Targeting the AKT Pathway as a Therapeutic Strategy for Hepatocellular Carcinoma

Targeting the AKT Pathway as a Therapeutic Strategy for Hepatocellular Carcinoma

*Oluseyi Adeboye Akinloye¹, Adewumi Kamarudeen Aremu^1,2, Ibukun Dorcas Akinloye¹, Jacob Kehinde Akintunde¹, Olajire Moshood Olaniyi³, Antiya Moses Ceaser¹.

¹Department of Biochemistry, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria

²Department of Basic Science, Kwara State College of Education, Oro, Kwara State, Nigeria

³Department of Veterinary Pathology, Federal University of Agriculture, Abeokuta, Ogun State, Nigeria

⃰ Corresponding Author

DOI: https://doi.org/10.51244/IJRSI.2024.1111005

Received: 10 October 2024; Accepted: 19 October 2024; Published: 25 November 2024

ABSTRACT

Hepatocellular carcinoma (HCC), one of the most common cancers of the liver has been on the rise globally and is complicated by drug resistance in treatment targets and pathway interactions such as the Akt pathway. Akt-1 protein also known as serine/threonine kinase is a good target for designing drugs for hepatocellular carcinoma. It controls cell development, survival, proliferation, glycogen consumption, and apoptosis. The overexpression of Akt-1 is a hallmark of various cancers including HCC, and plays a crucial role in tumorigenesis and cancer progression. The 3D crystal structure of human Akt-1 with an allosteric inhibitor was downloaded from a protein data bank. The preparation of ligands was done by downloading structures from the PubChem database and converted to 3D using Open Babel. In this study, phytochemicals (ligands) from some Nigerian medicinal plants were docked against the binding pocket of Akt 1 using virtual high throughput screening. Validation of docking results was done to determine the accuracy of the docking methods. The drug-like properties of the lead compounds and the standard drug were tested by employing the Lipinski rule of five. Silymarin has the highest docking score of -10.6 kcal/mol and was found to be the lead compound as a potential inhibitor with the best absorption, distribution, metabolism, and excretion properties. Silymarin interactions with key amino acid residues in the active sites were determined and compared with the reference drug, sorafenib. It was found that both silymarin and sorafenib exhibit similar interactions with the formation of hydrogen bonds, pi-cation, and pi-pi interactions. Silymarin has a relatively better inhibitory and pharmacokinetic profile than, vicenin, epig allocate chin-3-gallate, ginsenoside, and palstatin, thus it can be a useful therapeutic candidate in the treatment of HCC.

Keywords: Hepatocellular carcinoma, Akt, Silymarin, Molecular docking, Phytochemicals.

INTRODUCTION

A class of disorders known as Cancer is the uncontrolled growth of abnormal cells that have the potential to invade and destroy healthy tissues in the body. With a predicted 9.6 million fatalities in 2018, it is the second-leading cause of mortality worldwide and can spread throughout the body.

There are various types of cancer, including lung, prostate, colorectal, breast, cervical, and thyroid1.

Cancer development may be influenced by a mix of hereditary and environmental factors. For example, exposure to ultraviolet light and tobacco smoke from the sun can damage DNA, or inheriting damaged DNA from parents canal so cause DNA damage2. As weage, our body’s ability to destroy cells with damaged DNA before they become malignant reduces, which raises our chance of developing cancer at a later age3. About 80% of instances of liver cancer are attributed to hepatocellular carcinoma (HCC), the most prevalent kind of primary liver cancer. The development of HCC is closely linked to chronic liver disease, which is brought on by several risk factors such as alcohol use, metabolic syndrome, and persistent infections with the hepatitis B and C virus (HCV and HBV). Hepatitis B virus infection, which accounts for about 50% of cases, is the most significant risk factor for the development of HCC. The beginning and stimulation of many signaling pathways, including the PI3K/Akt pathway, are typically associated with the onset and progression of HCC. This system is crucial for regulating a cell’s metabolism, development, and survival; disruption of it has been connected to the genesis of several malignancies, including HCC4.

Silymarin, a bioflavonoid isolated from milk thistle plant (Silybum marianum) seeds, has been the subject of much research due to its possible anti-cancer effects including the inhibition of cell proliferation and the induction of apoptosis 5. It exhibits antioxidant, anti-inflammatory, hepatoprotective, anti-proliferative, pro-apoptotic, and anti-metastatic effects 6. In addition, silymarin delays the activation of hepatic stellate cells, which are important contributors to liver fibrosis, and encourages liver regeneration. The capacity of silymarin to scavenge free radicals and strengthen endogenous antioxidant defense systems accounts for much of its antioxidant action. Modifying multiple signaling pathways implicated in inflammation, like NF-kB and MAPK, reduces inflammatory reactions7. Silymarin has been shown to modulate the phosphorylation of Akt, thus influencing its activities such as inhibition of Akt activation, activate pro-apoptotic proteins like Bad and Bax, inhibit anti-apoptotic proteins like Bcl-2 and Bcl-xL, increase the release of cytochrome c from mitochondria, leading to the activation of caspases and cell death7,8
The Akt signaling pathway, also referred to as PI3K/Akt/mTOR pathway, is a critical regulator of cell growth, survival, and metabolism 8. It is activated by the phosphorylation of Akt at two key residues, Thr308 and Ser473, which are mediated by phosphoinositide 3-kinase (PI3K) and other upstream kinases 8. Growth factors including epidermal growth factor (EGF) and insulin-like growth factor 1 (IGF-1) link to their corresponding receptor tyrosine kinases (RTKs) to activate phosphatidylinositol 3-kinase (PI3K), which in turn activates this pathway. PIP3 is produced when PI3K phosphorylates PIP2 (phosphatidylinositol 4,5-bisphosphate), and PIP3 acts as a docking site for Akt at the plasma membrane. After being recruited, Akt is fully activated when phosphoinositide-dependent kinase 1 (PDK1) and mammalian target of rapamycin complex 2 (mTORC2) phosphorylate it at serine 473 and threonine 308 residues, respectively 9.

A typical characteristic of HCC is dysregulation of the Akt signaling system, which plays a role in the development, growth, and resistance to treatment of the tumor. In HCC, it is common to find upstream regulators such as RTKs and PI3K amplification, overexpression, or activating mutations, which cause Akt to remain activated over time. After activation, Akt stimulates the growth of HCC cells by phosphorylating and blocking a number of downstream targets that are involved in the regulation of the cell cycle, such as glycogen synthase kinase 3 beta (GSK3β), forkhead box protein O (FoxO), and inhibitors of cyclin-dependent kinases (e.g., p21 and p27)10. Furthermore, Akt signaling promotes the survival of HCC cells by preventing apoptosis by phosphorylating and deactivating pro-apoptotic proteins such as caspase-9 and Bad 11.

The Akt signaling pathway has gained attention as a potential therapeutic target for the treatment of HCC due to its crucial role in the pathophysiology of the disease. Preclinical research has shown that Akt inhibitors are effective in reducing the growth of tumors in animal models, causing apoptosis, and slowing the proliferation of HCC cells when used alone or in conjunction with other targeted treatments11.

METHODOLOGY

Retrieval and refining of target receptor proteins

The three-dimensional crystal structure of human AKT-1 with an allosteric inhibitor was downloaded from aprote in data bank (PDB:http://www.rcsb.org/pdb/) which has the PDBID code 3o96. The receptor was set up using Py MOLX 11 software. Before running docking simulations, water molecules, hetero atoms, and the co-crystallized inhibitors were taken out 12.

Ligands preparation

Sixty different compounds from 10 medicinal plants reported in the literature were obtained from the PubChem database (https://pubchem.ncbi.nlm.nih.gov) and downloaded in the Structure data format (SDF). These compounds were sorted based on chemical properties, such as molecular weight, H-bond donor, and H-bond acceptor. These compounds were chosen and open babel was used to convert them from their SDF formats to the protein data bank (PDB) formats.

Molecular docking

Phytochemicals were docked against the binding pocket of Akt 1 using virtual high throughput screening (vHTS), a computational method for screening a pool of chemical libraries against the target receptor. The MGL tools of the Auto Dock 4.2 version were used to create the pdbqt file for each protein. The rotatable bonds of the ligand were set to be free, whereas the protein target was considered a stiff body. All of the catalytically significant amino acid residues were included in grid maps that were 22.50, 22.50, and 22.50 (x, y, and z), with grid points spaced 12.82, 15.68, and 13.13 angstroms apart, respectively. In order to guarantee that the ligands occupy the same binding pocket, the grid center coordinates were exactly the same as those of the co-crystallized compounds. Auto dock Vina was used for molecular docking 13.

Validation of docking results

To check the accuracy of the docking methods, the co-crystallized ligand (IQO) was once again docked into the catalytic site of Akt 1. The deviation was accessed using root means square deviation (RMSD), a measure of the co-crystallized divergence from its initial geometry when re-docked. The docking approach was accurate and suitable for use in the docking of additional ligands to the receptor when the RMSD value for the re-docked pose and the co-crystallized pose is less than 2.0. As a general rule, a docking method can be declared valid if the RMSD value is ≤ 2.0Å13.

ADMET screening

Swiss ADME, a free online software developed by14 was adopted through the website http://SwissADME.ch/ to determine the physicochemical and pharmacokinetic properties of Akt inhibitors. The chemical structure of each ligand was submitted to Swiss ADME server in form of a canonical simplified molecular-input line-entry system (SMILES), which was obtained from PubChem. Upon calculation submission by clicking the “Run” button, the SMILES of each compound are canonicalized by Open Babel (version 2.3.0, 2012) and processed. This was used to examine and filter the molecules based on their adsorption, distribution, metabolism, and excretion (ADME) 15. These parameters were used to identify their drug-likeness and toxic nature. The compounds were strictly screened based on Lipinski’s rule16. Any compound that did not satisfy this rule was exempt from performing further experimental studies due to its limitations in being an ideal drug-like molecule. Also, the toxicity (mutagenicity and carcinogenicity) of nominated compounds was evaluated by using Discovery Studio.

Protein-Ligand Complex Conformational Analysis

Silymarin and co-crystallized (IQO) complexed with Akt 1 separately in the pdb format using PyRx and then submitted it on Discovery Studio, an online server. Discovery Studio automatically generated the PoseView (2D) diagrams of the macromolecular complexes.

RESULTS AND DISCUSSION

In this study phytochemicals such as glyzarin, Licoflavonol, Silymarin, Kumatakenin, Salvianolic acid, Podophyllotoxin, Psoralen, Paclitaxel, Actein, Vicenin, Amooranin, Procyanidins, Plumbagin, Schizophyllan, Saffron, Aspalathin, Anthocyanidins, Aloin, Phyllanthin, Quercetin, Epigallocate chin -3-gallate, Ginsenoside, Genistein, Combretastatins, Xanthatin, Palstatin, Orientin, and Bullatacin were docked against Akt-1 binding site revealed that silymarin, vicenin, epigallocate chin -3-gallate, ginsenoside and palstatin, having the highest binding scores of -10.6 kcal/mol, -10.4 kcal/mol, -10.4 kcal/mol, -10.5 kcal/mol and -10.4 kcal/mol respectively and are referred to as lead compounds as shown in table 1.

Table 1: Physicochemical properties of the phytochemicals of the lead compounds docked with Akt-1 as predicted by Swiss ADME

Phytochemicals	Docking Score (kcal/mol)	Mwt <500 (g/mol) 482.40	TPSA 140	Lipinski Violation	HBA ≤10	HBD ≤5	Log P ≤ 5
Silymarin	-10.6	482.4	155.14	0	10	5	-2.07
Vicenin	-10.4	594.5	271.2	3	15	11	-2.07
Epigallocate chin -3-gallate	-10.4	458.37	197.37	2	11	8	1.01
Ginsenoside	-10.5	785.01	218.99	3	13	9	2.45
Palstatin	-10.4	524.47	157.28	2	11	4	2.64

Figure 1: 3D structure of Akt 1with co-crystallised ligand, (green), (PDB. ID 3o96)

Figure 2: Validation of the docking protocol employed with co-crystallised ligand (red) before docking and after docking (blue). The co-crystallised ligands overlaps almost perfectly, with an RMSD of 0.8376 indicating accuracy in the docking protocol employed

Validation of the docking site

The validation of the docking site with co-crystallised ligand (red) before docking and after docking (blue). The co-crystallised ligand overlaps almost perfectly, with an RMSD value of 0.8376 indicating accuracy in the docking protocol employed as shown in figure 2. The validation was done to ensure that silymarin was actually docked into the active site of Akt-1

Lipinski’s rule of five (RO5)

According to Lipinski17 the RO5 is useful in assessing drug-likeness or determining whether a chemical molecule with a certain pharmacological or biological activity has characteristics that make it a potential orally active medication in humans. An oral active medication, in Lipinski’s opinion, shouldn’t break more than one of the following guidelines: Four requirements must be met: (1) a molecular mass of less than 500 Daltons; (2) a maximum of 10 hydrogen bond acceptors; (3) a maximum of 5 hydrogen bond donors; and (4) an octanol-water partition coefficient log-P not exceeding 5. As shown in Table 1, silymarin showed a docking score of -10.6 which did not violate Lipinski’s rule of five. It was reported that a more negative docking value indicates stronger interaction between ligands and protein which leads to inhibition of protein activity18 therefore this result proves that Silymarin has relatively better inhibitory activity and may pose lesser side effects than other phytochemicals.

Additionally, other significant properties such as total polar surface area (TPSA) and the number of rotatable bonds were also determined by Swiss ADME (Table 2). TPSA predicts drug potential for oral bioavailability and ability to reach target sites within the body, drugs with a TPSA greater than 150 Å2 or a number of rotatable bonds above 10 leads to lessened oral bioavailability and permeability 19. In addition, novel 1,2,3-triazolyl-pyridine derivatives showed promising anticancer activity against HepG2 HCC cells compared to the reference drug doxorubicin. However, 1,2,3-triazolyl-pyridine derivatives violate Lipinski’s rule of 5 with respect to molecular mass (>500 g/mol) and topological polar surface area (TPSA >140 Å2) 20. According to Veber21 TPSA is not the only factor that determines the bioavailability and permeability of drugs. Other factors include, lipophilicity (log P), based on Lipinski’s rule of five, log-P not greater than 5, such drugs have good oral and intestinal absorption22. A number of rotatable bonds are also an important determinant of drug oral bioavailability, such as hydrogen bond donors and acceptors are predictors of oral bioavailability23.

The findings of this study show silymarin has the lowest TPSA of 155.14 Å2 compared to other phytochemicals of the study and has several rotatable bonds of 4 which is good for oral bioavailability. Silymarin is an inhibitor of CYP3A4. Inhibition of CYP3A4 has previously been found to reduce the probability of drug clearance from the systemic circulation and improve drug bioavailability 24. Also, silymarin has a lower log P (log P 1.9) and therefore obeys the Lipinski rule. However, silymarin will have good permeability across cell membranes and potentially good oral bioavailability.

P-glycoprotein (P-gp)is an ATP-dependent trans membrane efflux pump that transport tsdrugsa way from the cytoplasm and cell membrane causing compounds to undergo farther metabolism and clearance, thereby limiting cellular uptake of drugs resulting in therapeutic failure because the drug concentration would be lower than expected. The result demonstrates that Silymarin is not a substrate of P-gp, which indicates that P-gp activity cannot affect its absorption, distribution, and elimination 25. Due to the non-substrate of silymarin to P-gp, silymarin is less likely to be subject to drug-drug interactions mediated by P-gp inhibition or induction 26.

Table 2: Pharmacokinetics properties of the lead phytochemicals as predicted by Swiss ADME

Parameters	Silymarin	Vicenin	Epigallocatechin -3-gallate	Ginsenoside	Palstatin
GI Absorption	low	low	low	low	low
P-gp substrate	No	Yes	No	No	No
Log Po/w	1.9	-1.98	0.95	6.75	2.59
Rotatable bonds	4	1	0	4	6
Lipinski	0	3	2	1	2
Oral Bioavalability	55%	17%	17%	55%	17%
CYP1A2 inhibitor	No	No	No	No	No
CYP2C19 inhibitor	No	No	No	No	No
CYP2C9 inhibitor	No	No	No	No	Yes
CYP2D6 inhibitor	No	No	No	No	No
CYP3A4 inhibitor	Yes	No	No	No	Yes

Figure 3: 2D interaction view of co-crystallized ligand (IQO) in the binding pocket of Akt -1 upon QM/MM assisted docking.

Figure 4: 2D interaction view of silymarin in the binding pocket of Akt -1 upon QM/MM assisted docking.

Figure 5: 2D interaction view of sorafenib (standard drug) in the binding pocket of Akt -1 upon QM/MM assisted docking.

Analysis of amino-acids interactions of the silymarin and standard compounds

The interactions with key amino acid residues in the active site are of great importance while influencing the inhibitory activity of Akt-1 inhibitors. The interactions of amino acid residues with the active site of Akt-1 are shown in Figures 3, 4, and 5 for the co-crystallized ligand, silymarin, and sorafenib (standard drug) respectively. Silymarin and Sorafenib exhibit similar interactions with the co-crystallized ligand (IQO) with the formation of hydrogen bonds, pi-cation, and pi-pi interactions. The protein-ligand interaction plays a prominent role in the structural-based drug design. The interaction between amino acid residues and ligands enables stability in the protein-ligands complex. The amino residues that participated in stabilizing the protein-ligand complex in silymarin and Akt 1 are Cys-296, Glu-85, and Val-271. These amino acid residues formed an H-bond with Akt-1, hydrogen bonds play an important role in the structural stability of many biological molecules and enzyme catalysis. Other molecular interactions between silymarin and Akt-1 are π–π stacking (Trp-80) and π-cation (Arg-273). The amino residues and sorafenib that are involved in interaction to make the complex stable are similar to silymarin/Akt- 1 interaction these are, H-bond (Tyr-272 and Val- 271), π–π stacking (Tyr-272 and Trp-80) and halogen bond (Glu-298). Also, co-crystallized ligand (IQO) interacted and stabilized with amino-acids residues such as Val-271 (H-bond), Trp-80 (π–π stacking) and Tyr-272, Arg-273 (π-cation). Therefore, silymarin could serve as a potential inhibitor of Akt-1in the treatment of hepatocellular carcinoma.

CONCLUSION

The results of this study have shown that silymarin binds and inhibits Akt-1. The compound has a relatively better inhibitory and pharmacokinetic profile than, vicenin, epigallocate chin -3-gallate, ginsenoside, and palstatin, thus it can be a useful therapeutic candidate in the treatment of HCC. In vivo and or in vitro assays are required to validate the docking result experimentally to demonstrate the potential of the compound in treating HCC.

Ethical Statement

No ethical issue is to be declared

Competing Interests

No conflicts of interest

ACKNOWLEDGEMENT

The authors wish to appreciate everyone who has contributed to the success of this study.

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