Therapeutic mechanistic study of novel indole derivatives as SIRTUIN3 modulators in Parkinson’s disease with in vitro evaluation

Abstract

SIRT3 modulates reactive oxygen species and helps to reduce oxidative stress, resulting in a neuroprotective effect in Parkinson’s disease. The work seeks to develop and synthesize novel, effective SIRT3 modulators, which will modify SIRT3 expression and generate the neuroprotective effect by downregulating reactive oxygen species. The synthesis of the new indole-based carboxamide derivatives IMW 1–16 was guided by molecular docking experiments with the SIRT3 wild-type protein. FT-IR, ¹H-NMR, ¹³C-NMR, and mass spectrometry analyzed the synthesized compounds. The efficacy of the compounds IMW 1–16 was assessed using in-vitro assays such as the MTT assay, neuroprotection assay, lactate dehydrogenase assay, superoxide dismutase assay, glutathione peroxidase assay, reactive oxygen species estimation, mitochondrial membrane potential, and real-time polymerase chain reaction estimation. The synthesized compounds IMFW-1, IMTW-5, and IM24DCW-16 were determined to have the lowest cytotoxicity and the most neuroprotective effect. They were found to downregulate the lactate dehydrogenase enzyme levels. The compounds efficiently increased the expression of superoxide dismutase and glutathione peroxidase. They were shown to be beneficial in reducing reactive oxygen species levels. The compounds also significantly increased the expression of SIRT3, PGC-1α, and FOXO3, which are crucial for detoxifying reactive oxygen species. The synthesized novel indole-based carboxamide derivatives were effective modulators of SIRT3. Upregulating SIRT3 expression also upregulated other supporting proteins such as PGC-1α, SOD2, GPx, and FOXO3, and these targets have been known to downregulate the reactive oxygen species levels. The study’s findings reveal the therapeutic potential of the developed and synthesized compounds and their involvement in such pathological situations; among all the compounds, IM24DCW-16 was discovered to be the most promising candidate for the modulation of the SIRT3 in PD.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-025-99534-3.

Introduction

Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by the loss of the dopaminergic neurons in the substantia nigra pars compacta (SNc) of the brain accompanied by mitochondrial dysfunctioning¹. PD remains the second-most common neurodegenerative disorder, followed by alzheimer’s disease (AD), which mostly targets the population of ≥ 65 years of age; the major common symptoms related to PD cardinal symptoms are rigidity, bradykinesia, and postural instability². The current medications used in PD therapies are palliative and symptomatic; early onset usage of these therapies can result in downregulation of the PD, and usage of the most effective medications such as levodopa, bromocriptine, ropinirole, tolcapone, and selegiline have been known to cause several cognitive dysfunctions and neurotoxicity^3,4. New strategies have come into play to bypass the existing concerns related to PD by targeting the specific pathogenesis of the disease. Synthesizing novel molecules can be a game changer in treating PD, as shown in Fig. 1^5,6. Indole-based compounds belong to heterocyclic compounds, which have bicyclic rings containing a six-membered and five-membered nitrogen and pyrrole ring; various indole-based molecules have been reported as anti-cancer⁷, Indole 3 propanoic acid and melatonin exhibit significant neuroprotective properties through various mechanisms, including antioxidant activity, anti-inflammatory effects, and enhancement of neuronal survival; NC009 series, particularly NC009-1, shows promise in reducing neuroinflammation and protein aggregation in models of neurodegeneration^8,9. These indole moiety-based compounds could serve as potential therapeutic agents for conditions like PD and AD. Sirtuin-3 (SIRT3) is a deacetylating protein located in the mitochondria that plays a significant role in mitochondrial biogenesis and balances reactive oxygen species (ROS) levels and oxidative stress¹⁰. SIRT3 has also been known to downregulate the progression of the dopaminergic neurons in the SNc by decreasing ROS production and oxidative stress¹¹. SIRT3 has various roles such as autophagy, apoptosis, inflammation, and oxidative stress; mitochondrial sirtuins have been widely studied in the context of nutritional stress and cellular energy metabolism; research on knocked-down SIRT3 mice model resulted in the decrease of enzymatic activity of SIRT3 and ATP levels¹². Mitochondrial dysfunctioning has resulted in the downregulation of the Peroxisome proliferator-activated receptor gamma coactivator 1- α (PGC-1α), superoxide dismutase (SOD2), forkhead box 3 (FOXO3), and glutathione peroxidase (GPx). In contrast, these genes are highly involved in the downregulation of the ROS species and oxidative stress, which mainly results in neuronal death^13,14. There are several kinds of pathophysiologies about how PD can be induced, such as protein aggregation (accumulation of the Lewy Bodies), mitochondrial dysfunctioning (responsible for the modulation of the reactive oxygen species and oxidative stress), neuroinflammation (activation of the microglia in the brain in terms of defense mechanism) and blood brain-barrier breakdown; among these pathophysiologies, our study highlights and explains mitochondrial dysfunctioning (responsible for the modulation of the reactive oxygen species and oxidative stress) role in the progression of PD and how our molecules could be effective towards the ROS and oxidative-based insults in cells¹⁵. Due to multifactorial physiopathology, such as genetic factors, protein aggregations, oxidative stress, neuroinflammation, environmental factors, dysfunctional proteostasis, or excitotoxicity, the proper therapies for PD remain vague and untreatable; our study utilizes hybrid heterocyclic molecules in the targeting of SIRT3 to attenuate the further loss of the dopaminergic neurons and provide mitochondrial biogenesis in PD. The compounds will be evaluated for their ability to modulate targets such as SOD2, GPx, and PGC-1α, resulting in reduced ROS and oxidative stress levels.

Fig. 1.

Indole-based molecules exhibiting antioxidant properties in various cell lines-based studies.

Material and methods protocols

Pharmacophore hypothesis

The study was developed using the Schrodinger Suite 2023–24 "Develop pharmacophore hypothesis," whereas 110 selective antioxidants and neuroprotective molecules were obtained from the various literature. The individual IC₅₀ values were collected for all the compounds and converted into pIC₅₀ values (http://www.sanjeevslab.org/tools.html). The pIC₅₀ values were further screened into “active” and "inactive" groups. The "multiple ligand pharmacophore model" was utilized to identify the pharmacophoric features¹⁶.

Ligand preparation

To attest to valid modeling, the “LigPrep” module in the Schrodinger suite 2023–24, maestro 13.4, was utilized, and the ionization parameters and pH conditions were kept to 7.0 ± 0.2. The OPLS 2004 was used as a forcefield to obtain a low-energy molecular geometry. The confirmations were generated using the addition of the hydrogen atoms and were minimized¹⁷.

Protein preparation

The protein was extracted from the RCSB protein data bank (http://www.rcsb.org/); the protein utilized was a human SIRT3 bound to Ac-Acs peptide and Carba-NAD (PDB ID: 4fvt), with a resolution of 2.47 Å; the protein was extracted using the X-ray diffraction method. The protein was prepared using the "protein preparation wizard tool". The protein was precisely evaluated for the missing amino acids, the hetero-atoms and the water molecules were removed; the necessities were excluded, and the missing residues were added¹⁸.

Molecular docking studies

The "receptor grid generation" module was utilized to form the grid box. The co-crystal Ac-Acs peptide was selected from the site centroid of the workspace ligand (protein). The "receptor grid generation" module was utilized to form the grid box. The co-crystal Ac-Acs peptide was selected from the site centroid of the workspace ligand (protein). The “ligand-docking” and "extra precision (XP)" modules were utilized to proceed with the molecular docking. Further, the "ligand interaction tool" was used to visualize the ligand/protein amino acid interactions¹⁹.

Prime MM/GBSA studies

The free energy calculations were performed for the designed ligands and the protein complex using the module "Prime-MMGBSA" in the Schrodinger suite 2023–24. The study was performed to align the top stable hit-ligands based on the ligand’s free binding energies and the VSGB solvation parameters along with forcefield OPLS4; further, the free energy (Δ_Gbind) was obtained for individual ligand/protein complex. The Δ_EMM was calculated using the OPLS4, which identified the free energy between the ligand–protein complex, whereas Δ_Gsolv (GBSA solvation energy) between the ligand and sirtuin 3, the complex was determined, Δ_GSA represents the surface area energy difference between the sirtuin 3 and the designed compounds. The overall sum between all these variables was calculated in one variable known as Δ_Gbind²⁰.

Absorption, metabolism, distribution and excretion studies (ADME)

The ADME studies were performed for all the designed compounds using the "QikProp" module in the Schrodinger suite 2023–24²¹.

Molecular dynamics simulation studies

The study was carried out to assess the binding stability of the ligand/protein complex in a virtual “TIP4P” solvent environment with a time interval of 100 ns (ns). The ligand/protein complex was prepared using the “system builder” with a forcefield OPLS4. The complex was minimized under the orthorhombic box size with a distance of 10/10/10 Å. The extended water molecules were removed, and the Na + ions were added to the complex; the simulation parameters, such as temperature, were kept at 311 K²².

Experimental section

Chemical procurement

All the chemicals and the reagents utilized in the synthesis were purchased from the carbino innovative chemical interchange pvt. ltd. The thin-layer chromatography (TLC) used a glass plate coated with silica gel-G as an adsorbent. The TLC was performed using a 10% ethyl-acetate and n-hexane solvent system. The spotting in the TLC plate was observed using the UV chamber. The melting points were determined using the Veego VMP-I melting point apparatus. The Fourier transform infrared (FT-IR) instrument was used to identify the functional groups present in the synthesized compounds from the 400 cm⁻¹ to 4000 cm⁻¹ range, including the fingerprint region 400–1400 cm⁻¹. Furthermore, to ascertain whether the synthesized compounds contained the required hydrogen/proton counts ranging from 0 to 15 δ parts per million and carbon counts ranging from 0–200 δ parts per million, the Nuclear Magnetic Resonance (¹H-NMR and ¹³C-NMR) method was used and the reference solvent used was dimethyl sulphoxide (DMSO_d6). The molecular weight was determined using a mass spectrometry instrument, and the spectra ranges were maintained between 0 and 500 Daltons. All these instrumental methods helped in the confirmation of the synthesized compounds. The FT-IR, NMR spectroscopy, and Mass spectrometry data are provided in the supplementary file (Spectra 1–44).

General procedures in the synthesis of IMW 1–16

Figure 2 depicts the general procedure involved in the synthesis of IMW-16; ethane-1,2-diamine (8 mL) 2 and 0.5 g of methyl 1H-indole-2-carboxylate 1 were reacted in a round bottom flask in a microwave at 210 watts to yield compound 3. The compound 3 (0.5 g) was further reacted with the substituted aromatic aldehydes 4 (0.5 g) to produce the final IMW 1–16 derivatives. The reaction was monitored for 16–30 min using the TLC (ethyl-acetate: n-hexane) techniques until the reaction was completed. Distilled water was added to the synthesis mixture and kept for cooling. After additional filtering with distilled water, the precipitates were set aside to dry at room temperature. The precipitates were further recrystallized using ethanol.

Fig. 2.

(A) General synthetic route of the designed ligands IMW 1–16; (B) Steps involved in synthesizing compound 3 and IMW 1–16; (C) Different types of substituted aromatic aldehydes were utilized in the synthesis of IMW 1–16.

Step I

Methyl 1H-indole-2-carboxylate 1 (0.5 g) was treated with 8 mL of ethane-1,2-diamine 2. Additionally, 0.2 mL of the acetic acid was added dropwise to the reaction mixture with 5 mL of ethanol to yield intermediate compound 3; the reaction was carried out in the microwave at 210 watts, and the byproduct formed was methanol (CH₃OH). The reaction condition was monitored using the TLC technique every 4 min for 16 min. The different TLC spots observed the completion of the reaction. Further, the reaction mixture was precipitated using distilled water, and the precipitates were further washed with an excess amount of distilled water to remove the unreacted amine. The precipitates were dried at room temperature and were recrystallized using ethanol.

Step II

The N-(2-aminoethyl)-1H-indole-2-carboxamide 3 (0.5 g) was added to the aromatic substituted aldehydes 4 (0.5 g) with a 15 mL of ethanol, a 0.2 mL of acetic acid was added to the reaction mixture to yield IMW 1–16. The reaction was carried out in a microwave at 210 watts. The reaction was monitored every 10 min for 30 min using TLC techniques. Further, the reaction condition was precipitated using cold distilled water. The precipitates were further dried at room temperature and were recrystallized using ethanol²³.

In-vitro studies

Neurotoxicity analysis using 2-(4,5-Dimethylthiazol-2-yl)-3,5-diphenyl-2H-tetrazol-3-ium Bromide (MTT method)

The SHSY-5Y cell line (human neuroblastoma cell line) was cultured in a 25 cm² flask containing dulbecco’s modified eagle medium (DMEM), which was enriched in 10% fetal Bovine Serum (FBS) and various antibiotics such as penicillin, streptomycin, and amphotericin B. The cultured cells were incubated at 37 °C with 5% CO₂. An inverted phase microscope was used to observe the % of cell viability. The cell seeding was performed in the 96 well plates, where the cell’s monolayers led to a dissociation process where the adhered cells were dissociated from the culturing flask. The compound stock solutions were prepared by mixing 1 mg of the compound in 0.1% dimethyl sulphoxide using the cyclomixer. The neurotoxicity evaluation was conducted at 24-h intervals using compound solutions mixed with DMEM solution. The solution mixture of 100 µg, 50 µg, 25 µg, 12.5 µg, and 6.25 µg in 500 µL of DMEM was added to the well plates in triplicates and incubated at 37 °C under 5% CO₂. The test and control groups received 30 µL of the prepared MTT reagent and were maintained at 37 °C with 5% CO₂ for 4 h. The absorbance was measured at 540 nm²⁴.

Neuroprotection analysis using (MTT method)

The human SHSY-5Y neuroblastoma cell lines were acquired from NCCS Pune and grown in a 25 cm² flask alongside DMEM containing 10% FBS with antibiotics such as penicillin, amphotericin B, and streptomycin. The DMEM solution was supplemented with L-glutamine and sodium bicarbonate solution, and the cells were maintained at 37 °C with 5% CO₂. The cell counts were measured using direct observation methods and an inverted phase microscope. The cell seeding was done using 96 well plates, where the cell’s monolayers led to a dissociation process where the adhered cells were dissociated from the culturing flask and were cultured at 37 °C under 5% carbon dioxide. The compound stock solutions were prepared using 1 mg of the compound in 1 mL of DMEM solution. The toxicity was induced using 1-Methyl-4-phenylpyridinium (MPP+) with a dosage concentration of 0.5 mM. To obtain a final concentration of 0.5 mM, 2.9 mg MPP⁺ iodide was precisely weighed, mixed in 1 mL of 0.1% DMSO to create a 12.55 mM stock solution, and then applied to the experimental wells in the proper volume (50 µL/mL). The MPP+ at a dosage concentration of 0.5 mM was added to the cells, whereas 5% of the prepared DMEM solution was diluted at a two-fold concentration. Five different dosage concentrations of 5% DMEM were prepared: 25 µg, 12.5 µg, 6.25 µg, 3.1 µg, and 1.5 µg. The cells were maintained and grown at 37 °C under 5% CO₂ after adding 100 µL of the prepared concentrations. 30 µL of the MTT solution was added to the control and test groups and incubated at 37 °C under 5% carbon dioxide for 4 h. The absorbance was taken at 540 nm using a microplate reader²⁵.

Estimation of the lactate dehydrogenase assay (LDH)

LDH is an enzyme marker that mainly indicates the disturbance of the cellular glycolytic process; LDH’s leakage indicates cell inflammation. The human SHSY-5Y neuroblastoma cells were grown in the 25 cm² flask containing DMEM solution and 10% FBS with antibiotics such as penicillin and streptomycin. The cells were maintained at 37 °C with 5% carbon dioxide. The cells were cultured in the 96 well plates. The MPP+ was added to induce the toxicity at a dosage concentration of 0.5 mM. The compounds’ 12.5 µg/mL dosage concentration was incorporated into the cells and maintained for 24 h. The control, treated, and toxicity-induced groups were incubated for 24 h. LDH levels were quantified using the DGKC method at 340 nm using a spectrophotometer²⁶.

Estimation of the superoxide dismutase (SOD2)

The human SHSY-5Y neuroblastoma cell lines were obtained from NCCS Pune and cultured in DMEM solution in a 25 cm² flask. The flask was supplemented with 10% FBS, L-glutamine, and antibiotics such as penicillin and streptomycin. The cells were maintained for 24 h at 37 °C with 5% CO₂. The cells were further cultured in 96 well plates, and the toxicity inducer MPP+ was administered to the cells at a dosage of 0.5 mM. Cells were treated with 12.5 µg/mL sample concentrations and incubated for 24 h. The 50 µL of cell lysate was obtained and was added to the mixture of 50 mM phosphate buffer, 5.3 mM of riboflavin, 45 µL of methionine, and 84 µM of potassium ferric cyanide; the tubes were incubated at 25 °C for 10 min. The absorbance was taken at 600 nanometres²⁷.

Estimation of the glutathione peroxidase (GPx)

The human SHSY-5Y neuroblastoma cell lines were obtained from NCCS Pune and cultured in a 25 cm² flask containing DMEM solution with 10% FBS and antibiotics such as penicillin, L-glutamine, and streptomycin. The DMEM solution was supplemented with L-glutamine and sodium hydrogen carbonate solution, and the cells were maintained at 37 °C with 5% CO₂ for 24 h. The cell seeding was done using 96 well plates, where the cell’s monolayers led to a dissociation process where the adhered cells were dissociated from the culturing flask and were cultured at 37 °C under 5% carbon dioxide. The toxicity was induced in the cells using MPP+ at a dosage concentration of 0.5 mM. The compounds were added to the cultured cells at 12.5 µg/mL and incubated for 24 h. The 50 μL of the cell lysate was added to the tubes containing 3 mL of the NADPH and 1 mM of the glutathione. The absorbance was further taken at 340 nanometres²⁸.

Reactive oxygen species estimation by flow cytometry

In a 25 cm² flask, SHSY-5Y neuroblastoma cell lines were cultured in DMEM solution containing 10% FBS, L-glutamine, sodium hydrogen carbonate, and antibiotics (streptomycin 100 µg/mL, penicillin 100 µg/mL, and amphotericin B 2.5 µg/mL). The toxicity was induced in the cultured cells by using MPP+ at a dosage concentration of 0.5 mM. The cultured cells were treated with the compounds IMFW-1, IMTW-5, and IM24DCW-16 with a 12.5 µg/mL dosage. The control group cells, toxicity-induced group cells, and treated cells were incubated for 24 h at 37 °C in a humidified 5% CO₂. The ROS levels were estimated using the reactive oxygen species (ROS) detection assay kit (catalog # K936-100, BioVision, Milpitas, CA, USA). The cells were centrifuged at 300 X g for 5 min at normal temperature; the adhered cells were harvested and centrifuged. The cell pellets were suspended using 1 X ROS label and were incubated at 37 °C for 30 min. The cells were then resuspended in 1 X ROS assay buffer containing 10% FBS and treated with samples. The mean fluorescent intensity was measured and compared between the control and treated groups²⁹.

Estimation of the mitochondrial membrane potential by flow cytometry

In a 25 cm² flask, SHSY-5Y neuroblastoma cell lines were cultured in DMEM solution containing 10% FBS, L-glutamine, sodium hydrogen carbonate, and antibiotics (streptomycin 100 µg/mL, penicillin 100 µg/mL, and amphotericin B 2.5 µg/mL). The cells that were grown were humidified with 5% CO₂ at 37 °C. The cells were induced toxicity via MPP+ at a dosage concentration of 0.5 mM; the cells were treated with the compounds IMFW-1, IMTW-5, and IM24DCW-16 with a 12.5 µg/mL dosage. The MUSE™ Mito-Potential dye 1:1000 in 1X buffer was used as a buffer medium. The cells were centrifuged and were suspended in the 1X buffer. The 95 µL of the Mito-Potential was incorporated with the suspended cells; the solutions were stirred at 37 °C and incubated at 5% CO₂ for 20 min. An additional 5 µL of the Muse Mito-Potential 7-AAD was incorporated into the wells and cultured for 5 min. The samples were further loaded on a flow cytometry (Millipore, USA)³⁰.

Gene expression in SHSY-5Y neuroblastoma cells by real-time PCR analysis

SHSYSY neuroblastoma cell lines were cultured using DMEM and 10% FBS, sodium bicarbonate, L-glutamine, penicillin (100 u/mL), streptomycin, and amphotericin B (2.5 µg/mL). The toxicity was induced in the cell line using the MPP+ at a dosage concentration of 0.5 mM. Further, the cells were treated with the compounds IMFW-1, IMTW-5, and IM24DCW-16 at a dosage concentration of 12.5 µg/mL and were maintained at 37 °C with 5% CO₂. The RNA was extracted from the total RNA isolation toolkit, and the TRIzol solution was added to it, where the disruption of the cells released the RNA. The isopropanol was used to precipitate the RNA, and 1 mL of the TRIzol solution was incorporated into the culturing well plates and maintained for 5 min. After adding 200 µL of chloroform at ambient temperature, the mixture was centrifuged for 15 min at 40 °C at 14,000 rpm. After adding 500 µL of isopropanol to the liquid layer, it was centrifuged for 15 min at 4 °C after being placed for 10 min at room temperature. The supernatant was discarded, and the pellets were collected and rinsed with 200 µL of 75% ethanol. The pellets were centrifuged at 14,000 rpm for 5 min at 4 °C and were dried and suspended using TE (Tris–EDTA) buffer. While the template complementary DNA was being synthesized using the cDNA preparation kit (G BIOSCIENCES, Product code: 786-5019 s, 786-5020, master premix for first-strand cDNA synthesis), the total RNA was isolated via TRIzol solution, and the quality and quantity were measured. The 5 µL of RT easy mix, 0.5 µL of oligo dT, and 2 µL of RNA template (0.5 µg of the total RNA) were added to the RNAse free tube. The thermal cycler was used in the cDNA synthesized at 42 °C for 20 min, and the number of cycles kept was 1. RT-PCR was done to analyze the gene expression using the SYBR green master mix solution. All the reactions were performed in triplicates, and the data was analyzed using the ΔΔCt method. Distributing potential throughout an electrolyte buffer solution produces the electric field. After the agarose had cooled to 45 °C, 6 µL of 10 mg/mL ethidium bromide was incorporated, and the mixture was transferred into the gel casting equipment that held the gel comb. Further, the comb was removed from the gel, and the electrophoresis buffer was poured into the gel tank, where the gel was loaded with the samples and was run at 50 V for 30 min. The stained gel was visualized using a gel documentation system and was viewed using the E-gel imager. The agarose gel electrophoresis method was utilized to separate the DNA fragments.

Statistical analyses

All the experiments were performed in triplicates, and the results were obtained in the form of Mean ± SD (standard deviations). They were analyzed using the one-way analysis of variance (ANOVA) and Dunnett’s test using the GraphPad prism 8.0. Significant statistical differences were considered at (p) < 0.05.

Results

Pharmacophore modeling studies

The 110 compounds were collected from various databases and were found to have good antioxidant or neuroprotective effects. For each compound, the IC₅₀ values were collected. These values were subsequently changed to pIC₅₀ values, which lowers the possibility of error and represents the data as a molar concentration. The study predicted various types of pharmacophoric features such as acceptor (A), donor (D), hydrophobic (H), negative-ion (N), positive ion (P), and aromatic ring (R). The compounds were further categorized into two groups: active and inactive. The compounds with a pIC₅₀ value of more than 5.10 were assigned to the active groups. In contrast, the compounds found to have a pIC₅₀ value of less than 4.0 were assigned to the inactive groups; overall, 70% of the compounds were kept in the active group, and 30% were kept in the inactive groups. Figure 3 depicts the phases of the pharmacophore study. The best pharmacophoric model was exhibited to be “AA_RRR”, meaning two acceptor and three aromatic rings. The model predicted five active confirmations and 1 in-active confirmation, and the pharmacophore model predicted quercetin with the highest "survival and fitness" scores of 4.49 and 3.00. The AA_RRR model also predicted the vector and volume scores of 1.00 and 1.0.

Fig. 3.

Pharmacophore model and hypothesis representing the pharmacophoric feature AA_RRR for the collected 110 molecules. The AA_RRR (two acceptors’ groups and three aromatic rings) suggested that the involvement of these pharmacophoric features in the compounds could enhance the biological efficacy of the compounds. (A) The top 110 molecules were selected based on their antioxidant or neuroprotective effect; (B) Multiple ligands pharmacophoric method was utilized to obtain the required pharmacophoric features.

Ligand preparation

The ligands were drawn in a 2-dimensional structure using Chem Draw 19.0 and saved using an MOL file. The MOL file was imported to the “LigPrep” module in the Schrodinger suite 2023–24 to minimize the ligands. The ligands were fused with multiple functional groups, such as aromatic rings, donors, and acceptors. The pharmacophoric model study predicted two acceptor groups and three aromatic rings, as provided in the supplementary file Figure S2.

Protein preparation

The raw SIRT3 (PDB ID: 4fvt) in Fig. 4 was retrieved from the RCSB data bank. Subsequently, the Schrodinger suite 2023–24’s "protein-preparation wizard tool" minimized the protein. The missing amino acid residues were added, and other heteroatoms and water molecules were removed. Further, the “ramachandran plot” also predicted that more than 90% of the amino acid residues were located on the plots’ positive side. Moreover, hydrophobicity, hydrophilicity, and aromatic regions were quantified for the prepared protein.

Fig. 4.

(1A) The raw protein (SIRT3) PDB ID: 4fvt; (1B) Prepared protein; (1C) Ramachandran plot visualizing 90% of the amino acids on the positive side; (2A) Hydrophobicity regions in the SIRT3 (blue: low hydrophobicity; white: intermediate hydrophobicity; brown: high hydrophobicity); (2B) Hydrogen bond regions (donor: pink (more favorable towards donor atoms); white: intermediate region; acceptor: green (more favorable towards acceptor groups); (2C) The aromatic region was obtained as edge-to-face (non-covalent interactions with the aromatic amino acid that can stabilize the protein folds).

Molecular docking studies

The compound IMOCW-13 was found to have a docking score of − 9.86 kcal/mol. The indole moiety with the five-membered and six-membered rings was found to have two hydrophobic interactions with the residue His 248, the NH of the indole moiety was found to have polar amino acid interaction with the residue Gln 228, C=O group was found to have a nonpolar amino acid interaction with the residue Ala 146, CH=N was found to have a polar amino acid interaction with the residue Ser 321. Figure 5 illustrates the substituted aldehyde with the aromatic ring, which was found to have a hydrophobic interaction with the amino acid Phe 157, and the methoxy group had an amino acid interaction with the Arg 345; the compound IMCW-4 was found to have a docking score of − 8.71 kcal/mol. The indole moiety with the five-membered ring and NH was found to have hydrogen bond amino acid interaction with the Gln 228. The imine CH=N in Fig. 5 was found to have a hydrophobic amino acid interaction with the Val 292. The substituted aldehyde aromatic group was found to have a pi-pi hydrophobic amino acid bond interaction with the Phe 294. The hydrophobicity range was between 1.8 and 4.5, involving amino acid residues such as His 248, and Phe 157. The hydrophilic range was obtained from − 0.4 to − 4.5 with amino acids such as Gln 228, and Phe 294. The 2D interactions and the docking scores of the designed compounds IMW1-16 have been illustrated in the supplementary file Figure S1 and Table S1.

Fig. 5.

(1) Amino acid interaction with the compound IMOCW-13; (2) Amino acid interaction with the compound IMCW-4; (A) 2-Dimensional structure of the protein and the ligand; (B) Hydrophobic regions of the protein; (blue: low hydrophobicity; white: intermediate hydrophobicity; brown: high hydrophobicity); (C) Hydrogen bond donor and acceptor regions; (donor: pink more favorable towards donor atoms); (white: intermediate region); (acceptor: green more favorable towards acceptor groups).

The compound IMFW-1 was found to have a docking score of − 7.87 kcal/mol. Figure 6 illustrates the indole moiety with the six-membered ring had hydrophobic amino acid interaction with the Phe 294, and the NH (secondary amine) had an amino acid interaction with the Gln 228. The carbonyl (C=O) was found to have a hydrophobic amino acid interaction with the Ala 146. The hydrophobicity range was between 1.8 and 4.5, involving amino acid residues such as Phe 294. The hydrophilic range was obtained from − 0.4 to − 4.5 with amino acid residues such as Ser 321; the compound IMNW-7 was found to have a docking score of − 6.75 kcal/mol. The C=O was found to have a polar amino acid interaction with the hydrophobic residue Ala 146. The imine CH=N group was found to have a hydrogen bond interaction with the Ser 321.

Fig. 6.

(1) Amino acid interaction with the compound IMFW-1; (2) Amino acid interaction with the compound IMNW-7; (A) 2-Dimensional structure of the protein and the ligands; (B) Hydrophobic regions of the protein; (blue: low hydrophobicity; white: intermediate hydrophobicity; brown: high hydrophobicity); (C) Hydrogen bond donor and acceptor regions; (donor: pink more favorable towards donor atoms); (white: intermediate region); (acceptor: green more favorable towards acceptor groups).

The compound IM24DCW-16 was found to have a docking score of − 6.46 kcal/mol. Figure 7 illustrates the indole moiety with the six-membered ring had hydrophobic amino acid interaction with the His 248, and the NH (secondary amine) had an amino acid interaction with the Gln 228. The carbonyl (C=O) was found to have a hydrophobic amino acid interaction with the Ala 146. The substituted six-membered aromatic aldehyde ring (2-dichloro benzaldehyde) was found to have hydrophobic amino acid interaction with the Phe 157. The hydrophobicity range was between − 3 and 3, involving amino acid residues such as Ala 146 and Phe 294. The hydrophilic interactions were observed with amino acid residues such as His 248 and Gln 228.

Fig. 7.

(1) Amino acid interaction with the compound IM24DCW-16; (A) 2-Dimensional structure of the protein and the ligands; (B) Hydrophobic regions of the protein; (blue: low hydrophobicity; white: intermediate hydrophobicity; brown: high hydrophobicity); (C) Hydrogen bond donor and acceptor regions; (donor: pink more favorable towards donor atoms); (white: intermediate region); (acceptor: green more favorable towards acceptor groups).

Molecular mechanics with generalized Born and surface area solvation

MMGBSA study predicted the free energy available between the protein and the ligands. The energies such as binding, coulomb, covalent, hydrogen bond, solvating, and Vander wall force were obtained. The highest MMGBSA dG bind score was exhibited by the compound IMDCW-8, which had a score of − 108.1. The compounds IMCW-4 and IM2BW-11 were found to have the MMGBSA dG bind score of − 106.1. The compound IMTW-5, IMOCW-13, IMCW-14, and INCW-3 were found to have an MMGBSA score of − 98.3, − 97.46, − 91.72, − 94.6 and − 91.72. The compound resveratrol, IMNW-10, IMBW-12, IMNW-10, and IMHW-2 were found to have an MMGBSA score of − 87.8, − 83.04, − 87.03, and − 82.31. The lowest MMGBSA score exhibiting compounds were found to be IMDCW-9, 2CH3-15, IM24DCW-16, IMNW-7, and IMFW-1 with an MMGBSA score of − 73.9, − 72.7, − 66.4, − 53.6, and − 46.56. The MMGBSA dg bind scores for different energies of the designed compounds IMW1-16 have been illustrated in the supplementary file Table S2.

Absorption, metabolism, distribution and excretion studies (ADME)

The results showed that all the designed compounds had a molecular weight of less than 500 daltons and were in the 250–350 dalton range. This suggests that the compounds also possessed lead-like characteristics, also known to increase membrane permeability and bioavailability. Since the developed compounds were supposed to have 2–3 hydrogen bond donors (NH or OH groups) and less than 10 hydrogen bond acceptor groups (N or O atoms), additional research on compounds also anticipated good bioavailability. Since more NH, OH, or O can have higher interactions with water groups, which can also hinder the compound’s potency to cross the blood–brain barrier, fewer hydrogen bond donors and acceptors limit the necessary polar interactions. The compounds’ QPlogP_o/w scores ranged from 4 to 5, indicating moderate to high lipophilicity and a favorable reason to cross the blood–brain barrier. The potential for cardiac toxicity was also examined using the QPlogHERG parameter. The compounds were effective in blocking the hERG potassium channel, which can disrupt cardiac electrical activity and cause QT syndrome and cardiac arrest. The ideal QPlogHERG ranges were greater than 5.0, indicating the compounds’ inhibitory activity. Additionally, the study included the compounds’ QPPCaco and QPlogBB assessments. The QPPCaco, a metric used to assess intestinal absorption, was found to be > 500–2504, suggesting that oral drugs had a higher absorption rate. The QPlogBB (brain/plasma concentration) metric was also examined, and the results showed that the compounds had a moderate blood–brain barrier permeability. The compounds scored 3 from the human oral absorption metric research, indicating that they had high absorptions when compared to oral delivery. The polar surface area of the compounds also suggested a range of < 60 to 140, suggesting more permeability towards the membranes and indicating good oral bioavailability. The overall ADME scores predicted that the designed compounds have better affinity towards absorption and permeability towards the membranes; the study also found no Lipinski rule of 5 violations, making these compounds more drug-like. The study’s findings are in the supplementary file Table S3.

Molecular dynamics simulation studies

The simulation study was performed to analyze the type of stability between the top docking and MMGBSA-scored ligands and the protein SIRT3 (PDB ID: 4fvt) in a given solvent virtual environment. The compound IMOCW-13 was found to have a root mean square deviation (RMSD) of 4.8 Å, whereas the RMSD for the protein was found to be 2.8 Å. The stability study was performed for 100 ns, whereas from 5 to 80 ns, there was no fluctuation between the ligand/protein complex; from 80 to 85 ns, a minor fluctuation was observed; from the 85 to 100 ns, no fluctuations were observed. The CH=N was found to interact with His 248, Phe 180, and Phe 294 with an intensity of 61%, 14%, and 21%. The amino acids such as Glu 177, Phe 180, Leu 199, His 248, Val 292, and Phe 292 were observed to be in continuous contact with the ligand from 0 to 100 ns. A total of 20 interactions were observed, whereas the hydrophobic interactions were seen with Pro 155, Arg 158, Ile 179, Phe 180, Leu 199, Ile 230, His 248, Val 292, Leu 298, and Val 324 amino acids. The amino acids such as Arg 158, Glu 177, Gln 228, His 248, Val 292, and Gly 295 were found to have hydrogen bond interactions. The compound IMCW-4 was found to have a structural deviation of 9 Å, whereas the RMSD for the protein was found to be 4.0 Å. The stability study was performed for 100 ns, whereas from 5 to 50 ns, there was a minor fluctuation between the ligand/protein complex; from 50 to 100 ns, no fluctuation was observed. The NH was found to have an amino acid interaction with Phe 294, Phe 180, and His 248 with an intensity of 36%, 42%, and 73%. The amino acids such as Arg 158, Phe 180, Leu 199, Gln 228, Ile 230, His 248, Val 292, and Phe 294 were observed to be in continuous contact with the ligand from 0 to 100 ns. A total of 25 interactions were observed, whereas the hydrophobic interactions were seen with Ala 146, Ile 154, Pro 155, Phe 157, Arg 158, Leu 164, Tyr 165, Leu 168, Phe 180, Leu 195, Leu 199, Tyr 204, Ile 230, His 248, Ile 291, Phe 294, Leu 298 and Val 324. The amino acids are Tyr 165, Glu 177, Tyr 204, Gln 229, Ile 230, His 248, and Val 292, as shown in Fig. 8. The compound IMFW-1 was found to have a structural deviation (RMSD) of 8 Å, whereas the RMSD for the protein was found to be 4.0 Å. The stability study was performed for 100 ns, whereas from 5 to 40 ns, there was a minor fluctuation between the ligand/protein complex; from 50 to 100 ns, no fluctuation was observed. The C=O group was found to have a hydrogen bond interaction with ILE 230 with an intensity of 65%. The amino acids are Leu 164, Phe 180, Gln 228, Ile 230, His 248, and Phe 294. The ligand IMFW-1 was found to have hydrophobic amino acid interactions with Ala 146, Ile 154, Pro 155, Arg 158, Pro 160, Leu 164, Tyr 165, Leu 168, Pro 176, Phe 180, Leu 199, His 248, Phe 251, Ile 291, Phe 294 and Val 324. The ligand was found to have few favorable hydrogen bond interactions with Arg 158, Gln 228, Asn 229, Ile 230, Asp 231, and His 248. The compound IMNW-7 was found to have a structural deviation (RMSD) of 8 Å, whereas the RMSD for the protein was found to be 5.4 Å. Minor fluctuation between the ligand/protein complex; from 25 to 40 ns, a minor fluctuation was observed. The amino acids such as Phe 157, Phe 180, His 248, and Phe 294 were found to have interactions with the ligand from 0 to 100 ns. The ligand IMNW-7 was found to have hydrophobic amino acid interactions with Ala 146, Phe 157, Arg 158, Ser 159, Tyr 165, Leu 168, Ile 179, Phe 180, Phe 192, Leu 195, Ala 196, Leu 199, Ile 230, His 248, Phe 251, Ile 291, Phe 293, Phe 294 and Val 324. The interactions also predicted a few hydrogen bond interactions with Pro 155, Phe 157, His 248, and Glu 323, as shown in Fig. 9.

Fig. 8.

Molecular dynamic study of the designed ligands with the SIRT3 (PDB ID: 4fvt); (1) IMOCW-13; (2) IMCW-4. (A) RMSD between the ligand and the protein. (B) Protein–ligand contact with the amino acids. (C) Hydrogen and hydrophobic amino acid interactions.

Fig. 9.

Molecular dynamic study of the designed ligands with the SIRT3 (PDB ID: 4fvt); (1) IMFW-1; (2) IMNW-7. (A) RMSD between the ligand and the protein. (B) Protein–ligand contact with the amino acids. (C) Hydrogen and hydrophobic amino acid interactions.

The compound IM24DCW-16 was found to have a root mean square deviation (RMSD) of 4.5 Å, whereas the RMSD for the protein was found to be 3.2 Å. The stability study was performed for 100 ns, whereas from 7 to 42 ns, there was no fluctuation between the ligand/protein complex; from 60 to 70 ns, a minor fluctuation was observed within a range of 3.0 Å to 3.5 Å; from the 71 to 100 ns, no fluctuations were observed. The C=O was found to interact with Ile 230 and Asp 231 with an intensity of 75% and 71%. The amino acids such as Phe 157, Ile 230, Asp 231, and His 248 were observed to be in continuous contact with the ligand from 0 to 100 ns. A total of 31 interactions were observed, whereas the hydrophobic interactions were seen with Ala 146, Ile 154, Pro 155, Phe 157, Leu 164, Pro 176, Ile 179, Phe 180, Leu 199, His 248, Phe 251, and Val 324 amino acids. The amino acids such as Asn 229, Ile 230, and Asp 231 were found to have hydrogen bond interactions, as shown in Fig. 10.

Fig. 10.

Molecular dynamic study of the designed ligands with the SIRT3 (PDB ID: 4fvt); (1) IM24DCW-16. (A) RMSD between the ligand and the protein. (B) Protein–ligand contact with the amino acids. (C) Hydrogen and hydrophobic amino acid interactions.

Chemistry

Methyl 1H-indole-2-carboxylate

FT-IR : 3330.79 (NH), 2953.0 (CH), 1687.76 (C=O).

N-(2-aminoethyl)-1H-indole-2-carboxamide (3)

The compound was prepared according to the general procedure and was obtained as off-white solid; M.P—178–180 °C, Yield: 85%; FT-IR: 337.49 (NH), R-NH (3301.74), 3048.38 (NH₂), 2975.07 (CH), 1618.14 (C=O); ¹H-NMR (400 MHz; DMSO-_d6): 1.83 (2H, NH₂); 2.70–2.73 (2H, d, J = 6.5 Hz, CH₂); 3.28–3.33 (2H, d, J = 6.1 Hz, CH₂); 7.01–7.05 (1H, t, Ar–H); 7.13–7.15 (1H, m, Ar–H); 7.17–7.19 (1H, m, Ar–H); 7.43–7.45 (1H, d, J = 8.2 Hz, Ar–CH); 7.59–7.62 (1H, d, J = 8.0 Hz, Ar–H); 8.43 (1H, s, NH); 11.55 (1H, s, NH); ¹³C-NMR (400 MHz; DMSO-_d6): 41.94, 43.19, 102.87, 112.76, 120.14, 121.9, 123.66, 127.61, 132.60, 136.87, 161.78; MS: 204.05 (M + 1).

(E)-N-(2-((4-fluoro benzylidene) amino) ethyl)-1H-indole-2-carboxamide (IMFW-1)

The compound was prepared according to the general procedure and was obtained as white solid; M.P—220–222 °C, Yield: 89%; FT-IR: 3066 (NH), 3238.53 (NH), C-H (2900.12), C=O (1633.49); ¹H-NMR (400 MHz; DMSO-_d6): 3.58–3.62 (2H, d, J = 6.4 Hz, CH₂), 3.76–3.79 (2H, d, J = 6.4 Hz, CH₂), 7.02 (1H, t, Ar–H), 7.12 (1H, s, Ar–H), 7.17 (1H, t, Ar–H), 7.25–7.29 (t, 2H, J = 8.7 Hz, Ar–H), 7.43–7.45 (2H, d, J = 8.0 Hz, Ar–H ), 7.59–7.61 (1H, d, J = 8.0 Hz, Ar–H), 7.80 (2H, s, Ar–H), 8.35 (1H, s, CH=N), 8.58 (1H, s, NH), 11.57 (1H, s, NH); ¹³C-NMR (400 MHz; DMSO-_d6): 60.19, 102.90, 112.75, 115.98, 116.19, 120.14, 121.92, 123.69, 127.55, 130.57, 130.35, 132.21, 133.15, 136.86, 131.18, 161.70, 162.77, 165.24; MS: 311.10 (M + 2).

(E)-N-(2-((4-methylbenzylidene)amino)ethyl)-1H-indole-2-carboxamide (IMCW-4)

The compound was prepared according to the general procedure and was obtained as yellow solid; M.P—220–222 °C, Yield: 75%; FT-IR: 3062.2 (R-NH), 3242.7 (NH), 2921.96 (CH), 1637.84 (C=O); ¹H-NMR (400 MHz; DMSO-_d6): 2.27 (3H, s, CH₃), 3.35 (1H, d, J = 5.8 Hz, CH), 3.74–3.77 (2H, d, CH₂), 7.02 (2H, d, J = 7.7, 3.5 Hz, CH), 7.09 (1H, s, CH), 7.12 (1H, s, CH), 7.15–7.16 (2H, d, J = 3.5 Hz, CH), 7.23–7.25 (1H, d, J = 7.7 Hz, CH), 7.42 (1H, dq, J = 8.1, 4.3 Hz,), 7.67–7.53 (2H, m, CH), 8.31 (1H, s, CH=N), 8.56 (1H, s, NH), 11.96 (1H,s, NH); ¹³C-NMR (400 MHz; DMSO-_d6): 60.27, 102.27, 120.14, 121.91, 123.67, 127.54, 127.63, 128.41, 129.02, 130.06, 132.22, 133.87, 136.84, 140.92, 141.13, 161.67, 162.30, 162.90; MS: 306.06 (M + 1).

(E)-N-(2-(benzylidene amino)ethyl)-1H-indole-2-carboxamide (IMBW-6)

The compound was prepared according to the general procedure and was obtained as creamy white solid; M.P: 232–234 °C; Yield: 70%; FT-IR: 3238.9 (R-NH), 2928.09 (CH), 1638.7 (C=O); ¹H-NMR (400 MHz; DMSO-_d6): 3.61–3.63 (2H, d, J = 6.1 Hz, CH₂), 3.78–3.79 (2H, d, J = 6.3 Hz, CH₂), 7.13 (1H, s, CH), 7.44–7.45 (1H, d, J = 6.3 Hz, CH), 7.60–7.62 (2H, m, J = 8.0 Hz, CH), 7.71–7.81 (m, 3H, CH), 7.80 (2H, m, CH), 8.33–8.37 (d, J = 13.3 Hz, 1H), 8.48 (1H, dd, CH), 8.61 (1H, s, CH=N), 8.76 (1H, s, NH), 11.31 (1H, s, NH); ¹³C-NMR (400 MHz; DMSO-_d6): 56.54, 60.19, 102.91, 112.77, 118.10, 120.16, 121.25, 123.70, 124.49, 126.62, 127.56, 128.98, 129.01, 131.14, 132.22, 135.22, 136.51; MS: 292.05 (M + 1).

(E)-N-(2-((4-(dimethylamino)benzylidene)amino)ethyl)-1H-indole-2-carboxamide (IMNW-7)

The compound was prepared according to the general procedure and was obtained as brown solid; M.P: 195–197 °C; Yield: 84%; FT-IR: 3179.7 (R-NH), 3300.79 (NH), 2929.79 (CH), 1611.27 (C=O); ¹H-NMR (400 MHz; DMSO-_d6): 2.95 (6H, s, CH₃), 3.37 (2H, s, CH₂), 3.68 (2H, s, CH₂), 5.68 (1H, s, CH), 6.55–6.58 (1H, d, J = 9.4 Hz, CH), 6.67–6.79 (5H, d, J = 11.0 Hz, CH), 7.01–7.03 (2H, d, J = 8.5 Hz, CH), 8.12 (1H, CH=N), 8.15 (1H, s, NH), 11.89 (1H, s, NH); ¹³C-NMR (400 MHz; DMSO-_d6): 59.95, 60.93, 102.85, 111.81, 127.55, 128.48, 129.17, 134.11, 136.84, 141.77, 150.70, 152.35, 161.64, 162.03; MS: 336.10 (M + 2).

(E)-N-(2-((2,3-dichlorobenzylidene)amino)ethyl)-1H-indole-2-carboxamide (IMDCW-8)

The compound was prepared according to the general procedure and was obtained as white solid; M.P: 204–206 °C; Yield: 75%; FT-IR: 3341.9 (R-NH), NH (3420.70), 2898.34 (CH), 1627.94 (C=O); ¹H-NMR (400 MHz; DMSO-_d6): 2.51 (1H, s, CH₂), 3.36 (3H, d, J = 0.6 Hz, CH₂), 7.55–7.58 (4H, dd, CH), 7.84–7.85 (1H, d, J = 7.7 Hz, CH), 7.89 (1H, s, CH), 8.16 (1H, s, CH), 10.33 (1H, s, NH), 11.29 (1H, s, NH); ¹³C-NMR (400 MHz; DMSO-_d6): 12.90, 128.86, 129.27, 133.27, 134.57, 136.18, 189.92; MS: 360.10 (M + 1).

(E)-N-(2-((4-bromobenzylidene)amino)ethyl)-1H-indole-2-carboxamide (IMBW-12)

The compound was prepared according to the general procedure and was obtained as yellow solid; M.P: 202–204 °C; Yield: 70%; FT-IR: 3066 (R-NH), 3217.37 (NH), 2923.2 (CH), 1642.32 (C=O); ¹H-NMR (400 MHz; DMSO-_d6): 3.45 (1H, s, CH₂), 7.08–7.09 (1H, d, J = 4.1 Hz, CH), 7.17–7.28 (2H, d, J = 44 Hz CH), 7.31–7.33 (2H, s, CH), 7.43–7.47 (4H, m, CH), 7.49–7.52 (1H, m, CH), 7.91–7.93 (2H, d, J = 8.0 Hz, CH), 8.01 (1H, s, CH=N), 8.03 (1H, s, NH), 11.58 (1H, s, NH); ¹³C-NMR (400 MHz; DMSO-_d6): 62.60, 112.78, 119.99, 120.14, 121.76, 126.66, 129.03, 129.22, 130.26, 131.30, 131.38, 131.84, 132.24, 142.47; MS: 367.10 (M + 2).

(E)-N-(2-((4-methoxybenzylidene)amino)ethyl)-1H-indole-2-carboxamide (IMOCW-13)

The compound was prepared according to the general procedure and was obtained as light yellow solid; M.P: 236–238 °C; Yield: 88%; FT-IR: 3071.2 (R-NH), 2926.40 (CH), 1649.73 (C=O); ¹H-NMR (400 MHz; DMSO-_d6): 3.33 (2H, s, CH₂), 3.50 (2H, s, CH₂), 3.78 (3H, s, CH₃), 6.65–6.71 (2H, d, J = 5.9 Hz, CH), 6.70 (1H, m, CH), 7.06–7.09 (1H, t, J = 6.9 Hz, CH), 7.12–7.27 (1H, m, CH), 7.39–7.40 (1H, d, J = 4.0 Hz, CH), 7.54–7.56 (2H, d, J = 8.0 Hz, CH), 7.68 (1H, dd, CH), 8.12 (1H, s, CH=N), 8.38 (1H, s, NH), 11.46 (1H, s, NH); ¹³C-NMR (400 MHz; DMSO-_d6): 55.42, 55.70, 59.89, 112.74, 113.82, 114.36,, 119.69, 122.17, 127.72, 128.57, 129.26, 129.99, 135.95, 157.96, 161.63, 161.68, 162.65; MS: 322.10 (M + 1).

(E)-N-(2-((2-methylbenzylidene)amino)ethyl)-1H-indole-2-carboxamide (2CH3-15)

The compound was prepared according to the general procedure and was obtained as a dark brown solid; M.P: 236–238 °C; Yield: 88%; FT-IR: 3064.5 (R-NH), 3251.66 (NH), 2973.6 (CH), 1633.40 (C=O); ¹H-NMR (400 MHz; DMSO-_d6): 2.22 (3H, s, CH₃), 3.59–3.61 (2H, d, J = 8.0 Hz, CH₂), 3.78–3.80 (2H, d, J = 6.4 Hz, CH₂), 6.90–7.13 (2H, m, CH), 7.19–7.27 (3H, dt, J = 8.6 Hz), 7.28–7.34 (1H, d, J = 7.5 Hz, CH), 7.37–7.47 (1H, d, J = 8.9 Hz, CH), 7.53–7.66 (1H, t, CH), 7.78–7.92 (1H, d, J = 7.9 Hz, CH), 8.55 (1H, s, CH=N), 8.60 (1H, s, NH), 11.56 (1H, s, NH); ¹³C-NMR (400 MHz; DMSO-_d6): 60.64, 61.51, 102.84, 112.76, 120.14, 121.91, 123.67, 126.39, 127.55, 127.11, 130.60, 131.19, 131.23, 132.33, 134.43, 136.87, 137.90, 161.26, 161.86; MS: 306.25 (M + 1).

(E)-N-(2-((2,4-dichlorobenzylidene)amino)ethyl)-1H-indole-2-carboxamide (IM24DCW-16)

The compound was prepared according to the general procedure and was obtained as white solid; M.P: 245–247 °C; Yield: 75%; FT-IR: 3250.9 (R-NH), 3414.9 (NH), 2916.7 (CH), 1641.24 (C=O); ¹H-NMR (400 MHz; DMSO-_d6): 3.60–3.64 (2H, d, J = 6.1 Hz, CH₂), 3.85–3.90 (2H, d, J = 6.1 Hz, CH₂), 7.01–7.05 (2H, d, J = 7.5 Hz, CH), 7.08–7.13 (2H, d, J = 1.7 Hz, CH), 7.15–7.21 (2H, d, J = 7.6 Hz, CH), 7.58–7.62 (2H, d, J = 7.6 Hz, CH), 7.72–7.76 (1H, dd, J = 8.0, 1.6 Hz, CH), 7.92–7.97 (1H, dd, J = 7.9, 1.6 Hz, CH), 8.56 (1H, s, CH=N), 8.69 (1H, s, NH), 11.56 (1H, s, NH); ¹³C-NMR (400 MHz; DMSO-_d6): 60.43, 102.90, 112.75, 120.14, 121.92, 123.69, 127.39, 127.39, 127.53, 128.77, 132.15, 132.32, 132.65, 132.75. 135.56, 136.86, 158.66, 161.73; MS: 360.00.

In-vitro studies

Neurotoxicity analysis using MTT method

The compound IMFW-1 was prepared at different dosage concentrations, and the control group (no treatment) group was found to have a % cell viability of 100%. The dosage concentrations of 6.25 and 12.5 µg/mL were found to have the highest % cell viability of 94.59 and 81.88%. The dosage concentration of 25 µg/mL was found to have medium cell viability of 70.67%. The minimum % cell viability was produced at 50 and 100 µg/mL, having 51.42 and 43.13%. The compound IMCW-4 was prepared at different dosage concentrations, and the control group (no treatment) group was found to have a % cell viability of 100%. The dosage concentrations of 6.25 and 12.5 µg/mL were found to have the highest % cell viability of 39.58 and 28.94%. The dosage concentration of 25 µg/mL was found to have medium cell viability of 22.65%. The minimum % cell viability was produced at 50 and 100 µg/mL, having 18.17 and 11.77%. The compound IMBW-6 was prepared at different dosage concentrations, and the control group (no treatment) group was found to have a % cell viability of 100%. The dosage concentrations of 6.25 and 12.5 µg/mL were found to have the highest % cell viability of 56.86 and 45.59%. The dosage concentration of 25 µg/mL was found to have medium cell viability of 24.60%. The minimum % cell viability was produced at 50 and 100 µg/mL, having 22.08 and 19.68%. The compound IMNW-7 was prepared at different dosage concentrations, and the control group (no treatment) group was found to have a % cell viability of 100%. The dosage concentrations of 6.25 and 12.5 µg/mL were found to have the highest % cell viability of 38.65 and 33.27%. The dosage concentration of 25 µg/mL was found to have medium cell viability of 29.32%. The minimum % cell viability was produced at 50 and 100 µg/mL, having 22.63 and 17.83%. The compound IMDCW-8 was prepared at different dosage concentrations, and the control group (no treatment) group was found to have a % cell viability of 100%. The dosage concentrations of 6.25 and 12.5 µg/mL were found to have the highest % cell viability of 77.98 and 65.84%. The dosage concentration of 25 µg/mL was found to have medium cell viability of 58.46%. The minimum % cell viability was produced at 50 and 100 µg/mL, having 39.82 and 19.67%. The compound IMBW-12 was prepared at different dosage concentrations, and the control group (no treatment) group was found to have a % cell viability of 100%. The dosage concentrations of 6.25 and 12.5 µg/mL were found to have the highest % cell viability of 70.57 and 44.70%. The dosage concentration of 25 µg/mL was found to have medium cell viability of 22.73%, as shown in Fig. 11.

Fig. 11.

Viable cells at different dosage concentrations for the top synthesized compounds. The cells were treated with the five dosage concentrations of 6.25, 12.5, 25, 50, and 100 µg/mL; the increased dosage concentrations were found to decrease the % viability of the cells.

The compound IMOCW-13 was prepared at different dosage concentrations, and the control group (no treatment) group was found to have a % cell viability of 100%. The dosage concentrations of 6.25 and 12.5 µg/mL were found to have the highest % cell viability of 79.51 and 54.93%. The dosage concentration of 25 µg/mL was found to have medium cell viability of 27.75%. The minimum % cell viability was produced at 50 and 100 µg/mL, having 18.57 and 14.77%. The compound 2-CH3-15 was prepared at different dosage concentrations, and the control group (no treatment) group was found to have a % cell viability of 100%. The dosage concentrations of 6.25 and 12.5 µg/mL were found to have the highest % cell viability of 54.15 and 35.59%. The dosage concentration of 25 µg/mL was found to have medium cell viability of 24.13%. The minimum % cell viability was produced at 50 and 100 µg/mL, having 22.71 and 19.61%. The compound IM24DCW-16 was prepared at different dosage concentrations, and the control group (no treatment) group was found to have a % cell viability of 100%. The dosage concentrations of 6.25 and 12.5 µg/mL were found to have the highest % cell viability of 92.98 and 87.12%. The dosage concentration of 25 µg/mL was found to have medium cell viability of 73.06%. The minimum % cell viability was produced at 50 and 100 µg/mL, having 66.07 and 37.01%. The compound IMTW-5 was prepared at different dosage concentrations, and the control group (no treatment) group was found to have a % cell viability of 100%. The dosage concentrations of 6.25 and 12.5 µg/mL were found to have the highest % cell viability of 80.66 and 70.47%. The dosage concentration of 25 µg/mL was found to have medium cell viability of 62.75%. The minimum % cell viability was produced at 50 and 100 µg/mL, having 51.49 and 42.87%, as shown in Fig. 12.

Fig. 12.

Graphical representation and % viable cells at different dosage concentrations for the top synthesized compounds IMOCW-13, 2CH3-15, IM24DCW-16, and IMTW-5. The cells were treated with the five dosage concentrations of 6.25, 12.5, 25, 50, and 100 µg/mL; the increased dosage concentrations were found to decrease the % viability of the cells; % viability (X-axis), different dosage concentrations (X-axis). One-way ANOVA and Dunnett’s test were performed to analyze data. ***p < 0.001 compared to control groups, **p < 0.01 compared to control groups.

Neuroprotection analysis using (MTT method)

The top 3 compounds in Fig. 13, IMFW-1, IMTW-5, and IM24DCW-16, were selected for neuroprotection studies; these compounds had higher % cell viability in the neurotoxicity studies. The compound IMFW-1 was prepared at a different dosage concentration of 1.5 µg/mL, 3.1 µg/mL, 6.25 µg/mL, 12.5 µg/mL, and 25 µg/mL. The control (no treatment) group was found to have a % cell viability of 100%. The toxicity was induced in the SHSY-5Y neuroblastoma cell lines using MPP+ at a dosage concentration of 0.5 mM. The induction of MPP+ decreased the cell viability from 100% to 47.77%. The dosage concentrations of 1.5 µg/mL and 3.1 µg/mL increased the cell viability to 52.66% and 55.29%. The dosage concentrations of 6.25 and 12.5 µg/mL increased the cell viability to 66.32% and 79.10%. The compound IMTW-5 was prepared at a different dosage concentration of 1.5 µg/mL, 3.1 µg/mL, 6.25 µg/mL, 12.5 µg/mL, and 25 µg/mL. The control (no treatment) group was found to have a % cell viability of 100%. The toxicity was induced in the SHSY-5Y neuroblastoma cell lines using MPP+ at a dosage concentration of 0.5 mM. The induction of MPP+ decreased the cell viability from 100% to 47.77%. The dosage concentrations of 1.5 µg/mL and 3.1 µg/mL increased the cell viability to 51.01% and 68.50%. The dosage concentrations of 6.25 and 12.5 µg/mL increased the cell viability to 79.22% and 70.92%. Cell viability decreased by 62.99% at a dosage concentration of 25 µg/mL. The compound IM24DCW-16 was prepared at a different dosage concentration of 1.5 µg/mL, 3.1 µg/mL, 6.25 µg/mL, 12.5 µg/mL, and 25 µg/mL. The control (no treatment) group was found to have a % cell viability of 100%. The toxicity was induced in the SHSY-5Y neuroblastoma cell lines using MPP+ at a dosage concentration of 0.5 mM. The induction of MPP+ decreased the cell viability from 100% to 47.77%. The dosage concentrations of 1.5 µg/mL and 3.1 µg/mL increased the cell viability to 53.31% and 61.50%. The dosage concentrations of 6.25 and 12.5 µg/mL increased the cell viability to 69.77% and 83.80%. Cell viability decreased at 74.70% at a dosage concentration of 25 µg/mL.

Fig. 13.

Neuroprotective effect of (1A) IMFW-1; (2A) IMTW-5; (3A) IM24DCW-16 in the SHSY-5Y neuroblastoma cell lines at different dosage concentrations. The % cell viability was evaluated in the three groups: control (untreated), toxicity-induced (MPP+ -0.5 mM), and the top compounds (treated); the 25 µg/mL dosage concentrations were determined to have the highest % cell viability. Y-axis (% cell viability), X-axis (compound concentrations). ***p < 0.001 contrast to non-treated groups, **p < 0.01 contrast to non-treated groups.

Enzyme estimations assays

Estimation of the lactate dehydrogenase assay (LDH)

The levels of LDH were measured in the SHSY-5Y neuroblastoma cell lines. In the control (no-treatment) group, LDH levels were found to be 140.386 U/mL (Fig. 14A). The toxicity was induced in the SHSY-5Y cell lines using MPP+ at a dosage concentration of 0.5 mM. LDH level was measured to be 643.53 U/mL due to MPP+. Further, the cells were treated with the compounds IMFW-1, IMTW-5, and IM24DCW-16 at 12.5 µg/mL. The compound IMFW-1 was found to decrease LDH levels to 375.82 U/mL. The compound IMTW-5 was found to maintain LDH levels to 304.90. The compound IM24DCW-16 was found to decrease LDH levels to 221.64 U/mL. IM24DCW-16 was the most promising compound in reducing LDH levels.

Fig. 14.

(A) LDH activity; (B) Enzyme units SOD2; (C) Fold change of GPx. The compounds IMFW-1, IMTW-5, and IM24DCW-16 were found to decrease LDH levels and were found to upregulate the expression of SOD2 and GPx; overexpression of the targets may result in the downregulation of ROS species. ***p < 0.001 compared to control groups, **p < 0.01 compared to control groups.

Estimation of the superoxide dismutase assay (SOD2)

The control group was found to have a SOD2 expression enzyme unit of 1.568724 U/mL, which indicated the normal functioning of SOD2 (Fig. 14B). The toxicity was induced by using MPP+ at a dosage of 0.5 mM. Due to the toxicity, the expression of SOD2 was reduced to 0.531791 U/mL. In the treated group with the IMFW-1 compound, the expression of SOD2 was increased to 1.062412 U/mL, which indicated an increase in SOD2 expression. The compound IMTW-5 was found to be mildly effective towards SOD2 expression, as the enzyme units obtained were 1.041141 U/mL, which suggested that compound IMTW-5 was less ineffective than IMFW-1. The compound IM24DCW-16 was found to have promising activity in increasing the expression of SOD2, whereas the compound was found to bring the expression of SOD2 to 1.295465 U/mL. Among the best promising compounds, IM24DCW-16 was found to have promising activity toward the SOD2 enzyme.

Estimation of the glutathione peroxidase (GPx)

In the control group (no treatment), the enzyme unit was 0.779486 U/mL (Fig. 14C). The toxicity was induced using the MPP⁺ with a dosage concentration of 0.5 mM; the enzyme unit was decreased to 0.318971 U/mL, which indicated the probable chances of activity reduction of the enzyme. The groups were treated with the compounds; the compound IMFW-1 was found to upregulate the fold-change to 0.430611 U/mL. The compound IMTW-5 was found to upregulate the fold-change to 0.41865 U/mL. The compound IM24DCW-16 was found to increase the enzyme concentration to 0.568167 U/mL.

Reactive oxygen species estimation by flow cytometry

The ROS levels were estimated for the control, toxicity-induced, and treated groups (Fig. 15) with compounds IMFW-1, IMTW-5, and IM24DCW-16 at 12.5 µg/mL. The control groups were found to have 96.30% of M1 cells (ROS negative cells) and 2.96% of M2 cells (ROS positive cells). Toxicity was induced in the cells by using MPP+ at a dosage concentration of 0.5 mM; M1 cells were found to be 40.21%, and MPP+ induction caused a decrease in the M1 cells; the level of M2 cells was found to be 58.16%, suggesting an upregulation of ROS species. The cells were treated with compounds IMFW-1, IMTW-5, and IM24DCW-16 at 12.5 µg/mL. The IMFW-1 and MPP+ induction was found to have 79.48% of M1 cells and 20.49% of M2 cells. The compound IMTW-5, along with MPP+ induction, was found to have 86.02% of M1 cells and 13.75% of M2 cells. The compound IM24DCW-16, along with MPP+ induction, was found to have 87.02% M1 cells and 12.78% M2 cells. IM24DCW-16 was found to be the active compound in controlling the ROS levels. The downregulation of ROS levels and oxidative stress could result in mitochondrial biogenesis and cell survival.

Fig. 15.

ROS level estimation using FACS; (A) ROS profile of control group; (B) ROS profile of MPP+ group; (C) ROS profile of compound IMFW-1; (D) ROS profile of the compound IMTW-5; (E) ROS profile of compound IM24DCW-16; (F) Graphical representation of ROS generation by FACS analysis and were analyzed using the Mean ± Standard Deviation.

Determination of mitochondrial membrane potential by flow cytometry

An electrochemical gradient energy produced during the electron transport chain process forms the mitochondrial trans-membrane potential (∆Ψm), which generally facilitates the formation of ATP. The collapse of these gradient channels results in the formation of pores, and the cytochrome c leaks, resulting in the apoptosis process. Figure 16 illustrates the mitochondrial membrane potential; in the control group, the depolarized live cells were found to be 4.76%, live cells were found to be 83.28%, and dead cells were found to be 11.94%. MPP+ (0.5 mM) toxicity-induced group, depolarized live cells were 11.18%, live cells were 59.97%, and dead cells were 28.55%. In the IMFW-1 treated group, depolarized live cells were found to be 4.60%, live cells were found to be 68.63%, and dead cells were found to be 26.67%. In the IMTW-5 treated group, the depolarized live cells were found to be 29.76%, live cells were found to be 62.91%, and dead cells were found to be 7.08%. In the IM24DCW-16 treated group, the depolarized live cells were found to be 3.71%, the live cells were found to be 72.07%, and the dead cells were found to be 24.12%.

Fig. 16.

Population profile and cell health profile of cells; (A) Population profile and cell health profile of control cells; (B) Population profile and cell health profile of toxicity induced with MPP+ (0.5 mM); (C) Population profile and cell health profile of MPP+ /IMFW-1 treated cell; (D) Population profile and cell health profile of MPP+ /IMTW-5 treated cells; (E) Population profile and cell health profile of MPP+ /IM24DCW-16 treated cells.

Gene expression in SHSY-5Y neuroblastoma cells by real-time PCR analysis

SIRT3 expression fold change for the control group was found to be 1; after inducing the toxicity to the gene with the MPP+ at a dosage of 0.5 mM, the fold change of the gene was reduced to 0.23766; the treatment was provided with compounds IMFW-1, IMTW-5, and IM24DCW-16 at a dosage of 12.5 µg/mL. The IMFW-1 was found to increase the expression of SIRT3 by 1.786399-fold change, whereas compound IMTW-5 was found to increase the expression of SIRT3 by 1.600124-fold change. The compound IM24DCW-16 was found to increase the expression of SIRT3 by 1.954842; the study presented that the compound IM24DCW-16 was the most active compound towards SIRT3. The PGC-1α expression fold change for the control group was found to be 1; after inducing the toxicity with MPP+ at a dosage of 0.5 mM, the fold change expression of the gene was reduced to 0.261914, treatment was given to the groups with compounds IMFW-1, IMTW-5, and IM24DCW-16 at a dosage concentration of 12.5 µg/mL. The IMFW-1 compound was found to increase the expression of PGC-1α by 1.87716-fold change, whereas compound IMTW-5 was found to increase the expression of PGC-1α by 1.898246-fold change, and IM24DCW-16 was found to increase the expression of PGC-1α by 1.927095. The FOXO3 expression fold change for the control group was found to be 1; after inducing the toxicity to the gene with MPP+ at a dosage concentration of 0.5 mM, the fold change of the gene was reduced to 0.261914, the treatment was given to the groups with the compounds IMFW-1, IMTW-5, and IM24DCW-16 at a dosage of 12.5 µg/mL. The IMFW-1 was found to increase the expression of FOXO3 by 1.119355-fold change, whereas compound IMTW-5 was found to increase the expression of FOXO3 by 1.252321-fold change, and compound IM24DCW-16 was found to increase the expression of FOXO3 by 1.302149-fold change (Fig. 17). The primer sequences used and gene expression amplification curves in SHSY-5Y neuroblastoma cells by Real-time PCR analysis have been provided in Figure S3, Tables S4, and S5 in the supplementary files. These findings indicated the overexpressing potency of the compounds in toxicity-induced conditions, and overexpression of these targets could also result in the detoxification of the ROS, leading to cellular survival.

Fig. 17.

(A) Photomicrograph of a typical 1.5% agarose gel depicting the gene expression pattern of PGC-1α, FOXO3, SIRT3, and housekeeping gene GAPDH in SHSY-5Y cells (i) untreated control (ii) 1-Methyl-4-phenylpyridinum (MPP+) (0.5 mM) treated Cells (iii) IMFW-1–12.5 µg/mL (iv) IMTW-5–12.5 µg/mL and (v) IM24DCW-16–12.5 µg/mL; (B) Gene expression levels with compounds IMFW-1, IMTW-5, and IM24DCW-16 at 12.5 µg/mL dosage concentrations.

Discussion

The current study investigated the efficacy of the novel synthesized indole derivatives as SIRT3 modulators in PD. The study included rigorous in-silico, synthesis, characterization, and detailed in-vitro studies, which provided a comprehensive understanding of the therapeutic efficacy of the compounds. SIRT3 is the nicotinamide adenine dinucleotide (NAD⁺), containing 275 residues that catalyze mutated proteins’ deacetylation; the 275 amino acid regions are mainly involved in the full enzymatic activity, which leads in the deacetylating function, moreover, binding of the molecules to this region could increase the activity of the SIRT3³¹. SIRT3 contains a catalytic core containing the Rossmann fold and zinc-binding domain, which binds to NAD⁺, with additional N and C terminals; these catalytic core domains also highlight conserved portions that are critical for catalytic activity with additional structural and enzymatic function of the protein; The C-terminal is primarily involved with protein stability and folding concerning other mitochondrial proteins. The synthesized compounds were found to modulate amino acids like Ala, Phe, Gln, Val, and Ser at the N-terminal, which contains a mitochondrial targeting sequence with 100–130 amino residues. This suggests that these allosteric sites are crucial for protein stability, and our compounds were found to have a strong interaction with these residues³². SIRT3 has been associated with the modulation of ROS levels and enhancing mitochondrial biogenesis, thereby providing neuronal survival against oxidative or cellular stress³³. According to a recent study, indole (NC001-8) was able to activate the expression of NRF2, a crucial governing body in the activation of the anti-oxidant mechanism, and reduce ROS levels in the MPP+-treated SH-SY5Y neuroblastoma cell line by 50% when compared to the control cells; the findings from our study predicted IM24DCW-16 (indole-based molecule) as the most active compound among the synthesized compounds as a significant modulator of SIRT3, where it also reduced the ROS levels in the stress conditions with the MPP⁺-treated condition; this outcome aligned the importance of indole molecules in targeting SIRT3 in producing mitochondrial biogenesis and its efficacy in the PD; SIRT3 has been found to modulate targets such as SOD2, FOXO3, GPx, and PGC-1α that downregulate ROS levels and oxidative stress via the mitochondrial process^34,35. The in-silico findings suggested altering the different biological active groups, which might be important in improving the compounds’ biological activity. Essential hydrophobic bonding residues like Phe, Val, His, and Ile, as well as essential hydrogen bonding residues like Gly, Glu, and Leu, were shown in the SIRT3 complex with AceCS-2 in the earlier study by Chiara et al. the study concluded that these amino acid residues are crucial for supplying the critical enzymatic function³⁶; Our findings from the in-silico studies further guided the synthesis of the novel indole-based derivatives; molecular docking studies provided an understanding of the essential pharmacophoric features, such as AA_RRR, essential in the constructive binding of the novel indole compounds with the SIRT3. The molecular docking studies predicted IMOCW-13, IMCW-4, and IMFW-1 compounds with the highest docking of − 9.86, − 8.71, and − 7.87 kcal/mol with the SIRT3, PDB ID: 4fvt; essential amino acid interactions such as His (decrease the enzymatic activity), Phe (downregulate the catalysis process of the enzyme), Arg (essential for the acetyl-lysine binding which facilitates the deacetylation of the enzyme) were visualized through this study. These results indicated the significance of the compounds in target modulation and their ability to promote SIRT3 expression and reduce ROS levels, which suggested that they might be further employed in novel pharmacological treatments to slow the progression of Parkinson’s disease. Recent research has also demonstrated the potential effectiveness of NC009 derivatives, which are an indole-based series, in reducing neuroinflammation and protein buildup in neurodegenerative diseases. The compounds with the highest binding affinity for SIRT3 demonstrated exceptional resilience of the complex in the solvent environment; the findings suggested that the molecules are hydrophobic, indicating blood–brain barrier permeability. The synthesized compounds IMFW-1, IMTW-5, and IM24DCW-16 revealed a considerably high percentage of cell viability and neuroprotective activity, implying that they could be employed as therapeutic agents in Parkinson’s disease. The cell toxicity study predicted that lower dosage concentrations of the compounds had less toxicity than higher dosages; in the MPP⁺-toxicity-induced stress condition, the compounds were found to halt further cellular damage, resulting in a higher percentage of cell viability in the SHSY-5Y neuroblastoma cell lines. SIRT3 has been known for its various activities, among which it has been known to modulate LDH activity, for instance in oxidative stress or dietary restriction, SIRT3 can shift its position to aerobic metabolism and decrease lactate production, and can decrease ATP production; overexpressed LDH levels into the cells can reveal the signs of tissue damage, our synthesized compounds illustrated the downregulation of the LDH levels in the stress condition which also suggest the mitochondrial biogenesis³⁷. The therapeutic efficacy of these compounds imparted useful study outcomes clarifying their involvement in mitochondrial biogenesis and cellular survival; additionally, these compounds could support the SIRT3 mechanism of action and its pathways involved in Parkinson’s disease progression. During the electron transport chain process, a few electrons escape from the membrane and convert into ROS and free radical groups; an overexpressed form of the ROS levels results in oxidative stress and neuronal death; SOD2 breaks the free radical groups (OH^-) into the weaker form of the radical group hydrogen peroxide, and the hydrogen peroxide (H₂O₂) is further broken down into the water molecule in the presence of the GPx enzyme³⁸, The results revealed that the produced compounds enhanced the expression of SOD2 and GPx, implying that they should further reduce ROS levels. Even though ROS species are involved in vital processes like immune responses and cellular signaling pathways like apoptosis, they are also closely linked to DNA, protein, and lipid damage. Overexpressed ROS species have been shown to impair mitochondrial functions, which suggests that suppressed antioxidant genes like SOD2, GPx, and FOXO3 may be the primary cause of these overexpressed ROS species. Among the synthesized compounds, IM24DCW-16 was found to overexpress these targets, and it was also found to decrease the ROS level in the groups that were exposed to MPP+ toxicity^39,40. Since mitochondria are the main source of ROS species and excessive production of these species has been known to damage mitochondrial species, including lipids and mitochondrial DNA, the compound IM24DCW-16 showed 72.07% of the live cells compared to the toxicity-induced group’s 59.97%; this suggested that the mitochondrial membrane permeability was enhanced and unharmed; It is known that oxidative stress reduces the potential of the mitochondrial membrane, releasing cytochrome c and triggering apoptosis⁴¹. The overexpression of these targets was significantly impacted by the compounds, with IM24DCW-16 being the most active of the produced compounds. PGC-1α has been shown to alter mitochondrial activity, improving several targets related to antioxidant defense systems and oxidative stress situations. It has been discovered that FOXO3 increases its ability to go to the nucleus, upregulating antioxidant targets that are crucial under oxidative stress, such as SOD2 and GPx. Overall, FOXO3 and SIRT3 work together to downregulate ROS levels and oxidative stress, with SIRT3 serving as the primary regulator of PGC-1α. The significance of PGC-1α in the regulation of mitochondrial metabolism and oxidative stress was demonstrated by a prior study by Bin et al. Pioglitazone and CoQ10 were found to increase PGC-1α expression in a swine model of hibernating myocardium, which downregulated ROS and improved cardiac and mitochondrial biogenies. The results of our study also showed that the compound IM24DCW-16 increased PGC-1α expression in stress conditions with MPP+-treated^42,43. PGC-1α and SIRT3 have been known to provide a protective axis that restores metabolic pathways, reduces inflammation, and promotes cellular recovery during oxidative stress conditions. Figure 18 illustrates the research and theory, offering insightful information about the molecule’s mechanisms and significance in successfully binding to SIRT3, which may aid in creating strong therapeutics that target SIRT3-mediated pathways in Parkinson’s disease.

Fig. 18.

The illustration represents the possible route mechanism involved in downregulating the ROS level and oxidative stress; this is a probable mechanism for the synthesized compounds.

Conclusion

The research has exhibited computational and experimental investigations within this framework that have produced encouraging outcomes for developing novel indole-based SIRT3 modulators. The in-silico studies identified the pharmacophoric features helping the compounds to yield more potent biological activities. Using the appropriate techniques, the designed compounds were synthesized and characterized. The novel indole carboxamide derivatives IMW 1–16 are presented in the study as influential modulators of the target SIRT3. The neuroprotective assay discovered that the most potent compounds were IMFW-1, IMTW-5, and IM24DCW-16. In the experimental validation, which included RT-PCR studies, IM24DCW-16 was the most potent compound. Here, we showed the effectiveness of the compound IMW 1–16 in modulating SIRT3 expression. Compared to the reference molecule resveratrol, the compounds were found to have less oral toxicity. These results highlight the synthesized novel molecules and their therapeutic potential in deacetylating SIRT3 and reducing ROS levels and oxidative stress. The research highlights the compound IM24DCW-16 as a potent molecule in advancing oxidative stress and SIRT3/ROS-based Parkinson’s disease.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Author contributions

Jagdish Chand: Funding acquisition, Methodology, Project administration, Software, Writing—original draft. Srikanth Jupudi: Investigation, Methodology, Project administration, Writing–original draft. Sheikh F. Ahmad: Investigation, Methodology, Project administration, Writing–original draft. Talha Bin Emran: Formal Analysis, Writing–review and editing. Gomathy Subramanian: Funding acquisition, Methodology, Supervision, Visualization, Writing–original draft, Writing–review and editing.

Funding

This research was supported by King Saud University, Riyadh, Saudi Arabia, Project Number (RSPD2025R709).

Data availability

The original contributions presented in the study are included in the article/Supplementary material, further inquiries can be directed to the corresponding authors.

Declarations

Competing interests

The authors declare no competing interests.