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. 2025 Aug 1. Online ahead of print. doi: 10.1039/d5md00345h

Novel small molecule derivatives improve survivability in the cellular model of Huntington's disease via improving mitochondrial fusion

Pradeep Kodam a, Vaishali Kumar a, Paramita Pattanayak b, Praharsh Vitta a, Tanmay Chatterjee b,✉, Shuvadeep Maity a,✉
PMCID: PMC12415771  PMID: 40927636

Abstract

Mitochondrial dysfunction is one of the primary cellular conditions involved in developing Huntington's disease (HD) pathophysiology. The accumulation of mutant huntingtin protein with abnormal PolyQ repeats resulted in the death of striatal neurons with enhanced mitochondrial fragmentation. In search of neuroprotective molecules against HD conditions, we synthesized a set of isoxazole-based small molecules to screen their suitability as beneficial chemicals improving mitochondrial health. Systematic characterization of one of these isoxazole derivatives, C-5, demonstrated improved mitochondrial health with reduced apoptosis via rebalancing fission–fusion dynamics in HD condition. Gene and protein expression analysis confirmed that C-5 treatment enhanced the expression of mitochondrial fusion regulators (MFN1/2) via transcriptional upregulation of PGC-1α, a transcriptional co-activator controlling mitochondrial biogenesis. Collectively, this novel fusion agonist can potentially become a new therapeutic alternative for treating PolyQ-mediated mitochondrial dysfunction, a hallmark of HD pathology.

Identification and characterization of a novel mitochondrial fusion agonist beneficial against Huntington's disease (HD) pathology.graphic file with name d5md00345h-ga.jpg

1. Introduction

Huntington's disease (HD) is an autosomal dominant disorder linked with the accumulation of polyglutamine expansion in the huntingtin protein (mutant huntingtin, mHTT) due to a CAG trinucleotide repeat (≥40 for complete penetrance) in the huntingtin gene (HTT).1,2 This is a late-onset neurodegenerative disease characterized by movement disorders and cognitive decline.3 Most importantly, in HD, the underlying mechanisms of the pathological condition are far more complicated beyond the accumulation of PolyQ repeats. Due to its multifactorial nature, despite enormous research, there is no cure for HD, and options for drug-based therapies are still very limited.4

Mitochondrial dysfunction is one of the pathophysiological conditions thought to be a major cause of selective degeneration of the striatum, with specific loss of efferent medium spiny neurons (MSNs) in HD.5 Mitochondria undergo fusion–fission cycles to maintain their dynamic structure often controlled by a set of fission (DRP1 (dynamin-related protein 1), FIS1 (fission, mitochondrial 1), MFF (mitochondrial fission factor)) and fusion proteins (mitofusin 1/2 (MFN1, MFN2) and optic atrophy 1 (OPA1)). The fission–fusion cycle is critical for maintaining mitochondrial homeostasis in response to metabolic or environmental stresses and is also linked to cellular division, apoptosis, and autophagic processes.6–8 Altered expression and functionality of these proteins lead to abnormal dynamics and pathological conditions. At the molecular level, mHTT was found to interact with fission protein (DRP1), leading to increased mitochondrial fragmentation via enhancing the enzymatic activity of DRP1.9–11 Similarly, mHTT repressed PGC1α (peroxisome proliferator-activated receptor gamma coactivator 1-alpha), which controls mitochondrial biogenesis and fusion gene (MFN1/2) expression, resulting in mitochondrial dysfunction and fragmentation.12 In addition, excessive mitochondrial fission in the HD striatum disrupted the mitochondria-ER associated membrane or MAM's integrity, followed by disturbances in Ca2+ and ROS (reactive oxygen species) homeostasis.13 Thus, improving mitochondrial fusion efficiency could be a potential therapeutic intervention for HD.

Small molecules that enhance mitochondrial fusion either by decreasing fission or increasing fusion are found to be effective in restoring mitochondrial health with subsequent recovery from disease symptoms. Interestingly, in an earlier study, small molecule M1 was found to be effective in optic nerve regeneration via enhanced expression of fusion proteins MFN1/MFN2.14 Similarly, teriflunomide (TFM), a known mitochondrial fusion activator, prevented mitochondrial shortening and fragmentation and preserved neuronal activity in brain slices exposed to oxidative stress.15 In small-cell lung cancer (SCLC), TFM along with chemotherapy was found to decrease mitochondrial fragmentation via modulating phosphorylation of DRP1.16 Another small molecule agonist, S89, restored mitochondrial dysfunction by enhancing MFN1-mediated fusion.17 These studies showed that small molecules improving mitochondrial fission–fusion balance can be an effective alternative for diseases linked to mitochondrial dysfunction.

In this study, we developed a new small molecule C-5 that can improve mitochondrial health in HD conditions with better efficacy. We utilized our in-house developed method to synthesize our target molecules to evaluate their effectiveness over different in vitro models of HD expressing mHTT with >35 PolyQ repeats.18 Our designed isoxazole-based molecule exhibited favorable pharmacokinetic properties in silico. The combined in silico and in vitro screening with the help of different molecular and biochemical assays identified the underlying mechanisms involved in rescuing cellular viability in HD conditions. Hence, our study identified a new mitochondrial fusion activator as a promising candidate offering the opportunity for further preclinical development of a new alternative therapeutics against HD.

2. Methods

2.1. Design strategy of target isoxazole-based molecules

As our primary focus is to develop small molecules for the treatment of HD via the improvement of mitochondrial health, we designed a series of isoxazole-based molecules, in particular, 4-(trifluoromethyl) isoxazoles through our in-house derived method. We noticed that the Food and Drug administration (FDA)-approved teriflunomide (TFM) and its precursor leflunomide (LM) have been reported to be effective in restoring mitochondrial dysfunction in brain cells. Notably, both TFM and LM contain a trifluoromethyl (–CF3) functional group, and it is well-documented that the presence of a –CF3 functional group can increase a molecule's lipophilicity by reducing hydrogen bonding and enhancing hydrophobicity, facilitating its incorporation or interaction with lipid membranes, which plays a critical role in improving drug-like properties. Keeping this in mind we created our novel molecules. Structurally, our synthesized isoxazole derivatives differ significantly from TFM. Unlike TFM, which is a non-heterocyclic molecule and contains cyano, enol, and amide groups, our compounds are heterocyclic and feature an aryl group at the 3rd position, a trifluoromethyl group at the 4th, and a methyl or hydrogen at the 5th position of the isoxazole ring (Fig. 2a and b). These distinctions clearly place our molecules in a novel chemical class. We designed compounds with lower molecular weight that could have a better ability to traverse the blood–brain barrier (BBB), a crucial determinant of central nervous system (CNS) efficacy. We designed a series of forty-two isoxazole-based molecules (Table S1). Out of the forty-two, we selected five molecules after in silico screening of their physical and pharmacokinetics properties (see section 2.2).

Fig. 2. Structure–function relationship of new isoxazole-based small molecules. a. Schematic of the design strategy for target small molecules (C-1 to C-5). Details of the synthesis can be accessed from Methods. b. The five derivatives under two broad structural categories (MTI and TI) show a structure–function (cell viability/cytotoxicity) relationship. The presence of the aryl ring and variation in the functional group influence the cytotoxicity. C-5 with minimum concentration achieves the maximum effectivity (IC50) compared to the rest of the molecules. c. IC50 value and dose–response curves of C-5 and teriflunomide (TFM) are generated after testing a range of concentrations (10 nM to 100 μM) on control (Q23) and HD (Q74) cells. Cell viability is measured by the MTT assay, and the cytotoxicity curves represents measurements of three independent experiments with four technical replicates for each drug concentration. Percent cell viability data represented as mean ± SD. See also the SI for Fig. S2.

Fig. 2

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We categorized these five molecules into two sets based on their structural features. The first category of TMs includes 5-m̲ethyl-3-aryl-4-(t̲rifluoromethyl) i̲soxazoles, i.e., MTI-1 to MTI-3 (C-1, C-2, and C-3), and the second category includes 3-aryl-4-(trifluoromethyl) isoxazoles, i.e., TI-1 (C-4) and TI-2 (C-5). A method developed by our group, i.e., metal-free, cascade regio-, and stereoselective trifluoromethyloximation, cyclization, and elimination strategy with readily available and inexpensive α,β-unsaturated carbonyls, was used to synthesize the small molecules.18 This method requires commercially available and cost-effective reagents such as CF3SO2Na (as the CF3 source) and tertiary butyl nitrite (TBN) as a multitasking reagent for the synthesis of a wide variety of 4-(trifluoromethyl) isoxazoles (Fig. 2a).

2.1.1. Synthesis of 5-methyl-3-aryl-4-(trifluoromethyl) isoxazoles (MTI, C-1–C-3)

5-Methyl-3-aryl-4-(trifluoromethyl) isoxazoles (MTI) were successfully synthesized via a two-step reaction, starting from the feedstock materials, i.e., benzaldehydes (1) and acetone (Scheme 1).

Scheme 1. A two-step experimental procedure for synthesizing 5-m̲ethyl-3-aryl-4-(t̲rifluoromethyl) i̲soxazoles (MTI) from commercially available feedstock materials, i.e., benzaldehydes and acetone.

Scheme 1

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Briefly, 5-methyl-3-aryl-4-(trifluoromethyl) isoxazoles (MTI) were successfully synthesized via a two-step synthetic route as described below:

Step 1: To a solution of appropriate benzaldehyde (10 mmol, one equivalent) in acetone (5 ml), an aqueous solution of NaOH (4 equivalents) was added and the mixture was stirred at room temperature for five hours. The progress of the reaction was monitored by thin layer chromatography (TLC). The resulting solution was extracted with ethyl acetate three times (3 × 10 ml), and the combined organic layer was washed with water (3 × 10 ml). The organic layer was dried with anhydrous Na2SO4, and the solvent was evaporated under reduced pressure to afford the crude product, which was purified by column chromatography to afford 2 in a quantitative yield.

Step 2: Freshly synthesized 2a (0.5 mmol, 1 equivalent) and CF3SO2Na (3 equivalent) were taken in an oven-dried 10 mL sealed tube, and dimethyl sulphoxide (DMSO) (1.25 mL, 0.4 M) was added to that mixture. Then TBN (4 equivalents) was added to the reaction mixture, and it was stirred at room temperature. The progress of the reaction was monitored by TLC, then the tube was closed very tightly and sealed properly, and the reaction mixture was heated at 120 °C in an oil bath for 30 min. Then resulting solution was extracted with ethyl acetate three times (3 × 10 ml), and the combined organic layer was washed with water (3 × 10 ml). The organic layer was dried with anhydrous Na2SO4, and the solvent was evaporated under reduced pressure to afford the crude product, which was purified by column chromatography to afford MTI, i.e., 5-methyl-3-aryl-4-(trifluoromethyl) isoxazoles (C-1 to C-3). The synthesized target molecules were characterized by 1H and 19F Nuclear Magnetic Resonance (NMR) spectroscopy and high-resolution mass spectrometry (HR-MS) (Fig. S1d under SI).

The molecules were characterized by 1H and 19F NMR spectroscopy, which matched the literature18 and HR-MS analysis. Analytical data of the synthesized final compounds are given in SI.

2.1.2. Synthesis of 3-aryl-4-(trifluoromethyl) isoxazoles (TI, C-4 and C-5)

3-Aryl-4-(trifluoromethyl) isoxazoles (TI) were synthesized via a one-pot reaction (details in Methods), starting from the readily available starting material, i.e., α,β-unsaturated aldehyde 3 (Scheme 2). At first, α,β-unsaturated aldehyde 3 (0.5 mmol, 1 equivalent) and CF3SO2Na (3 equivalents) were taken in an oven-dried 10 mL sealed tube, and DMSO (1.25 mL, 0.4 M) was added to that mixture. TBN (4 equivalents) was added to the reaction mixture, and it was stirred at room temperature. The progress of the reaction was monitored by TLC, then the sealed tube was closed very tightly and sealed properly, and the reaction mixture was heated at 120 °C in an oil bath for 30 min. The resulting solution was extracted with ethyl acetate three times (3 × 10 mL), and the combined organic layer was washed with water (3 × 10 ml). The organic layer was dried with anhydrous Na2SO4, and the solvent was evaporated under reduced pressure to afford the crude product, which was purified by column chromatography to afford 3-aryl-4-(trifluoromethyl) isoxazoles i.e.TI (C-4 and C-5). The synthesized target molecules were characterized by 1H and 19F NMR spectroscopy and high-resolution mass spectrometry (HR-MS).

Scheme 2. One-pot experimental procedure for synthesizing 3-aryl-4-(t̲rifluoromethyl) i̲soxazoles (TI) from α,β-unsaturated aldehydes.

Scheme 2

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2.2. In silico screening of the isoxazole-based derivatives for blood–brain barrier (BBB) permeability assessment

We initiated the in silico screening process utilizing the diverse library of the designed forty-two 4-(trifluoromethyl) isoxazoles and their derivatives. The SMILES of all the designed molecules were entered into the Swiss-ADME workspace (http://www.swissadme.ch), followed by the estimation of the ADME properties (Absorption, Distribution, Metabolism, and Excretion) through Swiss-ADME tools.19 Swiss-ADME is a widely used computational tool that provides detailed insights into the pharmacokinetics, drug-likeness, and medicinal chemistry suitability of small molecules. It draws on a comprehensive database supported by literature reviews, patent analyses, and cross-referenced chemical data. Notably, it includes a reference library of 660 compounds—567 classified as well-absorbed and 93 as poorly absorbed. One of Swiss-ADME's key features is the BOILED-Egg model (Brain Or IntestinaL EstimateD permeation), which offers a reliable prediction of a compound's gastrointestinal absorption and brain penetration. This model estimates permeability by calculating key physicochemical properties, particularly lipophilicity and polarity, using a robust set of parameters.19,20 Our compounds were analyzed to evaluate their ability to traverse the BBB, a crucial determinant of central nervous system (CNS) efficacy. The blood–brain barrier (BBB) permeability of the compounds was evaluated using SwissADME, which utilizes the BOILED-Egg model—a widely accepted predictive tool based on molecular lipophilicity and polarity. This model generates a BOILED-Egg plot by plotting the water partition coefficient (WLOGP) against the topological polar surface area (TPSA, in Å2). TFM, an FDA approved drug, was used as a positive control during the in silico assessment of our target molecules.

Our analysis revealed that five compounds (MTI and TI) exhibited satisfactory BBB permeability and drug-likeliness (see details in Table S2). Thus, the selected five compounds served as our target molecules (TMs) for IC50 determination (Fig. 2c and S2).

2.3. Cell culture of in vitro HD model cells

We used recombinant HEK293 cell lines expressing exon 1 of the human HTT gene with 23 CAG (control cells; Q23) or 74 CAG (HD cells; Q74) repeats fused to green fluorescent protein (GFP) (a generous gift from David C. Rubinsztein's lab, University of Cambridge, UK).21 Recombinant HEK293 cell lines were cultured in poly-l-ornithine coated plates using Dulbecco's modified Eagle's medium (DMEM, Gibco, Thermo Fisher Scientific, USA) supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher Scientific), a combination of 10 000 units per ml penicillin, and 10 mg ml−1 streptomycin (Gibco, Thermo Fisher Scientific), hygromycin (150 μg ml−1), and blasticidin (5 μg ml−1), and maintained at 37 °C with 5% CO2. At 60–70% confluency, doxycycline (1 μg ml−1) was added to induce PolyQ HTT formation for 48 hours in the case of recombinant HEK293 cell lines (Fig. 3a).

Fig. 3. C-5 mediated restoration of mitochondrial dynamics in HD condition. a. Experimental outline for gene/protein expression studies after C-5 and TFM treatment on two different cell lines. Left, HEK293 cells are stably expressing PolyQ23 (control) or PolyQ74 (HD) repeats. Right, Neuro2a (N2A) cells transiently transfected with plasmid carrying either PolyQ23 (control; N2AQ_Con) or PolyQ74 (HD; N2AQ_HD) are used. All data from N2AQ_Con and N2AQ_HD are presented in SI figures. b. Representative confocal images of mitochondrial morphology (MitoTracker-Red-stained) show rescue of elongated mitochondrial morphology in HD cells after C-5 treatment. The inset shows a magnified portion of the marked area. All representative images are with scale bar 25 m. c. Box–whisker plot represents the mitochondrial footprint area in control, HD and HD with small molecule treated cells (n = 20, all four conditions). C-5 treatment restores elongated, tubular network footprint from small fragmented mitochondrial footprint in HD (Q74) significantly. d. RT-qPCR represents the expression patterns of fission (DNM1L/DRP1, FIS1 and MFF) and fusion (MFN1) genes showing significant upregulation of fission genes in HD condition, while C-5 treatment reverts the expression. C-5 treatment enhances expression of MFN1 significantly in HD condition. For the bar plot, data represents mean ± SD of three independent experiments (n = 3). For all graphs, p-values are expressed relative to two groups (marked with line). *p < 0.05, **p < 0.005, and ***p < 0.0005 indicate statistically significant difference between two groups estimated via paired t-test. e. Western blot analysis for selected fission/fusion proteins confirms similarity with gene expression. C-5 enhanced the expression of the fusion proteins (MFN1/2) while reducing the expression for fission proteins (DRP1, FIS1). The bar graph below the Western blot image shows the quantification of fusion/fission protein abundance relative to β-tubulin and relative to the DMSO sample. For the bar plot, data represents mean ± SD of three independent experiments (n = 3). See also the SI for Fig. S3. DMSO – control treated with solvent DMSO; C-5 – target molecule; TFM – teriflunomide.

Fig. 3

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During validation experiments, we also utilized the pARiS system for transiently expressing Q23/Q100 repeats in Neuro2a (N2A) cells. The pARiS plasmids were a generous gift from the Frédéric Saudou lab. Transient expression was performed using a jetPRIME kit as per the previously described protocol.22 Briefly, Neuro2a cells were cultured under standard cell culture conditions at 37 °C with 5% CO2 in DMEM supplemented with 10% fetal bovine serum. At 70% confluency, cells were transiently transfected with respective plasmids using a jet PRIME transfection kit (Cat. No: 101000027/0.1 ml) and incubated for four hours, then replaced with new media. For small molecule treatment, C5 and TFM were added to the new media and incubated for 48 hours. Finally, cells were collected and used for expression analysis (Fig. 3a).22 All cell lines tested negative for mycoplasma contamination using the EZdetect PCR kit (HiMedia Laboratories Private Limited) (Fig. S1b and c). We will use “control” and “HD” to denote Q23 and Q74/Q100 expressing cells, respectively.

2.4. Cell viability assay and IC50 determination

Cell viability assays were conducted using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. The MTT assay protocol involved incubation of the cells with the MTT reagent (HiMedia Laboratories Private Limited, India) for three hours in a 37 °C incubator. The entire medium was then discarded, and DMSO was added to dissolve formazan crystals. Cell viability was quantified by measuring the absorbance at 570 nm in a plate reader (Spectramax iD3, USA). To assess the cytotoxic effects of 4-(trifluoromethyl) isoxazoles and their derivatives on Q23 and Q74 cell lines, 10 000 cells were seeded in 96-well plates and allowed to attach for 24 hours. At 60% confluency, doxycycline was added to induce PolyQ HTT formation, followed by adding the test compounds at various concentrations (10 nM, 100 nM, 1 μM, 10 μM, 100 μM) for 48 hours. MTT assays were performed after 48 h, as indicated above. The experiment was performed in triplicate with minimum three technical replicates for each test condition and the IC50 values were calculated using a Hill-slope model.23X values were transformed using X = log(X). The values were then normalized to 100, and IC50 values were calculated using non-linear regression (curve fit). Data analysis and visualization were performed using GraphPad Prism, and the results were shown as a dose–response curve.

2.5. Confocal live imaging and mitochondrial morphology analysis

Cells were plated in confocal dishes at a density of 1.8 × 106 cells per dish and incubated at 37 °C with 5% CO2. At 60–70% confluence, the cells were induced with doxycycline (1 μg ml−1) for 6 hours. This induction step was followed by treatment with small molecules at their calculated IC50 values for 48 hours. Before imaging, the confocal dishes were transferred to a temperature- and CO2-controlled chamber attached to a Leica confocal microscope. The cells were stained with Mitotracker Red (100 nM, Invitrogen, Thermo Scientific) for a period of 5 minutes at 37 °C. This staining step allowed for the visualization of the mitochondria within the cells. Fluorescence images were captured using the Leica confocal microscope with excitation/emission settings of 581 nm/644 nm for Mitotracker Red and 475 nm/509 nm for GFP. Bright-field images were also acquired to provide reference for the fluorescence images. Multiple fields were imaged using identical settings to ensure consistency and accuracy. To quantify mitochondrial network morphology, confocal fluorescence microscopy images were analyzed using the MiNA (Mitochondrial Network Analysis) plugin in ImageJ/Fiji. From each image, a single representative cell was selected and converted to 32-bit grayscale. The image scale was calibrated using the ‘Set Scale’ function to convert pixel dimensions to microns based on microscope metadata. Image pre-processing was performed within the MiNA workflow to enhance quality and minimize noise, following the recommended protocol: an unsharp mask (radius = 1–2 pixels, mask weight = 0.6), contrast enhancement using CLAHE (block size = 127, histogram bins = 256, maximum slope = 3), and a median filter (radius = 1 pixel). These steps improved the signal-to-noise ratio and edge definition of mitochondrial structures. The processed image was then analyzed using MiNA's analysis tool, which generates a CSV output containing quantitative parameters. Different features of mitochondria were analyzed using MiNA as per the developer's (StuartLab, https://github.com/stuartlab) instruction24 using the same number of cells (n = 16) from each condition indicated. Key metrics such as mitochondrial footprint (total area occupied by mitochondria) and summed branch length (cumulative length of all mitochondrial branches) were extracted to assess mitochondrial content and network complexity, respectively. The data points were plotted through a box–whisker plot where a paired t-test was performed between two conditions using the R-based analysis as described in the statistical section. These parameters were used to infer alterations in mitochondrial morphology under experimental conditions.

2.6. Determination of mitochondrial membrane potential and ROS

Control and HD cells were seeded in 12-well plates and incubated for 24 hours under appropriate growth conditions. Following the initial incubation, doxycycline was added at a 1 μg ml−1 concentration to induce PolyQ expression. After 6 hours of doxycycline induction, cells were treated with IC50 dosage of the selected compounds. Cells were further incubated for 48 hours to allow for sufficient treatment exposure. Following the 48-hour incubation period, cells were trypsinized, washed with phosphate-buffered saline (PBS), and resuspended in PBS containing 5 μM MitoSOX Red (M36008, Invitrogen). The cell suspension was then incubated in the dark for 15 minutes at 37 °C to allow for MitoSOX Red staining. Subsequently, the fluorescence intensity was measured using a BD-FACSAria flow cytometer at 488 nm and 585 nm for excitation and emission wavelengths. The fluorescence of MitoSOX Red in compound-treated control and HD cells was assessed compared to the negative control (1% DMSO-treated cells). The increase or decrease in fluorescence intensity was determined, and the data (intensity from n = 10 000 cells) was plotted using FlowJo software to visualize the changes in mitochondrial ROS levels. Statistical differences were calculated using median intensity (ROS amount) from three independent biological experiments (n = 3).

To measure mitochondrial membrane potential, cells were stained with 100 nM tetramethylrhodamine ethyl ester (TMRE) for 20 minutes at 37 °C, after TMRE staining as described earlier. Briefly, cells were trypsinized after removal of media and PBS wash, followed by collection through centrifugation. Cells were resuspended in PBS after 2 times PBS wash. Cells from different treatment conditions (positive control, negative control, and treated with small molecules) were subjected to BD-FACSAria to measure the fluorescence intensity of TMRE to assess mitochondrial membrane potential. To prepare the positive control for the membrane potential estimation experiment, cells were treated with 10 μM carbonyl cyanide-p-trifluoromethoxy phenylhydrazone (FCCP) for 1 hour at 37 °C before collection. The fluorescence of TMRE in compound-treated control and HD cells (n = 10 000) was assessed in comparison to the negative control (1%-DMSO-treated cells). The data was analyzed using FlowJo software to visualize changes in the membrane. Statistical differences were calculated using the median intensity of three independent biological experiments (n = 3).

2.7. RT-qPCR and Western blotting

Total RNA was isolated from control and HD cells using the Trizol method (RNAiso Plus-Takara), while the quantity and quality of RNA were determined using NanoDrop (Thermo Scientific). cDNA was prepared using iScript™ cDNA Synthesis (Bio-Rad). RT-qPCR was carried out in a CFX96-real-time PCR machine (Bio-Rad) using SYBR green master mix (TB Green, Takara) with specific primers for DNM1L (DRP1), MFN1, MFF, FIS1, and PGC1α-Exon1a25 (primer details in Table S3 under SI). Three technical replicates for each gene were determined from three independent biological experiments. Relative gene expression was quantified using the ΔCt method for all the primers using beta-actin as the normalizing gene. We used the ΔΔCt method to determine the fold changes between treatment and control groups where fold change = 2−ΔΔCt, where ΔCt (cycle difference) = Ct (target gene) − Ct (control gene) and Δ(ΔCt) = Ct (treated condition) − Ct (control condition).26

For Western blotting, proteins were extracted using RIPA lysis buffer with continuous agitation for 1 minute and left on ice for 5 minutes. The lysate was centrifuged to remove cell debris, and the supernatant was collected. The concentration of the protein was measured using a Pierce BCA protein assay kit (Cat No 23227, Thermo Scientific). Total proteins (75 μg) were separated by SDS-PAGE and transferred to a polyvinylidene fluoride (PVDF) membrane (Immobilon PVDF, IPVH00010, Millipore). The membrane was blocked in 8% fat free skimmed milk for one hour, followed by primary antibody incubation overnight at 4 °C with agitation. The following antibodies were used: DRP1 (1 : 1000, ABclonal, A21968), mitofusin-1/MFN1 (1 : 1000, ABclonal, A21293), mitofusin-2/MFN2 (1 : 1000, CST, D2D10), DHODH (1 : 1000, ABclonal, A6899); β-tubulin (1 : 1000, ABclonal, AC008). The next day, after washing the blots with Tris-buffered saline with 0.1% Tween 20 detergent (TBST), a secondary antibody was added and incubated for one hour at room temperature with agitation. The secondary antibody was an anti-rabbit IgG, HRP-linked antibody (1 : 10 000, CST, 7074S). The signal intensities of the bands were captured using the Fusion Pulse gel documentation system (Eppendorf, USA). For all the proteins, expression was measured from three independent biological experiments (n = 3). Raw ImageJ software was used to quantify the band intensities and used for plotting the quantitative data. Using ImageJ software, the protein expression was measured and normalized to the protein intensity of the housekeeping gene.

2.8. Apoptosis assay

Control (Q23) and HD (Q74) cells were seeded in 12-well plates and incubated for 24 hours. After 24 h, the cells were incubated for six hours following the induction of doxycycline (1 μg ml−1). The cells were treated with the IC50 dosage of our target compound and TFM separately. The cells were then incubated for 48 hours. After the treatment period, apoptosis was measured using Annexin V-Elab Fluor Red 780/propidium iodide (PI) assay (E-CK-A23, Elab Bioscience, Texas, USA) following the manufacturer's protocol. Briefly, cells were collected and washed in PBS. An appropriate number of cells (1–5 × 105) were then suspended in 100 μL of 1× annexin V binding buffer. The supernatant was discarded, and the cells were rewashed with PBS to remove any remaining debris. The cells were then gently resuspended and counted to determine the total number of cells. The cell suspension was then split into tubes, with 1–5 × 105 cells per tube and centrifuged at 300 × g for 5 minutes to pellet the cells. The supernatant was discarded, and the cells were washed with PBS. The cells were then resuspended in 100 μL of 1× annexin V binding buffer. 2.5 μL of annexin V-Elab Fluor Red 780 and 2.5 μL of PI were added to respective tubes followed by incubation at room temperature for 15 minutes in the dark. After incubation, 400 μL of 1× annexin V binding buffer was added to each tube, and the samples were gently mixed to dilute them. The cells (n = 10 000) were then analyzed immediately using a flow cytometer (BD-FACS Aria) to capture the intensity of annexin V-Elab Fluor Red 780 (625/765 nm) and PI (488/610). The data, represented as dot plots, were then analyzed using BD software to determine the changes in apoptosis under different treatment conditions.

2.9. Molecular docking using Schrödinger Suite 2025-1

Preparation of protein crystal structure using protein preparation wizard:

The initial coordinates of the huntingtin protein were obtained from the RCSB protein databank (PDB), which was resolved using NMR with accession ID 6N8C.27 An insightful study by Yoo Ji-Na et al. described the limitation of the XRC due to the intrinsic flexibility and dynamic structural transitions of HttEx1 and demonstrated concentration-dependent structural transitions and their relationship to aggregation via NMR.28 We thus used NMR-resolved structure for our docking study. The protein preparation wizard of Glide software (Schrödinger Suite 2025-1) was used to assign possible missing hydrogen atoms in the PDB structure, and other options were left as default. Full energetic optimization was performed in the final refinement step using the OPLS4 forcefield. Ligand minimization: the two compounds C-5 and curcumin were imported in the Maestro module, and further optimization of ligands was carried out using LigPrep with a biological pH of 7.4 ± 2. LigPrep generated the 3D structure of ligands in their lowest energy conformation using the OPLS4 forcefield, and other parameters were left default. Grid generation and ligand docking: following the minimization of ligands and proteins, the grid was generated using the entire protein. The “Receptor Grid Generation” module was used by setting the coordinates of X: 0.002, Y: 0.12, Z: −0.12 with a grid size of 25 Å. The Maestro module's Glide application was used for ligand docking with standard precision (SP). The ligands C-5 and curcumin were docked into the huntingtin protein grid to determine their binding pose and docking score.

Docking using AutoDock Vina: molecular docking study of C-5 and curcumin with huntingtin proteins. Preparation of protein and ligands: the structure coordinates of the C-5 molecule and curcumin molecule were obtained from the PubChem Database in SDF file format. The ligands were prepared for docking using Open Babel 2.4.1 and AutoDock Tools 1.5.7. The initial coordinates of the huntingtin protein (PDB ID: 6N8C) were obtained from the RCSB PDB database. The structure was optimized using AutoDock Vina software by removing all water molecules and adding Kollman charges and nonpolar hydrogens. Preparation of huntingtin protein grid preparation: the grid box center coordinates and dimensions for the huntingtin protein were set as follows: X = 0.002, Y = 0.011, Z = 0.006, and grid dimensions: 40 × 40 × 40. Molecular docking: molecular docking of the prepared C5 and curcumin molecules with the huntingtin protein was performed using AutoDock Vina. The ligand–protein complex with the lowest binding energy and RMSD of 0.00 was selected as the optimal interaction position. Molecular interaction studies were performed using Discovery Studio Biovia. The SwissDock webserver was used to confirm the results from AutoDock.

2.10. Statistical analysis

Post-processing data analysis and statistical analyses were done in R version 4.3.3 (https://www.R-project.org) using specific packages. The CRAN R packages: “tidyverse” and “devtools” were used for basic data manipulation, visualization, and statistical analysis. These packages were allowed access to ‘rstatix’ (statistical calculations), ‘ggpubr’, ‘ggplot2’, ‘dplyr’, ‘tidyr’, ‘readr’, ‘purrr’, ‘tibble’ libraries for data analysis and visualization. Statistical significance was calculated by paired t-test with Holm's correction.29P-Values of less than 0.05 were considered statistically significant; P-value significance was denoted by asterisk (*) (* represents the following, * = p < 0.05; ** = p < 0.001; *** = p < 0.0001; NS, non-significant).

3. Results

3.1. In silico screening identified potential small molecule derivatives

We first employed an in silico screening using Swiss-ADME to assess the drug-likeness of our compounds based on several defined criteria, including their physicochemical properties (Fig. 1a). We considered passive gastrointestinal absorption (human intestinal absorption; HIA) and BBB penetration as principal criteria in the in silico screening. As our final goal is to identify new small molecules that should cross the BBB, we included FDA-approved TFM, as a positive control, to ensure that the new molecules should be clustered together with TFM. This in silico screening, thus, helped us to reduce pharmacokinetics-related failure during in vivo testing and clinical trials at a later stage.

Fig. 1. Design, synthesis, screening and characterization of small molecules. a. Schematic representation showing the design, in silico filtering, synthesis, and characterization (NMR & HR-MS) of target small molecules. SWISS-ADME based in silico analysis identifies the five best small molecules suitable for their drug-likeness based on multiple parameters including blood–brain barrier (BBB) permeability. Five compounds (C-1 to C-5) along with FDA-approved drug teriflunomide (TFM) are synthesized, characterized and subjected to downstream in vitro screening. b. Bioavailability radar chart for C-5 and TFM shows the physiochemical properties of isoxazole derivatives C-5 and TFM. The pink zone represents physiochemical space for oral bioavailability, while the red line indicates oral bioavailability. c. BOILED-Egg plot of isoxazole derivatives (C-1 to C-5) and TFM represents the passive gastrointestinal absorption (HIA) and blood–brain barrier (BBB) properties of molecules in WLOGP-versus-TPSA. See also the SI for Fig. S1, NMR data and radar charts of other compounds.

Fig. 1

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Out of our 42 compounds (see Table S1 under SI), five compounds (C1–C5) met the filtering criteria of BBB penetration. These five compounds adhered to Lipinski's rule of five30 (Fig. 1b and S1a, Table S2). Furthermore, these compounds also passed Ghose, Veber, Egan, and Muegge rules for drug-likeliness. These molecules have a molecular weight (MW) ranging from 227–271 g mol−1, fulfilling the recommended size (range between 150–500 g mol−1) for a good drug candidate (Table S2). The topological polar surface area (TPSA) of these compounds was also within the recommended range of 20–130 Å (Table S2). Lipophilicity is an essential indicator of permeability across cell membranes and the oral bioavailability of drug molecules.31 The five shortlisted drugs were within the standard range (between −0.7 and +5), signifying their suitability to cross the cell membrane. Importantly, in the bioavailability diagram (radar chart), all the derivatives are highly comparable with the FDA-approved TFM (Fig. 1b, also see Fig. S1 under SI).

We then assessed our molecules based on the Brain Or IntestinaL EstimateD permeation predictive model (BOILED-Egg) using Swiss-ADME.19,20 The generated graph between WLOGP (considering lipophilicity) and TPSA identified the most suitable small molecules to potentially cross the BBB. Out of the forty-two, five small molecules were found to be BBB permeable with TPSA < 80 Å and WLOGP ≦ 6. All these five compounds were inside the yellow zone with TFM, indicating their ability to be good BBB permeants and non-p-gp substrates (i.e., cells will not efflux the small molecule out) (Fig. 1c, Table S2 under SI).

Overall, the in silico screening allowed us to identify five potential small molecules to proceed with our in vitro test to understand their effect over mitochondrial dysfunction in HD condition.

3.2. Cytotoxicity and structure–activity-relationship (SAR) analysis of the synthesized target molecules (TMs)

First, we determined the cytotoxicity of our synthesized molecules (C1–C-5, Fig. 2a) over control and HD cells. Among these five molecules, C-5 was required at the lowest amount to reach its IC50 (Fig. 2b and c and S2 under SI). With respect to the obtained IC50 values of C-1 to C-5 against HD cells, we tried to understand the structure–activity-relationship (SAR) (Fig. 2b). With the increase of the electron density of the aryl ring at the 3rd position of 5-methyl-4-(trifluoromethyl) isoxazoles (C-1 to C-3), the toxicity increased linearly (entries one to three) with respect to their sensitivity towards the drug amount, whereas cell viability was inverse (C-3 to C-1). In the absence of a –CH3 functional group on the 5th position of isoxazoles (C-4vs.C-1 and C-5vs.C-3), the cell toxicity increases (entry four vs. entry one, and entry five vs. entry three) subsequently in the presence of methyl group on the 5th position of isoxazoles, the cell viability increases. Interestingly, in the presence of a methoxy (–OCH3) functional group in the para-position of the aryl-substituent present at the 3rd position of 4-(trifluoromethyl) isoxazole (C-5vs.C-4), the cytotoxicity increases 14 times (entry five vs. entry four). All observations were also the same for control cells. Compared to TFM, except for C-1, all of these molecules required lower concentration to reach similar cytotoxicity, and C-5 required the lowest concentration to achieve the maximum effect compared to other newly synthesized derivatives. To decipher the mechanism of its action, we thus primarily focused on the isoxazole derivative, C-5.

3.3. Small molecules act as mitochondrial fusion agonists, restoring the mitochondrial network in HD cells

The striking phenotypes of mitochondrial dysfunction are excessive fragmentation and altered mitochondrial network. Interestingly, striatal neurons which degenerated in HD condition exhibited severe mitochondrial fragmentation.10,13,32 Conversely, conditions that improve mitochondrial fusion are known to be beneficial in protecting cells from apoptosis by improving mitochondrial function.33 Thus, we first tested whether our targeted molecules had any effect on improving mitochondrial dynamics.

We used confocal imaging to visualize mitochondrial morphology and distribution using mitochondria targeted fluorescent dye, MitroTracker Red, in the presence and absence of the target molecule. Like earlier studies our in vitro models (Fig. 3a) exhibited the characteristic fragmented or punctate morphology in HD condition (Fig. 3b). Interestingly, C-5 restored the elongated mitochondrial morphology with subsequent increase in mitochondrial footprint (total area occupied by mitochondria) in HD condition. C-5 even worked better than the known FDA drug (Fig. 3c). Like mitochondrial footprint, we also estimated mitochondrial branch length changes through the MiNA image analysis tool. Branch length data also supported the same observation, i.e.C-5 effectively restored the branch length which was altered in HD cells (Fig. S3d). Together these observations confirmed the fusion agonist property of the newly synthesized molecule C-5. Though we mainly focused on C-5 for detailed characterization, it is also noteworthy to mention that other derivatives (C-1 to C-4) also showed enhanced fusion, as evidenced by confocal imaging through MitroTracker red (Fig. S3a under SI).

3.4. C-5 mediated downregulation of fission genes and enhanced expression of fusion genes improved the mitochondrial network

The balance between mitochondrial fission and fusion is regulated by a coordinated expression of fission/fusion proteins depending on cellular conditions. Either enhanced expression of fission genes or reduced expression of fusion genes results in fragmented mitochondria. Previous studies reported increased expression and activity of DRP1 or impaired expression of mitochondrial fusion and biogenesis in HD.10,13 To understand the underlying mechanisms of our fusion agonist, we systematically dissect whether C-5 mediated restoration was caused by the inhibition of mitochondrial fission or the promotion of mitochondrial fusion.

Initially, we examined the expression profile of a set of major fission genes (DRP1, FIS1, MFF) in our cellular models. We observed enhanced expression of fission genes under HD conditions (PolyQ74) corroborating the enhanced fission observed in confocal imaging (Fig. 3d). Similar to the observed confocal images, treatment with C-5 restored elongated mitochondrial dynamics by reducing the expression of fission genes (Fig. 3d). Conversely, we found that expression of fusion gene, MFN1, was significantly downregulated in HD conditions, whereas C-5 treatment significantly enhanced MFN1 expression (Fig. 3d). Additionally, we checked the protein expression pattern of selected fission and fusion proteins which recapitulated the similar expression pattern observed at the transcript level (Fig. 3e). We also found the similar expression pattern of fission–fusion genes by using transiently expressing plasmid Q23 or Q100 in neuronal cells (neuro2a) supporting its robust activity irrespective of background of the cells (Fig. S3b). C-5 treatment rescued MFN1 expression, confirming its efficacy across cell lines (Fig. S3c). The reduction of fission genes was also observed. These results demonstrated that C-5 is a compelling fusion agonist that significantly mitigated the PolyQ-mediated fragmentation as shown using confocal imaging (Fig. S3d) via enhanced expression of fusion genes and decreased fission genes.

3.5. C-5 treatment restored mitochondrial health in HD cells via improving membrane potential and reduction of mitochondrial ROS and cell death

For any neurological diseases including HD, disruption of bioenergetics is one of the hallmarks of the mitochondrial dysfunction. Bioenergetic stress is characterized by the loss of mitochondrial membrane potential, which may release apoptotic factors that lead to neuronal death. Multiple studies using HD patients, mouse models, and neuronal HD cell lines have reported a decrease in mitochondrial membrane potential with enhanced sensitivity to calcium (Ca2+) induced permeability transition.34,35 Mitochondrial fragmentation is also linked to decreased membrane potential (ΔΨm).36 Therefore, we first estimated the change in membrane potential in the presence and absence of our target molecule (C-5). Like improved fusion, C-5 significantly restored mitochondrial membrane potential (Fig. 4a), indicating a transition towards healthy mitochondria. Interestingly, the loss of mitochondrial membrane potential (ΔΨm) is coupled with oxidative stress. Moreover, classical studies from postmortem brains of HD patients found a significant increase in the level of oxidative damage, whereas different HD model studies implicated oxidative stress as a key player in the pathogenesis of HD.37,38 In HD conditions, production of excessive ROS, elevated expression of antioxidant enzymes, and increased cell death are observed.39 Similarly, activation of the intrinsic mitochondrial apoptotic pathway was observed in HD patients and in R6/2 HD mice.40 Conversely, mitochondrial elongation is known to protect against apoptosis.41,42 Considering the above-described fact, we expected to observe similar pathogenic conditions in the HD cellular model. We confirmed a significant increase in mitochondrial ROS using the fluorescence red dye indicator, MitoSOX Red (Fig. 4b). Similarly, increased apoptosis was confirmed in HD cells (Fig. 4c). If C-5 induced mitochondrial fusion restored mitochondrial homeostasis, then we should observe reduction of ROS and apoptotic cell death. Indeed, C-5 reduced ROS and apoptosis significantly, corroborating improved mitochondrial health (Fig. 4b and c). Thus, C-5 is a potent small molecule that works against mitochondrial dysfunction and is effective against HD conditions.

Fig. 4. Restoration of mitochondrial membrane potential, reduction of ROS accumulation and reduced apoptosis in the presence of C-5. a. TMRE-based measurement indicates loss of mitochondrial membrane potential (ΔΨm) in HD (Q74) cells. C-5 treatment restores the membrane potential. Both HD (Q74) and control (Q23) cells are treated with FCCP (positive condition-membrane potential lost), negative (DMSO-untreated), C-5 (treatment), and TFM (FDA drug) and corresponding fluorescence intensities are represented by histogram plots (left). Bar plot of median fluorescence intensity is represented as mean ± SD from three independent experiments (n = 3). P-Values between two groups are estimated via paired t-test with *p < 0.05, **p < 0.005, and ***p < 0.0005. b. C-5 reduces mitochondrial reactive oxygen species (mtROS) in HD cells. Histogram plot of MitoSOX fluorescence shows ROS accumulation in both control and HD cells under different conditions (untreated control, C-5, and TFM). Bar plot of median fluorescence intensity is represented as mean ± SD from three independent experiments. P-Values between two groups are estimated via paired t-test with *p < 0.05, **p < 0.005, and ***p < 0.0005. c. Apoptosis measurement using annexin/PI double staining assay shows significant reduction of cell death after C-5 treatment. Dot plots represented n = 10 000 cells where each dot corresponds to a single cell. Dot plots with four quadrants (Q1 – necrotic cells, Q2 – late apoptosis, Q3 – live cells, Q4 – early apoptotic cells) exhibit the better rescue effect of C-5 over TFM.

Fig. 4

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3.6. Mitochondrial fusion agonist C-5 acts through PGC1α

So far, we have demonstrated that C-5 significantly increased fusion gene expression with subsequent improvement of mitochondrial homeostasis. It restored membrane potential and reduced ROS accumulation and apoptosis. We observed C-5 mediated transcriptional level changes in fusion genes. PGC-1α played a critical role in mitochondrial biogenesis by directly regulating MFN1/2 gene transcription.43 Transcription of MFN1 is known to be activated through the PGC-1α–TFAM (transcription factor A, mitochondria) axis.44 An earlier report showed that mutant HTT aggregates can inhibit transcription of PGC-1α.45 In our in vitro system HD cells exhibited decreased expression of PGC-1α and C-5 treatment restored the expression of PGC-1α as well as TFAM indicating the activation of the PGC-1α signaling axis for mitochondrial fusion (Fig. 5a and b).

Fig. 5. C-5 enhances PGC1α expression by potential removal of mutant HTT mediated repression through direct interaction. Bar graph represents the enhanced expression pattern of a. PGC1α and b. TFAM from three independent experiments. C-5 treatment of HD cells enhances expression of PGC1α, a co-factor for transcriptional activation of fusion genes. Data are mean ± SD (n = 3). P-Values are expressed relative to the untreated group where *p < 0.05, **p < 0.005, and ***p < 0.0005 indicate statistically significant effects of C-5. c. Receptor–ligand docking analysis (2D) of compounds – HTT (PDB-6N8C) with C-5 (left) and HTT (PDB-6N8C) with curcumin (positive control known to interact with HTT, right) shows strong association. See also the SI for Fig. S4.

Fig. 5

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We also explored whether fusion–fission gene expression is controlled through the DHODH mediated pathways. Earlier studies on cancer and neuronal cell lines observed reduced expression of DHODH by TFM or its precursor, leflunomide, linked with DRP1 or MFN expression.16,46,47 We found no significant decrease of DHODH at the transcript as well as protein level (Fig. S4a). Unlike TFM/LFM, C-5, thus, does not control DRP1expression via the DHODH mediated pathway. Thus, C-5 mediated control of fusion–fission genes is potentially through PGC-1α but not through DHODH.

As C-5 treatment enhanced PGC-1α transcription, we checked the in silico probability of HTT protein and C-5 interaction. Our in silico docking study using C-5 and huntingtin protein predicted its binding capacity. Receptor–ligand docking analysis via standard precision (SP) showed that compound C-5 represented a docking score of −6.092 kcal mol−1, and the docking score of positive control curcumin was −5.398 kcal mol−1 (Fig. 5c, S4 under SI). Furthermore, the investigation of binding interaction revealed that C-5 had hydrophobic interactions with LEU6; MET7: PHE10 in the D chain; PHE10; MET7; LEU6 in the A chain; MET7; PHE10 and positively charged interaction with LYS14 in the C chain. Curcumin had similar hydrophobic interaction to the C-5 molecule but included a few more interactions, such as THR2 of the A chain and LYS14 of the C chain with hydrogen bonding interactions (Fig. 5c and S4b). This result indicated the potential interaction of C-5 with HTT.

Overall, C-5 mediated transcriptional activation effectively controls downstream genes responsible for improving mitochondrial fusion and balancing the mitochondrial dynamics to maintain mitochondrial homeostasis in HD conditions.

4. Discussion

Mitochondrial dysfunction with altered dynamics and severe fragmentation is a major pathophysiological event observed in neurodegenerative diseases. In HD, electron microscopic analysis of lymphoblasts from HD patients revealed abnormal clustering of mitochondria,48 indicating defects in the ultrastructure and mitochondrial network. Similarly, in vitro cells expressing mutant huntingtin displayed severe fragmentation of the mitochondrial network, alteration in the cristae structure, reduced ATP levels, and enhanced apoptosis. Further study identified that the death of HD neurons is linked with the mitochondrial fission protein DRP1, which plays an essential role at the crossroad between modulation of mitochondrial structure and dynamics.31 In HD, neuronal death has been found to be linked with enhanced activity or expression of fission proteins DRP1, impairing mitochondrial fission–fusion balance.10 Conversely, downregulation of pro-fusion proteins (MFN1, MFN2, OPA1) was also observed in HD patients. Thus, improving mitochondrial fusion or decreasing fission will be an effective way to restore mitochondrial health and treat neuronal diseases.

Indeed, mitochondrial fragmentation and dysfunction in HD mouse and cellular models were found to be mitigated with genetical correction through expressing the fusion genes OPA1 and MFN1 or by reducing DRP1 activity and translocation to mitochondria.49 Overexpression of the fusion gene (MFN2) in HeLa cells harboring PolyQ74 repeats showed a reduction of mitochondrial fragmentation with increased cell survival.9 Similarly, increasing fusion indirectly by knocking down of fission protein was also found to be beneficial in preventing neuronal death.50 In addition to genetic approaches intervention with small molecules showed its potential as a therapeutic intervention for neurodegenerative diseases. A selective inhibitor of DRP1, P110-TAT, was reported as an effective molecule against HD condition by improving mitochondrial dysfunction through the inhibition of mitochondrial fission (fragmentation).51 Similarly, mitochondrial fusion activators, such as teriflunomide (TFM, an FDA-approved drug), have shown increased fusion through decreased DRP1 expression and function, offering promising therapeutics for cancer and other conditions.16,52 Although inhibition of fission is promising, long-term inhibition can lead to increase mutation in mitochondrial DNA (mtDNA), potentially causing mitochondrial dysfunction.53 Thus, a fusion agonist has an advantage over inhibition of fission. A compelling study by Guo et al.17 demonstrated the therapeutic potential of a mitochondrial fusion agonist, S89, which activates a key outer mitochondrial membrane protein, MFN1. This compound was shown to protect against mitochondrial damage and ischemia/reperfusion injury in a mouse model. It also restored mitochondrial and cellular function impaired by mitochondrial DNA mutations, oxidative stress (e.g., paraquat), ferroptosis inducers (e.g., RSL3), and CMT2A-associated mutations by enhancing endogenous MFN1 activity. While MFN1 plays a critical role in mitochondrial fusion, both MFN1 and MFN2 are required for the complete and efficient execution of the fusion process. Importantly, these two mitofusins have distinct yet complementary functions, as demonstrated in previous studies.54,55 Though the effect of S89 is yet to be tested in other neuronal diseases including HD its effect might be limited due to selective activation of MFN1 only. Currently, there are limited drugs (FDA-approved drugs, which are primarily vesicular monoamine transporter 2 (VMAT2) inhibitors) used against chorea (involuntary movements) to treat HD. Strikingly, there is no drug available to prevent striatal neuron degeneration. Therefore, it is crucial to develop new therapeutic avenues in treating HD. Towards this effort, we designed, synthesized, and validated the effectiveness of a novel molecule, a mitochondrial fusion agonist, that can reduce mitochondrial dysfunction and apoptosis even in HD condition. Our synthesized molecule increased fusion of mitochondria through transcriptional activation of fusion genes (MFN1/2) and enhanced cell survivability even in the presence of polyglutamine (PolyQ) repeats, a hallmark of the HD condition. We utilized an in-house developed novel synthesis method to create a set of mitochondrial fusion agonists. Utilizing in silico followed by in vitro testing we confirmed C-5 as a promising mitochondrial fusion agonist effective against HD condition.

The PGC1α–TFAM axis which controls fusion as well as mitochondrial biogenesis is linked with HD pathology.56 Several other studies indicated that PGC1α is crucial cofactor that maintains mitochondrial biogenesis and fusion by interacting with transcription factors like NRF1. The PGC1α/NRF axis regulates the expression of nuclear-encoded mitochondrial genes, including oxidative phosphorylation and other antioxidant genes.57 Additionally, the PGC1α signaling pathway influences the expression of a key enhancer TFAM, which governs the expression of mitochondrial DNA encoded genes.56,58 An intriguing study by Cui et al. demonstrated that mutant HTT (mHTT) aggregates suppressed PGC1α transcription, leading to an altered expression of fusion gene and mitochondrial biogenesis.45 In this study, we observed transcriptional upregulation of PGC1α and TFAM after C-5 treatment. Additionally, we also found that C-5 led to an increase in the transcriptional upregulation of fusion gene (MFN1/2) expression and decrease in DRP1expression. Earlier Dabrowska et al. (2015) demonstrated that PGC1α alone can regulate mitochondrial dynamics, via controlling DRP1 and MFN1expression levels. Neuronal cells overexpressing PGC1α exhibited significantly increased expression of MFN1 (mRNA) with subsequent decrease of DRP1 (mRNA).59C-5 treatment recapitulated the similar expression pattern, where, along with increased PGC1α expression the fusion gene (MFN1) expression was increased with subsequent decrease of fission gene (DRP1) expression (Fig. 3d and e). Taken together, our detailed analysis revealed that HD cells treated with C-5 restored transcription of PGC1α and TFAM strongly supporting the potential activation of the PGC1α pathway by C-5 treatment. Changes in mitochondrial dynamics were observed to be regulated through DHODH pathways.16,46 We also examined whether alteration of pyrimidine biosynthesis was activated via decreased expression of mitochondrial enzyme DHODH. Since C-5 treatment showed unaltered DHODH expression, it suggests that the reduced expression of DRP1 is potentially via PGC1α as discussed earlier (Fig. S4).

Finally, the docking study exhibited potential affinity between HTT and C-5, leading us to propose that C-5 mediated sequestrations of mutant HTT results in the release of inhibition on PGC1α. The upregulation of PGC1α subsequently activated enhanced expression of fusion genes as well as transcription of TFAM. The expression of MFN1/2 restored mitochondrial fusion, while TFAM contributed to mitochondrial biogenesis. Overall, the restoration of the mitochondrial network improved cell viability even under HD condition. Our results suggest that C-5 is an effective mitochondrial fusion agonist and underscore how enhanced fusion can serve as a potential therapeutic strategy against HD (Fig. 6).

Fig. 6. Working model of C-5 mediated rescue of mitochondrial function and cellular viability. PGC1α regulates mitochondrial gene expression to maintain energy homeostasis. In HD, mHTT oligomers repress PGC1α transcription by interacting with other transcriptional regulator proteins (CREBP, TAF4, etc.) (Cui et al., 2006).45 Inhibition of PGC1α limits expression of nuclear and mitochondria encoded genes controlling mitochondrial biogenesis, fusion, antioxidant genes. Together it results in mitochondrial fragmentation, imbalance in the fission–fusion cycle, accumulation of ROS, drop in membrane potential, and enhanced apoptosis – hallmarks of HD pathology. Interaction of C-5 with HTT leads to removal of repression of PGC1α transcription which in turn enhances expression of downstream genes including fusion genes MFN1/2. Enhanced fusion restores mitochondrial dynamics and mitochondrial membrane potential with subsequent reduction in ROS accumulation. Restoration of mitochondrial homeostasis improves cellular viability.

Fig. 6

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Despite the therapeutic potential of our findings, the current study has some limitations. For instance, predictions of Swiss-ADME prioritize the passive transport over active transport. Similarly, an immediate in vivo testing is essential in near future to establish its efficacy at the organism level. Live imaging of mitochondria in mice might be beneficial for evaluating direct effects. Also, the current study used the NMR solved structure to test the binding of the C-5 molecule with the HTT-Exon1 PolyQ region via docking. Conducting biophysical assays need to perform to assess the direct binding of C-5 and mutant HTT aggregates. The biophysical approach will provide more insight about its mechanism of action.

In summary, the present study demonstrates that (1) pharmacological promotion of mitochondrial fusion holds promise as a therapeutic strategy for HD; (2) C-5 mediated restoration of mitochondrial dynamics via transcriptional activation of fusion gene reduces apoptosis and ROS in HD conditions. These results confirm the beneficial role of mitochondrial fusion agonists and pave the way for in vivo and clinical trials in the future to validate this treatment strategy in HD patients.

Author contributions

S. M. conceptualized and supervised the overall completion. TC supervised the synthesis of the compounds. SM, TC, PK, VK, and PP contributed in writing the original draft of the manuscript. PK, VK, SM, PP, and TC performed experiments. VK, PK, and PV contributed in image analysis. All authors have read and agreed to the final version of the manuscript.

Conflicts of interest

There is no conflict of interest to declare.

Supplementary Material

MD-OLF-D5MD00345H-s001
MD-OLF-D5MD00345H-s002
MD-OLF-D5MD00345H-s003
MD-OLF-D5MD00345H-s004
MD-OLF-D5MD00345H-s005
MD-OLF-D5MD00345H-s006
MD-OLF-D5MD00345H-s007
MD-OLF-D5MD00345H-s008

Acknowledgments

We thank David C Rubinsztein's lab, University of Cambridge, UK, for providing Recombinant HEK293 cell lines (containing exon 1 of the human HTT gene, with either 23 CAG (control; Q23) or 74 CAG (HD; Q74) repeats, fused to GFP under the control of a doxycycline-inducible promoter). We also thank Prof Frédéric Saudou for providing the pARiS plasmids as a generous gift. S. M. acknowledges BITS-Pilani (Hyderabad Campus) for OPERA fellowship (ID -1022) and CDRF-II (C2/24/283), ICMR ad hoc research grant (Project ID 2021-13896), BITS-SPRKLE (SPARKLE/23/H30) initiative. V. K. acknowledges BITS-Pilani (Hyderabad Campus) for institutional doctoral fellowship and P. K acknowledges UGC (UGC 191620023107) for doctoral fellowship. PV acknowledges BITS-PILANI SPRKLE initiative for supporting undergraduate students research activities. The instrumentational facilities, at the central analytical laboratory (CAL) of BITS-Pilani, Hyderabad campus, are also gratefully acknowledged.

Data availability

Supplementary information: 1H NMR, 19F NMR, and HRMS spectra of C-1 to C-5, molecular structures of the designed 42 molecules with SWISS-ADME assessment of blood brain permiability, in silico pharmacokinetic assessment of C-1 to C-5, primer details, supplementary figures (S1–S4). See DOI: https://doi.org/10.1039/D5MD00345H.

All relevant data are within the manuscript and its ESI files.

Notes and references

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

MD-OLF-D5MD00345H-s001
MD-OLF-D5MD00345H-s001.pdf (4.6MB, pdf)
MD-OLF-D5MD00345H-s002
MD-OLF-D5MD00345H-s002.pdf (228.6KB, pdf)
MD-OLF-D5MD00345H-s003
MD-OLF-D5MD00345H-s003.pdf (134.1KB, pdf)
MD-OLF-D5MD00345H-s004
MD-OLF-D5MD00345H-s004.pdf (102.4KB, pdf)
MD-OLF-D5MD00345H-s005
MD-OLF-D5MD00345H-s005.pdf (269KB, pdf)
MD-OLF-D5MD00345H-s006
MD-OLF-D5MD00345H-s006.pdf (199.3KB, pdf)
MD-OLF-D5MD00345H-s007
MD-OLF-D5MD00345H-s007.pdf (336.4KB, pdf)
MD-OLF-D5MD00345H-s008
MD-OLF-D5MD00345H-s008.pdf (255.2KB, pdf)

Data Availability Statement

Supplementary information: 1H NMR, 19F NMR, and HRMS spectra of C-1 to C-5, molecular structures of the designed 42 molecules with SWISS-ADME assessment of blood brain permiability, in silico pharmacokinetic assessment of C-1 to C-5, primer details, supplementary figures (S1–S4). See DOI: https://doi.org/10.1039/D5MD00345H.

All relevant data are within the manuscript and its ESI files.

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