In silico approach towards the Anti-Obesity effects of Flaxseed Components Alpha Linolenic Acid and Secoisolariciresinol Diglucoside by the inhibition of SOCS3 – The inhibitor of Leptin Signalling Pathway

doi:10.21203/rs.3.rs-3573136/v1

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In silico approach towards the Anti-Obesity effects of Flaxseed Components Alpha Linolenic Acid and Secoisolariciresinol Diglucoside by the inhibition of SOCS3 – The inhibitor of Leptin Signalling Pathway

https://doi.org/10.21203/rs.3.rs-3573136/v1

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Objective

- Medications used as adjuncts to assist obese and overweight individuals in losing weight often come with numerous adverse effects and primarily target absorption and hormone levels, without addressing the underlying signalling issue. The aim of this paper is to focus on studying the potential role of flaxseed compounds, particularly Secoisolariciresinol diglucoside and α-Linolenic acid in obesity prevention and management.

Methods

- Molecular docking studies using Glide were performed to assess the interactions of with the target protein SOCS3. The stability of the binding was evaluated through Molecular Dynamics Simulation studies using Desmond.

Results

- The study revealed that the compounds Secoisolariciresinol diglucoside (SDG) and α-Linolenic acid shows high binding affinity with the target with a lest docking score of -9.00 and − 8.32 Kcal/mol respectively when compared with the control ligand Zoledronic acid which has a binding energy of -6.03 Kcal/mol.. The SOCS3-SDG complex exhibited a higher number of hydrogen bonds compared to the control complex, SOC3-Zoledronic acid complex, indicating greater stability of the complex.

Conclusion

- In- silico studies conducted demonstrate that both SDG and ALA exhibited favourable and stable interactions with the target protein SOCS3. This suggests that these compounds may impede the binding of leptin to SOCS3 by binding to the SH2 domain of the protein, hence exhibiting promising potential as nutraceuticals for obesity prevention

Obesity

Secoisolariciresinol diglucoside

α-Linolenic acid

Flaxseed

Nutraceutical

SOCS3

Obesity is a chronic disease caused by an imbalance in energy intake and expenditure, resulting in the accumulation of excess body fat (1). It is a complicated condition with multiple causes, including genetic, environmental, and behavioural factors, and is linked to serious health risks such as cardiovascular disease, type 2 diabetes, and certain types of cancer (2). Obesity has emerged as a major global public health concern, 39% of adults were reported to be overweight and 18% of children between the ages 5–19 were reported to be either overweight or obese in 2016 (3).

Obesity has a strong genetic component, with estimates ranging from 40–70% heritability (4). Researchers have identified several genes associated with an increased risk of obesity, including those involved in appetite regulation, metabolism, and energy expenditure (5). For instance, mutations in the melanocortin-4 receptor gene (MC4R) have been linked to severe early-onset obesity in both humans and animals (6) However, genetic factors alone cannot fully account for the current obesity epidemic, as obesity prevalence has risen dramatically in recent decades, suggesting the importance of environmental and behavioural factors (7). Obesity is associated with variety of health problems, such as cardiovascular disease, type 2 diabetes, certain cancers, and musculoskeletal disorders (8). The increased risk of these diseases in obesity is mediated by multiple physiological processes, including insulin resistance, chronic inflammation, and dyslipidemia (9).

Leptin is a hormone produced by adipose tissue that regulates energy balance and body weight. It signals the brain to reduce food intake and increase energy expenditure, while also playing a role in glucose metabolism, reproductive function, and immune function (10). In obesity, there is a decrease in sensitivity to Obesity is characterised by leptin resistance, or a decreased sensitivity to leptin known as leptin resistance, caused by chronic exposure to leptin, desensitizing the leptin receptor and impairing its signalling (11). Leptin resistance can lead to increased food intake and decreased energy expenditure, which may contribute to the development of obesity. Factors such as inflammation, oxidative stress, and changes in the gut microbiome contribute to leptin resistance (12). Inflammation and oxidative stress inhibit leptin receptor function, while changes in the gut microbiome impair leptin signalling (13,14). The JAK2-STAT3 pathway is involved in leptin signalling, and excessive or chronic stimulation of this pathway can lead to the development of leptin resistance (15).

SOCS3 (Suppressor of Cytokine Signalling 3) is a protein that negatively regulates leptin signalling and helps maintain metabolic homeostasis. It inhibits leptin signalling by binding to and inhibiting the activity of JAK2, the signalling protein. This prevents the activation of downstream transcription factor STAT3, which is necessary for the expression of genes involved in food intake and energy expenditure regulation. SOCS3 plays a role in limiting the signalling response to leptin, preventing the development of leptin resistance by inhibiting JAK2 (16). SOCS3 has been linked to the development of leptin resistance and obesity in mice. SOCS3 deletion or knockdown in the hypothalamus, for example, improves leptin sensitivity and protects against diet-induced obesity (17). Overexpression of SOCS3 in the hypothalamus, on the other hand, causes leptin resistance and obesity (18,19). These findings highlight the significance of SOCS3 in regulating leptin signalling and the development of metabolic disorders.

Although diet and exercise are the primary treatments for obesity, medications can be used as a supplement to aid in weight loss. Some of the drugs used to treat obesity include orlistat, liraglutide, phentermine, and naltrexone/bupropion (21). However, these medications often come with various side effects such as blood pressure fluctuations and flatulence (22). These drugs only act on the level of absorption and at the level of hormone itself but do not address the root issue of the signalling. The potential health benefits of flaxseeds in relation to obesity have received particular attention. Flaxseeds are small brown or golden seeds high in fibre, omega-3 fatty acids, lignans, and other bioactive compounds. They have been used for thousands of years for medicinal purposes and have been shown to have a variety of health benefits, including improved digestive health, reduced inflammation, and lower cholesterol levels. Flaxseeds have also been investigated for their potential role in obesity prevention and management (23). Keeping this in view, we have taken the flaxseed compounds as the ligands of study. It is important to note that these medications primarily act on absorption or hormone levels and do not address the underlying signalling issue. Taking this into consideration, our study focusses on the investigation of flaxseed compounds as potential ligands.

The flax seed compounds ALA, also known as α-linolenic acid and Secoisolariciresinol diglucoside (SDG) were reported to have health benefits. ALA is a naturally occurring antioxidant found in foods such as spinach, broccoli, and potatoes. Some studies suggest that ALA may have weight loss and obesity benefits by improving insulin sensitivity. However, more research is needed to fully understand its effects, determine optimal dosages, and treatment duration (24). SDG is a compound found in flaxseeds and other plant-based foods. It has been studied for its potential role in weight management. There is evidence suggesting that SDG may aid in weight management for obese individuals. For instance, one study found that SDG supplementation led to reductions in body weight, BMI, and waist circumference in overweight and obese postmenopausal women. However, further research is necessary to validate these findings and establish optimal dosages and treatment durations for SDG supplementation (25). Hence this study aims to study the anti-obesity activity of flaxseed components especially Secoisolariciresinol diglucoside (SDG) and Alpha Linolenic acid (ALA) through in silico methods by assessing the binding capability of the selected compounds with the target protein SOCS3.

Molecular docking studies.

The Protein Data Bank (PDB) was used to obtain the X-ray crystal structure of the target protein SOCS3 (PDB ID: 2HMH) (26). The ligand molecules SDG (CID: 9917980), ALA (CID: 5280934) and the control ligand Zoledronic Acid (CID: 68740) were retrieved from Pubchem. Maestro was used to visualize the downloaded structures (Schrödinger LLC, 2022-2). The Protein Preparation Wizard of Schrödinger suite was used to prepare the protein for molecular docking. Similarly, the ligand molecules were prepared using LigPrep. The glide grid file was created using the receptor grid generation panel, where the co-crystallized ligand situated at the protein's active site was selected to uncover the corresponding x, y, and z coordinates (-31.02, 21.89, and 30.09) automatically. The molecular docking was done with Glide software (27). The 2D plot of the interactions were plotted with the help of LigPlot + v.2.2.8. software.

Molecular Dynamics Simulation.

All simulations were run with the OPLS-AA force field using the academic version of the MD simulation software, Desmond 6.9 (28). Desmond uses a specific neutral territory technique dubbed the midpoint approach to effectively take advantage of a high level of parallelism in the computation. The initial system for the MD simulation was set using the system builder panel. The complexes were built in the orthorhombic box and solvated with SPC water models (29). The systems were equilibrated using the NPT ensemble, based on the default desmond options. The systems were then subjected to a 150ns simulation production run with a relaxation time of 2ps. During the simulation, 1 atm pressure and 26.85°C temperature of the system were set using a Martyna-Klein barostat (30) and Nose-Hoover chain thermostat(31)

To evaluate the potential effects of flaxseed compounds in managing obesity and diabetes, molecular docking and molecular dynamics simulations were conducted with the target protein SOCS3.

Molecular Docking Studies.

The molecular docking studies of SOCS3 with the flaxseed components especially Alpha-linolenic acid (ALA) and Secoisolariciresinol diglucoside (SDG) was done and Zoledronic acid, an inhibitor of SOS3 was taken as control. The study revealed that the compounds SDG and ALA shows high binding affinity with the target with a least docking score of -9.00 and -8.32 Kcal/mol respectively than that of the control ligand Zoledronic acid which has a binding energy of -6.03 Kcal/mol. The active site residues include Leu93, Leu104, Gln105, Ser106, Asp107, Arg109, Ser110, Gln112, Val114, Pro115, Tyr127, Arg140, Tyr142, Tyr143, Tyr145, Leu153. The residue Arg140 has been identified as playing a significant role in establishing hydrogen bonds with the oxygen atoms of compound ALA. Additionally, SDG (specific compound) formed five hydrogen bonds with the target, involving the residues Asp107, Leu104, Tyr143, Tyr145, and Tyr127. It is worth noting that the tyrosine residues contribute significantly to both the formation of hydrogen bonds and hydrophobic interactions with SDG. The residues interacting with the ligands forming hydrogen bonds and other interactions for each complex are shown in Table 1. The 2D plot of these interactions are shown in Fig 3, 4 and 5. Hence, from these results it can be inferred that the compounds ALA and SDG may have the potential for the inhibition of SOCS3 protein and thereby may have anti-obesity properties.

Table 1. Docking Results of SOCS3 with control and flaxseed components.

S.No.	Complex	Docking Score (Kcal/mol)	Interacting Residues (H Bonds)	Interacting Residues (Others)
1	2hmh – Zoledronic Acid (Control)	-6.03	Tyr107, Ser110, Tyr143, Lys150.	Ser106, Tyr127, Tyr142.
2	2hmh – Alpha-linolenic acid	-8.32	Arg140 (2)	Ser106, Asp107, Arg109, Ser110, Gln112, Pro115, Tyr127, Tyr142, Tyr143, Tyr145.
3	2hmh – Secoisolariciresinol diglucoside	-9.00	Leu104, Asp107, Tyr127, Tyr143, Tyr145.	Leu93, Gln105, Ser110, Gln112, Val114, Tyr142, Ile144, Leu153.

Molecular Dynamics Simulation

To evaluate the stability of the top docked complex, we performed molecular dynamics (MD) simulations on SOC3-SGD complex. Additionally, MD simulations of SOC3-Zoledronic acid complex was performed for comparison purposes. The simulations were conducted for a duration of 150 nanoseconds to capture potential conformational changes and system equilibration, ensuring a thorough assessment of the stability and dynamics of the complexes. The Root Mean Square Deviation (RMSD) of the targets in conjunction with the ligand molecules was monitored throughout the simulations. The RMSD indicates the extent of deviation of the atoms within the complexes from their initial reference structure. A high RMSD value suggests significant instability in the complex. The RMSD of the SGD bound complex exhibited a low value initially, and a slight increase was observed after 30 ns of the simulation. However, this increase in RMSD was subsequently found to stabilized. Comparatively both the control and SGD showed similar deviations. The Root Mean Square Fluctuation (RMSF) was utilized to assess the flexibility of the residues in the target proteins within the chosen complexes. Fluctuations were observed in 100 to 115 residues, which was also observed in the control, which might be due to the flexible nature of the residues.

Fig 6: RMSD of the complexes

Fig 7: RMSF of the protein upon binding of A) Secoisolariciresinol diglucoside and B) Zoledronic acid

Fig 8: Interaction histogram of the complexes of A) SOCS3 – Secoisolariciresinol diglucoside and B) SOSC3 – Zoledronic acid

The hydrogen bonds, hydrophobic interaction and salt bridges were calculated and the hydrogen bonds were assessed to validate the stable interactions from the docking results throughout the simulation time. The SOCS3-SDG complex exhibited a higher number of hydrogen bonds compared to the control complex, indicating greater stability of the complex.

SOCS3 is composed of an N-terminal region, a central SH2 domain, and a C-terminal SOCS3 box. The SH2 domain plays a crucial role in inhibiting signaling proteins either directly or competitively. Leptin, a hormone produced by fat cells, plays a crucial role in regulating hormonal and metabolic responses to changes in nutritional status by binding to and activating the long form of the leptin receptor (LRb) found in the hypothalamus. When a ligand binds to LRb, it triggers the activation and tyrosine phosphorylation of Jak2, which leads to the subsequent tyrosine phosphorylation of Tyr985 and Tyr1138 on LRb. The administration of leptin prompts the activation of STAT3-mediated transcription, resulting in the induction of SOCS3, which in turn, inhibits LRb signaling. In cell models of leptin resistance and certain obese animals, the levels of cellular SOCS3 are elevated, suggesting its role as a likely mediator of feedback inhibition on the leptin receptor and a potential contributor to physiological leptin resistance (18)

Both the molecules were found to be deeply fit in the active pocket occupying the SH2 domain (46–142 residues). The docking results indicate that both compounds, ALA and SDG, have a strong binding affinity with the target compared to the control drug. It is hypothesized that this binding could prevent the binding of leptin to SOCS3, thereby enabling leptin to bind to its receptor and inhibiting leptin resistance. Upon ligand binding, no changes in conformation were observed, and there were no significant fluctuations, indicating the stability of the bound complexes. SDG exhibited a greater number of interactions compared to ALA. Based on these results, both SDG and ALA can be considered as potential nutraceuticals for combating obesity.

Flaxseed has earned attention for its anti-obesity effects. While it has been utilized in traditional medicine for its therapeutic properties, there is a lack of studies investigating the specific mechanisms through which it combats obesity. Therefore, this in silico study was conducted to assess the potential activity of two compounds found in flaxseed, SDG and ALA, in regulating obesity via Leptin signalling. The results of this study demonstrate that both SDG and ALA exhibited favourable and stable interactions with the target protein SOCS3. This suggests that these compounds may impede the binding of leptin to SOCS3 by binding to the SH2 domain of the protein. Consequently, these compounds hold promise as potential lead compounds for obesity control. It is important to note that further research is needed to validate these findings and determine the optimal utilization of flaxseed components in combating obesity. By addressing the underlying signalling issues associated with obesity, these compounds could offer a promising approach for future obesity prevention and management strategies.

Authorship Contribution:

Concept- M.A.; Design- M.A.; Supervision- M.A., F.A.J.; Materials- S.S.; Data collection and processing- S.S., B.M.M.; Analysis and interpretation- S.S., B.M.M.; Literature search- S.S; M.S.R; Writing- M.A., S.S., B.M.M.; M.S.R; Critical review- M.A., F.A.J.

World Health Organization. Obesity and overweight [Internet]. World Health Organization. 2022 [cited 2023 Apr 27]. Available from: https://www.who.int/news-room/fact-sheets/detail/obesity-and-overweight.
Centre for Disease control and Prevention. Adult Obesity Facts | Overweight and Obesity. 2020.
World Health Organisation. Obesity and Overweight. 2022.
Visscher TLS, Seidell JC. The public health impact of obesity. Annu Rev Public Health [Internet]. 2001 [cited 2023 Apr 27];22:355–375. doi: 10.1146/ANNUREV.PUBLHEALTH.22.1.355. Cited: in: : PMID: 11274526.
Cawley J, Meyerhoefer C. The medical care costs of obesity: An instrumental variables approach. J Health Econ. 2012;31:219–230. doi: 10.1016/j.jhealeco.2011.10.003.
Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults--The Evidence Report. National Institutes of Health. Obes Res. 1998;6 Suppl 2:51S-209S. Cited: in: : PMID: 9813653.
Puhl RM, Heuer CA. Obesity Stigma: Important Considerations for Public Health. Am J Public Health. 2010;100:1019–1028. doi: 10.2105/AJPH.2009.159491.
Malik VS, Willett WC, Hu FB. Global obesity: trends, risk factors and policy implications. Nat Rev Endocrinol. 2013;9:13–27. doi: 10.1038/nrendo.2012.199.
Haslam DW, James WPT. Obesity. The Lancet. 2005;366:1197–1209. doi: 10.1016/S0140-6736(05)67483-1.
Friedman J. 20 YEARS OF LEPTIN: Leptin at 20: an overview. Journal of Endocrinology. 2014;223:T1–T8. doi: 10.1530/JOE-14-0405.
Chen J, Haase N, Haange S-B, Sucher R, Münzker J, Jäger E, Schischke K, Seyfried F, von Bergen M, Hankir MK, et al. Roux-en-Y gastric bypass contributes to weight loss-independent improvement in hypothalamic inflammation and leptin sensitivity through gut-microglia-neuron-crosstalk. Mol Metab. 2021;48:101214. doi: 10.1016/j.molmet.2021.101214.
Qatanani M, Lazar MA. Mechanisms of obesity-associated insulin resistance: many choices on the menu. Genes Dev. 2007;21:1443–1455. doi: 10.1101/gad.1550907.
Bjorbaek C. Leptin Signaling in the Central Nervous System and the Periphery. Recent Prog Horm Res. 2004;59:305–331. doi: 10.1210/rp.59.1.305.
Davis CD. The Gut Microbiome and Its Role in Obesity. Nutr Today. 2016;51:167–174. doi: 10.1097/NT.0000000000000167.
Sahu A. Leptin signaling in the hypothalamus: emphasis on energy homeostasis and leptin resistance. Front Neuroendocrinol. 2003;24:225–253. doi: 10.1016/j.yfrne.2003.10.001.
Kwon O, Kim KW, Kim M-S. Leptin signalling pathways in hypothalamic neurons. Cellular and Molecular Life Sciences. 2016;73:1457–1477. doi: 10.1007/s00018-016-2133-1.
Howard JK, Flier JS. Attenuation of leptin and insulin signaling by SOCS proteins. Trends in Endocrinology & Metabolism. 2006;17:365–371. doi: 10.1016/j.tem.2006.09.007.
Bjørbæk C, Lavery HJ, Bates SH, Olson RK, Davis SM, Flier JS, Myers MG. SOCS3 Mediates Feedback Inhibition of the Leptin Receptor via Tyr985. Journal of Biological Chemistry. 2000;275:40649–40657. doi: 10.1074/jbc.M007577200.
Zhang X, Zhang G, Zhang H, Karin M, Bai H, Cai D. Hypothalamic IKKβ/NF-κB and ER Stress Link Overnutrition to Energy Imbalance and Obesity. Cell. 2008;135:61–73. doi: 10.1016/j.cell.2008.07.043.
Lubis AR, Widia F, Soegondo S, Setiawati A. The role of SOCS-3 protein in leptin resistance and obesity. Acta Med Indones. 2008;40:89–95. Cited: in: : PMID: 18560028.
Yanovski SZ, Yanovski JA. Long-term Drug Treatment for Obesity. JAMA. 2014;311:74. doi: 10.1001/jama.2013.281361.
Myers MG, Leibel RL, Seeley RJ, Schwartz MW. Obesity and leptin resistance: distinguishing cause from effect. Trends in Endocrinology & Metabolism. 2010;21:643–651. doi: 10.1016/j.tem.2010.08.002.
Goyal A, Sharma V, Upadhyay N, Gill S, Sihag M. Flax and flaxseed oil: an ancient medicine & modern functional food. J Food Sci Technol. 2014;51:1633–1653. doi: 10.1007/s13197-013-1247-9.
Fiore G, Pascuzzi MC, Di Profio E, Corsello A, Agostinelli M, La Mendola A, Milanta C, Campoy C, Calcaterra V, Zuccotti G, et al. Bioactive compounds in childhood obesity and associated metabolic complications: Current evidence, controversies and perspectives. Pharmacol Res. 2023;187:106599. doi: 10.1016/j.phrs.2022.106599.
Dodin S, Cunnane SC, Mâsse B, Lemay A, Jacques H, Asselin G, Tremblay-Mercier J, Marc I, Lamarche B, Légaré F, et al. Flaxseed on cardiovascular disease markers in healthy menopausal women: a randomized, double-blind, placebo-controlled trial. Nutrition. 2008;24:23–30. doi: 10.1016/j.nut.2007.09.003.
Bergamin E, Wu J, Hubbard SR. Structural Basis for Phosphotyrosine Recognition by Suppressor of Cytokine Signaling-3. Structure. 2006;14:1285–1292. doi: 10.1016/j.str.2006.06.011.
Friesner RA, Banks JL, Murphy RB, Halgren TA, Klicic JJ, Mainz DT, Repasky MP, Knoll EH, Shelley M, Perry JK, et al. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy. J Med Chem. 2004;47:1739–1749. doi: 10.1021/jm0306430.
Bowers KJ, Sacerdoti FD, Salmon JK, Shan Y, Shaw DE, Chow E, Xu H, Dror RO, Eastwood MP, Gregersen BA, et al. Molecular dynamics---Scalable algorithms for molecular dynamics simulations on commodity clusters. Proceedings of the 2006 ACM/IEEE conference on Supercomputing - SC ’06. New York, New York, USA: ACM Press; 2006. p. 84.
Mark P, Nilsson L. Structure and Dynamics of the TIP3P, SPC, and SPC/E Water Models at 298 K. J Phys Chem A. 2001;105:9954–9960. doi: 10.1021/jp003020w.
Martyna GJ, Tuckerman ME, Tobias DJ, Klein ML. Explicit reversible integrators for extended systems dynamics. Mol Phys. 1996;87:1117–1157. doi: 10.1080/00268979600100761.
Hoover WG. Canonical dynamics: Equilibrium phase-space distributions. Phys Rev A (Coll Park). 1985;31:1695–1697. doi: 10.1103/PhysRevA.31.1695.

No competing interests reported.

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In silico approach towards the Anti-Obesity effects of Flaxseed Components Alpha Linolenic Acid and Secoisolariciresinol Diglucoside by the inhibition of SOCS3 – The inhibitor of Leptin Signalling Pathway

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