INTRODUCTION:

Stroke is the world’s second leading cause of death and the third leading cause of serious disability in adults, caused by a lack of oxygen delivery to the brain for more than or equal to 24 hours, or leads to death and causing focal or global disturbance of cerebral function¹. Infarction or rupture of blood vessels based on pathological or imaging evidence of persistent retinal, brain, or spinal cord cell death, with or without the presence of clinical symptoms, is defined as a vascular etiology^2,3.

Strokes can be classified into two types. Ischemic stroke, which is the most frequent type of stroke is caused by a disruption in the blood supply to a part of the brain, resulting in a rapid loss of function, whereas hemorrhagic stroke is caused by a blood vessel rupture⁴.

Lack of oxygen in the brain cells is one of the stimuli that triggers the activation of Phosphatidyl-inositol 3-kinase (PI3K) signaling pathways to further trigger cellular activity such as proliferation, differentiation, cell survival, and apoptosis for neuroprotection⁵. The PI3K/AKT signaling pathway activation modulates cellular activities like synaptic plasticity, neuronal cell proliferation, and migration. Hypoxia can induce PI3K/Akt/mTOR signaling pathway, which can modulate Hypoxia-inducible factor-1 alpha (HIF-1α) protein synthesis and inhibits prolyl hydroxylase enzyme activity, which can stabilize HIF-1 and activate the HIF-1a transcriptional response^6,7.

Hypoxia-inducible factor-1 alpha (HIF-1α) is the member of the HIF complex that consists of HIF-1α/β. HIF-1α promotes the expression of genes involved in cell survival mechanisms, such as glucose metabolism, angiogenesis, and erythropoiesis as a response to hypoxia and ischemia⁸. Hypoxia inhibits the activity of prolyl hydroxylase (PHD) enzyme and von Hippel−Lindau (VHL) protein, which is one of the identified components of an E3 ubiquitin-protein ligase, induces the accumulation of HIF-1α and boost its transcriptional activity⁹. Furthermore, the HIF complex interacts with transcription co-activator p300/CPB and binds with Hypoxia-response Elements (HRE) in DNA 5’-G/ACGTG-3’ sequence to further bind to a hypoxia-response gene promoter and stimulate the transcription of the hypoxia-response gene and regulate adaptive response to hypoxia by promoting cell survival, anaerobic metabolism, and angiogenesis, including endothelial nitric oxide (eNOS), erythropoietin (EPO), glucose transporter 1 (GLUT1), heme oxygenase 1 (HO-1), and vascular endothelial growth factor (VEGF)^10-14.

Treatment for ischemic stroke is limited to tissue plasminogen activator administration. The increasing prevalence of ischemic stroke, along with a lack of effective treatments, emphasizes the urgent need for more research to find safe and effective neurotherapeutics that affect the pathophysiological pathways and assist a wider number of stroke patients. The development of stroke therapeutics is focused on preventing neuronal death and improving recovery from ischemic injury, and there are still no medicines that can provide neuroprotection after an ischemic stroke. Preclinical studies use biological compounds that have neurotrophic activities against ischemic stroke injury, such as 7,8-dihydroxyflavone (DHF). Naturally, 7,8-DHF present in a wild plant such as Godmania aescufolia, Tridax procumbens, and various species of Primula, which acts as a protein kinase stimulant, a prolyl hydroxylase inhibitor, and also VHL protein inhibitor^15,16. These mechanisms have the potential to activate an endogenous adaptive response to low oxygen levels, making them a therapeutic target for ischemic stroke^1,8,15.

Drug design research studies take a long time, but screening to determine the potential of a compound as a drug candidate at this time can be done using the in silico approach¹⁷. In silico simulation of drug design interactions can predict an interaction mechanism between the ligand and the target protein, as well as the bond energy that occurs¹⁸. Through an in-silico approach, this study will predict the molecular interaction mechanism that occurs between 7,8-DHF and the protein target in order to develop an ischemic stroke treatment.

MATERIALS AND METHODS:

Ligand and Protein Preparation:

The ligand used in this experiment was 7.8-dihydroxyflavone (DHF), which was obtained from PubChem (http://pubchem.ncbi.nlm.nih.gov/) as a .sdf file. While the proteins Phosphoinositide 3-Kinase Gamma (PDB ID: 1E8X) and prolyl hydroxylase 2 (PDB ID: 5OX6) were obtained from RSCB website ((https://www.rcsb.org/search)¹⁹. The OpenBabel in PyRx software was used to carry out the ligand minimization process, which allowed the ligands to be more flexible, then change the file structure data format (sdf) into protein databank format (pdb)²⁰. Protein stabilization was carried out to adjust to the body's physiology using BIOVIA Discovery Studio by removing water and hydrogen atoms²¹. The control ligands used downloaded from RSCB in the form of .sdf. control ligands used was Adenosine-5'-Triphosphate for Phosphoinositide 3-Kinase Gamma and Vadadustat for prolyl hydroxylase 2.

Drug-likeness and Biological Activity Prediction:

The ligand compound of 7,8-DHF was analysed pharmacokinetically as a drug candidate through SwissADME webserver (http://swissadme.ch) and followed by drug-likeness analysis using the Lipinski Rule of Five through the website Lipinski Rule of Five (scfbio-iitd.res.in)^22,23,24. Furthermore, Quantitative structure-activity relationship is determined by running Way2Drug Prediction of Activity Spectra for Substance (http://www.pharmaexpert.ru/passonline/) to screen the bioactivity of the ligands as a neuroprotector agent²⁵. The expected bioactivity is a protein kinase stimulant.

Molecular Docking:

The binding energy value formed as a result of the ligand interactions with its receptor is determined through molecular docking^21,26. In this work, specific docking refers to comparing the binding energies of 7,8-DHF and control ligand with the same binding site. To perform molecular docking simulations, this work employs Vina Wizard in the PyRx program. The PDBQT file format was used to examine the receptor and ligand files^20,27.

Protein-ligand Interaction Analysis:

Molecular visualization of docking results showed in the PyMol and BIOVIA Discovery Studio software. Analysis of protein-ligand bonds was conducted based on the interaction and type of bond of the 7.8-DHF that binds to target proteins^28,29. The Discovery Studio program was used to analyze the ligand-protein interactions. Following docking with the previous VinaWizard, the program will provide a representative 2D schematic representation of the complex bond between the ligand and the receptor^30,31.

RESULT:

Drug-likeness and The Biological Activity Prediction of 7,8-dihydroxyflavone

The pharmacokinetic study was conducted using SwissADME webserver to predict Human Intestinal Absorption (HIA), ligand capacity to cross the Blood Brain Barrier (BBB), and plasma protein binding. The HIA, BBB, and plasma protein binding result displayed on (Table 1).

Table 1. Pharmacokinetic Study of 7,8-dihydroxyflavone

HIA	BBB	Plasma Protein Binding
92.63%	0.93	93.02%

The Lipinski Rule of Five is used for checking compounds for drug similarity based on criteria. 7,8-DHF compound was downloaded in 3D with .sdf format from the PubChem website (http://pubchem.ncbi.nlm.nih.gov/) to further checked on the Lipinski Rule of Five website (http://www.scfbio-iitd.res.in/software/drugdesign/lipinski.jsp). The drug likeness prediction result shown in (Table 2).

Furthermore, the 7,8-DHF compound was analyzed to study the bioactivity of the ligands as a neuroprotector agent using PASS (http://www.pharmaexpert.ru/passonline/). The result of biological activity prediction shown in (Table 3).

Molecular Docking:

The prediction of the ability of 7,8-DHF compounds is carried out through the molecular docking method. The binding energy value formed when the ligands interact with the receptor is shown in table 4. The molecular docking results showing a comparison of bond energy formed between 7,8-DHF compound and the native ligand with its targeted protein.

Protein-ligand Interaction Analysis:

The active site of the protein receptor can be known from the analysis of the bond between the protein receptor and the native ligand. Molecular docking was conducted to determine the bond strength of 7,8-DHF in the previously known active site of the target protein. The active site of the protein receptor and the docking grid center shown in (Table 5). The docking grid center is used to adjust the ligand-binding position to the target domain.

Table 2. Lipinski Rule of Five Test Results of 7,8-dihydroxyflavone

Compound	Molecular weight	Hydrogen bond donor	Acceptor bond donor	LogP	Molar refractivility	Lipinski Rule
7,8-DHF	254 g/mol	2	4	2.71	69.15	Yes

Table 3. Biological activity prediction of 7,8-dihydroxyflavone on PASSOnline

Compound	Probable activity	Probable inactivity	Biological activity
7,8-DHF	0.377	0.004	Protein kinase stimulant

Table 4. Binding Affinity of Protein Target and Ligand

Protein Target	RSCB ID	Binding Affinity (Kcal/mol)
Protein Target	RSCB ID	Native Ligand		7,8-DHF
PI3K	1E8X	Adenosine-5’-Triphosphate	-7.5	-8.6
HIF Prolyl Hydroxylase	5OX6	Vadadustat	-7.5	-7.5

Table 5. Dimension, active site, and docking centre grid of the docking with 7,8-dihydroxyflavone

Protein Target

Native Ligand

Active Site

Dimension

Docking Center Grid

PI3K

Adenosine-5’-Triphosphate

Asp161

Asp164

Asp358

Met804

Ser806

Lys807

Lys808

Pro810

Ile831

Lys833

Asp836

Tyr867

Glu880

Val882

Asp884

Asn951

Met953

Ile963

Asp964

X: 25 Amstrong (Å)

Y: 25 Amstrong (Å)

Z: 25 Amstrong (Å)

X: 21.441

Y: 61.673

Z: 20.633

HIF Prolyl hydroxylase

Vadadustat

Gln243

Ser247

Asp250

Trp258

Lys262

Gly288

Ser289

Tyr290

Met299

Tyr303

Tyr310

Arg312

His313

Asp315

Tyr329

Leu343

Glu357

Lys359

Trp367

Arg371

Pro373

His374

Val376

Arg383

Arg396

Arg398

Val401

Gly409

X: 50 Amstrong (Å)

Y: 50 Amstrong (Å)

Z: 50 Amstrong (Å)

X: 14.196

Y:123.269

Z: -0.296

Figure 1. Visualization of molecular interaction between protein target to the 7,8-DHF and its native ligand. (A) interaction between ligands and PI3K (PDB ID: 1E8X) and (B) interaction between ligands and prolyl hydroxylase enzyme (PDB ID: 5OX6)

(A) (B)

Figure 2. Type Bond formed between ligands and the protein target. (A) bond formed between 7,8-DHF and PI3K (PDB ID: 1E8X); (B) bond formed between Adenosine-5’-Triphosphate and PI3K (PDB ID: 1E8X).

(A) (B)

Figure 3. Type Bond formed between ligands and the protein target. (A) bond formed between 7,8-DHF and prolyl hydroxylase enzyme (PDB ID: 5OX6); (B) bond formed between Vadadustat and prolyl hydroxylase enzyme (PDB ID: 5OX6).

DISCUSSION:

The utilization of the compound 7,8-dihydroxyflavone (DHF) as a neuroprotective agent that is expected to play a role in ischemic stroke therapy that has a complex pathophysiology can be done by computational methods for screening its feasibility to be developed as a usable drug. Pharmacokinetic testing (Table 1) showed that the 7,8-dihydroxyflavone compound was worth developing as a neuroprotective agent and being administered orally, evidenced by good levels of absorption. The HIA test results are classified as low (0-20%), moderate (20-70%), or high (70-100%), depending on the percentage rating. 7,8-DHF compound had a high potential to be absorbed in the intestine, showing a 92.63 percent rating in HIA result³². The BBB penetration is classified as high-category compounds having a value of > 2.0, moderate-category 0.1-2.0, and low-category compounds having a value of less than 0.1. So that, the result of the BBB test was 0.93, indicating that the ligands' ability to penetrate the BBB was moderate. The plasma protein binding test measures the strength of the ligand-plasma protein relationship, with values greater than 90% suggesting a strong bond and less than 90% indicating a weak bond. The plasma protein binding test results were 93.02%, which showed that the ligands' ability to bind to plasma proteins was strong, indicating that the ligands were able to move through the cell membrane properly, and also beneficial to protect the compound itself from oxidation, minimize toxicity, increase the half-life and lipophilicity of the compound so that it is able to penetrate the BBB and trigger the desired effect on the site of action^32,33.

According to the drug-likeness prediction results (Table 2), 7,8-DHF compounds are predicted as potential drug molecules because they meet the Lipinski Rule of Five Criteria, such as a hydrogen bond acceptor value is <10, the hydrogen bond donor value is <5, the molecular weight is <500 Dalton, the H₂O partition coefficient (logP) value is <5, and the molar refractivity is 40-130 Å. Biological activity testing (Table 3), showed the 7.8-DHF compound triggered cell survival through activation of kinase protein signaling pathways with probable activity values greater than probable inactivity values, which probable activity value >0.3 indicating medium probability to activate the biological activity^25,34.

The visualization outcomes of molecular docking demonstrate that protein-ligand interaction with the lowest energy bond can affect proteins’ biological activity (Figure 1). Based on (Table 4), increasingly negative binding affinity values indicate the more stable bonds formed between ligands and receptors, implicating the role of ligands, both 7,8-DHF or native ligands, in stimulating protein kinase and signaling pathways below them, such as inhibiting prolyl hydroxylase activity to stimulate expression of HIF-1α protein as cell survival mechanisms. The same result of inhibition of prolyl hydroxylase activity between 7,8-DHF and its native ligand shows that the 7,8-DHF compound still has the ability to stimulate the cell survival mechanism through the PI3K/Akt/mTOR signaling pathway and stimulates HIF-1 transcription.

Hypoxia-inducible Factor 1α (HIF-1α) is not hydroxylated in hypoxic condition, inducing the up-regulation of its protein through phosphoinositide-3-kinase/AKT signaling pathway, and further preventing its interaction with pVHL, subsequent ubiquitination and degradation. HIF-1α is a main regulator of cellular response to hypoxia and maintain oxygen homeostasis in the cells, regulating the expression of many target genes including VEGF, EPO, GLUT1, and HO-1^35,36. This study result are in line with several studies that have shown molecular interactions between 7,8-DHF and TrkB and VEGFR2 receptors. The 7,8-DHF compound play a role in protecting retinal ganglion cells from excitotoxicity and oxidative stress that causes degeneration through TrkB activation and pro-survival cascade induction via PI3K/Akt and MAPK/Erk pathways^37,38. Research on myocardial infarction shows that activation of protein kinase signaling pathways triggers pro-survival and cardioprotective effects by inducing anti-apoptosis proteins through the activation of CRYAB and Nrf2 to further regulates the expression of antioxidant proteins, such as HO-1 and NQO-1³⁹.

Furthermore, 7.8-DHF compounds are able to inhibit the activity of prolyl hydroxylase enzymes via the PI3K/AKT signaling pathway to activate the HIF-1α gene and its underlying signaling pathways to further activate the mechanism of adaptation to hypoxia and minimize the volume of post-ischemia infarction⁴⁰. Akt phosphorylation causes activation of Bcl-2, FoxO3a, mTOR, and glycogen synthase kinase-3 to inhibit the occurrence of apoptosis⁷. Studies that analyzed the capacity of the zinc active chemical compound to inhibit the interaction between VHL protein and HIF-1α, resulting in suppression of mitochondrial function and upregulation of glucose metabolism have been demonstrated. This concludes that inhibition of VHL protein and HIF-1α interaction can be applied as an alternative way to treat anemia³⁵.

The result of binding affinity was also affected by amino acid residues in the binding domain of the target protein and the type of chemical interaction (Table 5), (Figure 2)⁴¹. Pi-anion and Pi-cation bonds are used as receptors to bind to ligands and as easily modified places, for example, for adding or reducing ions to maximize the effect of a tethered compound⁴². Pi-sulfur bonds play a role in supporting the stability of structures formed by ligand bonds and target proteins that may be related to biological activity, as well as facilitating ligand binding in target proteins⁴³. Pi-alkyl bonds play a role in the stability of bonding structures. While T-shaped pi-pi bonds commonly occur in interactions between phenyl rings in benzene elements of ligands and target proteins⁴⁴. The interaction between Pi-sulfur, Pi-Alkyl, and Pi-sigma supports the stability of ligand and receptor bonds and normalizes the dipole moment when energy transfer occurs with surrounding amino acids⁴⁵. Furthermore, the hydrogen bond is a non-covalent bond that plays a major role in determining the binding affinity and reactivity value of compounds^46,47. Many or at least hydrogen bonds determine the lipophilicity of the compound and its ability to penetrate brain blood cells to then work on the site of action. The amount of hydrogen bonding is associated with high lipophilicity and is inversely proportional to the resulting binding affinity value. A lot of hydrogen bonds in ligand and protein interaction lead to increased inhibition activity compared to their original function. Carbon-hydrogen bonds are covalent bonds that aid structural stability and are related to hydrogen bonds⁴⁸.

CONCLUSION:

The 7,8-DHF compound has a neuroprotective effect by stimulating protein kinase activity through activation of the PI3K signaling pathway and inhibit the prolyl hydroxylase enzyme as well as its native ligand to stimulate the downstream signaling pathway and increase the expression of HIF-1α as an adaptive response to hypoxia due to ischemic stroke.

CONFLICT OF INTEREST:

The authors declare that there is no conflict of interest.

ABBREVIATIONS:

Akt : alpha serine/threonine-protein kinase

BBB : Blood Brain Barrier

CBP : CREB Binding Protein

CRYAB : small heat shock protein a-crystallinB

eNOS : Endothelial Nitric Oxide

EPO : Erythroprotein

GLUT1 : Glucose Transporter-1

HIA : Human Intestinal Absorption

HIF-1α : Hypoxia Inducible Factor-1α

HO-1 : Heme Oxygenase -1

HRE : Hypoxia Response Elements

MAPK : Mitogen-Activated Protein Kinase

mTOR : Mammalian Target Of Rapamycin

NQO-1 : NAD(P)H dehydrogenase [quinone]1

Nrf-2 : NF-E2-related factor 2

PHD : Prolyl Hydroxylase

PI3K : Phosphatidyl-inositol 3-kinase

TrkB : Tyrosine Reseptor Kinase B

VEGF : Vascular Endothelial Growth Factor

VHL : von Hippel-Lindau

ACKNOWLEDGMENTS:

This work was funded by grants from the Maulana Malik Ibrahim State Islamic University of Malang and we thank all the Biomedical Laboratory staff of Brawijaya University for their excellent assistance during this study.

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Received on 25.08.2021 Modified on 08.11.2021

Research J. Pharm. and Tech 2022; 15(9):3980-3986.

DOI: 10.52711/0974-360X.2022.00667