen bonding.

Dysfunction of the proteasome leads to the accumulation of misfolded proteins, triggers ER stress, and induces autophagy.²³ Accumulation of misfolded proteins in the ER results in activation of certain specific proteins such CHOP by unfolded protein response (UPR). CHOP expression can promote apoptotic cell death. Thus, the inhibition of CHOP expression by niclosamide and the synthesized compounds is an indication of reducing cell apoptosis induced by proteasome inhibition in neural cells. Also, we tested p-JNK expression, which is one of the mitogen-activated protein kinase (MAPK) signaling pathways. Usually, activation of JNK (p-JNK) could induce apoptosis. As shown in Fig. 1, niclosamide 1 and compounds 2, 6, 7, 11 and 13 inhibited CHOP expression, and this is consistent with cleaved PARP expression. Along with niclosamide 1, compounds 2, 6, 7, 11 and 13 reduce apoptosis because these compounds suppress ubiquitination. This indicates that niclosamide and these compounds protected against MG-132-induced apoptosis.

The Wnt/β -catenin signaling pathway is required for neural stem cell development, particularly, involved in cell proliferation, differentiation, and migration. The expression of cyclin D, one of β-catenin downstream targets, were tested in SH-SY5Y cells with niclosamide 1 and the derivatives 2–13. In the presence of the active niclosamides 2, 6, 7, 11, and 13, the protein ubiquitination was increased, and cyclin D protein expression was almost completely suppressed after 24-hour treatment (Fig. 1). Interestingly, these active compounds 2, 6, 7, 11, and 13 also demonstrated anti-glioblastoma activity by ubiquitination and inhibition of other cell signaling pathways on human U87 cell in our previous study.¹⁷ These opposing effects of niclosamide on U87 and MG132-induced protein ubiquitination suggest that there are different mechanisms of action of niclosamide in different neuronal cell types.

p53 plays an important role in regulating many cellular processes, and it is also known as an apoptotic inducer which is activated in response to diverse cellular stresses. Activated p53 promotes cell cycle arrest to allow DNA repair and/or apoptosis to prevent the propagation of cells with serious DNA damage through the transactivation of its target genes. High levels of p53 were observed in the brains of PD patients as well as PD animal models; it indicates that p53 activation and the degeneration of dopaminergic neurons in PD could be linked.²⁴ MG132 alone (lanes 3 and 13, Fig. 1) showed a high level of p53 expression while niclosamide 1 inhibited expression of p53 and protected against MG132 induced apoptosis. In addition to niclosamide 1, compound 2, 6, 7, 11 and 13, also inhibited p53 expression. Interestingly, compound 4, which was not active on other markers, also inhibited p53 expression. Also, 10 and 12 showed weaker inhibition. This indicates that the effect on the expression of p53 by these compounds are through a different mechanism of inhibition.

In conclusion, all the bioassays showed that niclosamide 1 and derivatives 2, 6, 7, 11, and 13 can have a neuroprotective function. While there is no evidence for the direct binding of niclosamide and their derivatives to any specific proteins, the results indicate that the phenol OH at R¹ (2, 6, 7, 13) plays an important role, and the compounds 2 and 13, without chloride at 2′-Cl (R³) or 5-C1 (R²), does not diminish the effect in the neuroprotective activity. This means the drug structure can be simplified without Cl. 4′-NO₂ (R⁴) can be replaced with N₃ or CF₃ whereas NH₂ significantly decreased the neuroprotective function, which may indicate that additional hydrogen bonding causes increased structure changes in the target protein. Yet, when there is no substituent at R⁴ (9 and 12), there is no significant activity. This suggested the substituent at 4′ plays some role in binding. The inhibition of p53 expression by these compounds may have a different mechanism of action, and further investigation will be required in the future. Based on the results of the present study, niclosamide and its derivatives may represent new molecules for prevention of Parkinson’s disease (PD) and other neurodegenerative diseases.

Abbreviations:

SAR

structure–activity relationship

PD

Parkinson’s Disease

ER stress

Endoplasmic reticulum stress

FDA

U.S. Food and Drug Administration

UPS

Ubiquitin-Proteasome System

ER

Endoplasmic Reticulum

UPR

Unfolded Protein Response

NMR

Nuclear magnetic resonance

HPLC

High performance liquid chromatography

DMSO

dimethyl sulfoxide

GAPDH

glyceraldehyde

PARP

Poly(ADP-ribose) polymerase

CHOP

C/EBP homology protein

Appendix A. Supplementary data

Supplementary data to this article can be found online at https://doi.org/10.1016/j.bmcl.2023.129498.

Data availability

Data will be made available on request.