Elucidating the mechanism of anthocyanidins in selected axonal regeneration pathways In silico

John Sylvester Nas; Iris Kate Del Callar; Katlyn Keila Mendoza; Precious Dianne Verde; Francheska Anne Carapatan; Lana Gabrielle Abesamis; Jasmine Grace Hermano; Angelica Navarette; Jorlyn Anne Baldovino

doi:10.4103/jpdtsm.jpdtsm_2_23

ORIGINAL ARTICLE

Year : 2023 | Volume : 2 | Issue : 1 | Page : 43-54

Elucidating the mechanism of anthocyanidins in selected axonal regeneration pathways In silico

John Sylvester Nas¹, Iris Kate Del Callar², Katlyn Keila Mendoza², Precious Dianne Verde², Francheska Anne Carapatan², Lana Gabrielle Abesamis², Jasmine Grace Hermano², Angelica Navarette², Jorlyn Anne Baldovino¹
¹ Department of Biology, College of Arts and Sciences, University of the Philippines Manila, Manila, Philippines
² Department of Medical Technology, Institute of Arts and Sciences, Far Eastern University, Manila, Philippines

Date of Submission	03-Nov-2022
Date of Decision	26-Jan-2023
Date of Acceptance	08-Feb-2023
Date of Web Publication	13-Mar-2023

Correspondence Address:
John Sylvester Nas
Department of Biology, College of Arts and Sciences, University of the Philippines Manila, Manila
Philippines

Source of Support: None, Conflict of Interest: None

DOI: 10.4103/jpdtsm.jpdtsm_2_23

Abstract

BACKGROUND: Anthocyanidins are plant pigments known for their protective effect against inflammation, cancer, and neurodegenerative diseases. Axonal degeneration has been a hallmark of several neurodegenerative and neuropathic illnesses.
AIM AND OBJECTIVE: Recently, several studies have attempted to stimulate axonal regeneration by targeting the mammalian target of rapamycin (mTOR), Nogo, and transforming growth factor (TGF) pathways.
MATERIALS AND METHODS: To illuminate an understanding of the potential of anthocyanidins to promote axon regeneration, we investigated anthocyanidins' physicochemical properties, binding affinity, and noncovalent interactions with enzymes downstream of mTOR, Nogo, and TGF beta (TGF-β) pathways that are known to inhibit axonal regeneration.
RESULTS: We discovered that the six anthocyanidins we examined have favorable blood-brain barrier permeability and high estimated oral bioavailability. Most of the anthocyanidins exhibited the highest binding affinity with GSK3, Ret4, and TGF-βR1 in the mTOR-, Nogo-, TGF-β pathway. These compounds demonstrated a high number of hydrophobic interactions and hydrogen bonds with the selected proteins, which may explain the high binding affinity.
CONCLUSION: Although our findings are inconclusive due to the limitation of the in silico study, the binding affinity of anthocyanidins with these inhibitory enzymes may modulate them. However, it does not ensure axonal regrowth, necessitating additional in vivo and in vitro research.

Keywords: Anthocyanidins, axonal regeneration, mammalian target of rapamycin pathway, Nogo pathway, Transforming growth factor beta pathway

How to cite this article:
Nas JS, Del Callar IK, Mendoza KK, Verde PD, Carapatan FA, Abesamis LG, Hermano JG, Navarette A, Baldovino JA. Elucidating the mechanism of anthocyanidins in selected axonal regeneration pathways In silico. J Prev Diagn Treat Strategies Med 2023;2:43-54

How to cite this URL:
Nas JS, Del Callar IK, Mendoza KK, Verde PD, Carapatan FA, Abesamis LG, Hermano JG, Navarette A, Baldovino JA. Elucidating the mechanism of anthocyanidins in selected axonal regeneration pathways In silico. J Prev Diagn Treat Strategies Med [serial online] 2023 [cited 2023 Mar 31];2:43-54. Available from: http://www.jpdtsm.com/text.asp?2023/2/1/43/371633

Introduction

The nervous system contains a complex of connections between nerve cells that allow interactions, regulations, and various activity patterns needed for normal function.^[1] This system can adapt to environmental changes, even from physiological experiences, injuries, and certain neurodegenerative disorders.^[2] Failure of the axon to regenerate leads to paralysis and loss of sensation.^[3] Several factors antagonize axonal regeneration, particularly its surrounding environment, wherein lesions may inhibit the growth of the axons.^[4] Axon regeneration is controlled and regulated by various pathways, such as the transforming growth factor beta (TGF-β), the Nogo, and the mammalian target of rapamycin (mTOR) pathway.

The mTOR is responsible for regulating cell proliferation, metabolism, and survival and uniting intracellular and extracellular signals.^[5] It is activated by the removal of negative regulators, such as phosphatase and tensin homolog, hamartin (TSC1), and tuberin (TSC2), which helps increase regeneration in both the peripheral nervous system and central nervous system (CNS).^[6] Meanwhile, the Nogo pathway is for plasticity, axonal growth, and regeneration after an injury.^[7] Enzymes that inhibit axonal regrowth by preventing neurite outgrowth and inducing growth cone collapse are Nogo, chondroitin sulfate proteoglycans, myelin-associated glycoprotein (Mag), and oligodendrocyte myelin glycoprotein (Omgp).^[6] The two major classes responsible for inhibiting CNS regeneration are myelin-associated inhibitors and chondroitin sulfate proteoglycans.^[8],[9] Meanwhile, the TGF-β pathway primarily inhibits the growth of a tumor in its early stages and acts as a tumor promoter in its late stages.^[9] Its ability to influence the cell's primary function plays a huge role in cellular processes, especially during axonal regeneration.^[10] During axonal injuries, the TGF-β pathway reduces inflammation in the CNS, which allows a suitable growth environment for the axon.^[10] Hence, developing small molecular compounds targeting these pathways to enhance axonal regeneration is impossible.

Many small molecular anticancer drugs, such as taxol, epothilones, and more, have influenced these pathways.^[11] Studies have revealed that several anticancer medications substantially impact axonal regrowth.^[12]

Anthocyanidins are small molecular compounds found in various fruits and vegetables. They are well-known for their antioxidant, anti-inflammatory, antiviral, and anticancer properties.^[13],[14] Anthocyanidin has a basic chemical structure of flavylium cation (2-phenyl-1-benzopyrylium), as shown in [Figure 1], which links either both hydroxyl (-OH) or methoxyl (-OCH3) groups, but with the absence of sugar moiety. Anthocyanidins have variants, namely pelargonidin, cyanidin, delphinidin, petunidin, peonidin, and malvidin, commonly seen in vegetables and plants. They are differentiated depending on the number and position of hydroxyl and methoxyl groups. Several studies have shown that anthocyanidins affect neurodegenerative diseases such as Alzheimer's disease and dementia.^[15] Despite this connection, no existing evidence demonstrates the effect of anthocyanidins on axonal regeneration.

Figure 1: The structure of the different anthocyanidin compounds. (a) Cyanidin, (b) delphinidin, (c) malvidin, (d) pelargonidin, (e) peonidin, and (f) petunidin

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This study aims to hypothesize a possible mechanism of action for the potential of anthocyanidin compounds to promote axonal regeneration through modulating axonal regeneration inhibitory enzymes.

Materials and Methods

Preparation of the ligands and enzymes

Information about the different anthocyanidins, specifically cyanidin (CID: 128861), delphinidin (CID: 68245), malvidin (CID: 159287), pelargonidin (CID: 440832), peonidin (CID: 441773), and petunidin (CID: 441774), were gathered from PubChem (www.pubchem.ncbi.nlm.nih.gov). The crystal structures of the different inhibitory enzymes were from Protein Data Bank (www.rcsb.org/pdb/). In the mTOR pathway, the inhibitory enzymes are Bcl2-xL (PDB ID: 2W3 L), 4E-BP (PDB ID: 3HXG), and GSK3 (PDB ID: 3F88). In the Nogo pathway are RhoA (PDB ID: 3MSX), Rock2 (PDB ID: 2F2U), and Ret4 (PDB ID: 2VN8). Finally, in the TGF-β pathway are TGF-βR1 (PDB ID: 3KCF), and TGF-βR2 (PDB ID: 5E91). The inhibition of these pathways prevents the binding of various proteins necessary for axon regeneration.

Evaluation of the bioavailability of anthocyanidins in the central nervous system

The data of the physicochemical properties of anthocyanidins were collected from SwissADME (http://swissadme.ch/) and further evaluated for oral bioavailability using the Lipinski's Rule of Five.^[16]

Virtual molecular docking of anthocyanidin compounds and known inhibitory ligands

Before the docking experiment, the selected enzymes were prepared by adding hydrogen and Gasteiger charges. The charges were merged first followed by removing nonpolar hydrogen, lone pairs, water molecules, and nonstandard residues as described in a previous study.^[17] The different classes of anthocyanidins were docked on the binding centers of the selected enzymes using Mcule (https://mcule.com).

The binding centers of the enzymes are as follows: Bcl2-xL (X: 39.4679, Y: 27.0653, Z: 12.3721), 4E-BP (X: 36.5905, Y: 20.6409, Z: 0.7315), GSK3 (X: 33.4424, Y: 102.9242, Z: 12.8716), RhoA (X: 32.8526, Y: 29.7085, Z: 21.8721), Rock2 (X: 48.3623, Y: 95.6048, Z: 123.7467), and Ret4 (X: 74.6735, Y: 4.8041, Z: 181.642), TGF-βR1(X: 57.9888, Y: 48.6006, Z: 126.483), and TGF-βR2 (X: 24.332, Y: 0.231, Z: 11.375). The software predicted the docking score of the ligand to the target enzyme. Afterward, the docked crystal structure pose with the most negative docking was were considered in the ranking of the anthocyanidins in each enzyme.

Assessment of the binding affinities of the anthocyanidin compounds and known inhibitory ligands

The docked protein-ligand complex was visualized using protein–ligand interaction profiler (https://projects.biotec.tu-dresden.de/plip-web), which identified the different binding interactions. The different binding interactions of the anthocyanidin compounds were compared with each other.

Results

Predicted oral bioavailability of anthocyanidins

Among all the anthocyanidin compounds, delphinidin has the only violation of Lipinski's rule of five with greater than 5 H bond donors, as shown in [Table 1]. Despite having a violation, it is within the acceptable number of violations. Hence, all six anthocyanidin compounds followed Ro5, indicating high oral bioavailability. Similarly, the anthocyanidin's distribution coefficient (Log D) at pH 7.4 ranges from 1.35 to 2.02, suggesting high solubility in the blood–brain barrier environmental condition.

Table 1: Predicted bioavailability of anthocyanidins using Lipinski's rule of five

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Binding affinity of anthocyanidin with axonal regeneration-associated proteins

[Table 2] shows the ranking of the highest docking scores of the anthocyanidins in the various inhibitory enzymes found in the axonal regeneration pathways. In the mTOR pathway, anthocyanidins have a high binding affinity with the enzyme GSK3. The top-binding anthocyanidins in the mTOR pathway are peonidin for Bcl2-xL, delphinidin for 4E-BP, and peonidin for GSK3. In the Nogo pathway, the Ret4 inhibitory enzyme showed high binding affinities of anthocyanidins. In addition, delphinidin in RhoA and Rock 2 is the top-binding anthocyanidin, while in Ret4 is cyanidin. Finally, in the TGF-β Pathway, anthocyanidins have high binding affinities in the enzyme TGF-βR1; the top-binding anthocyanidin is the delphinidin both in the TGF-βR1 and TGF-βR2.

Table 2: Ranking of the highest docking scores of the anthocyanidins in various inhibitory enzymes present in axonal regeneration pathways

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Binding interaction of anthocyanidin with mammalian target of rapamycin-associated enzymes

Bcl2-xL

In Bcl2-xL, anthocyanidins show hydrophobic interactions and hydrogen bonds, as shown in [Figure 2] and [Table 3], mainly the peonidin, which has three hydrogen bonds in glu71, arg81, and ala84 and four hydrophobic interactions with val68, glu71, leu72, and phe88. On the other hand, Cyanidin shows four hydrogen bonds with glu71 (2) and arg81 (2) and four hydrophobic interactions with val68, glu71, leu72, and phe88. Delphinidin shows two hydrogen bonds with glu49, phe88, and four hydrophobic interactions with phe39, asp46, phe47, and ala84. Pelargonidin has five hydrogen bonds in glu71 (2), arg81 (2), ala84, and six hydrophobic interactions with phe39, val68, glu71, leu72, ala84, and phe88. Malvidin shows two hydrogen bonds with asp46 and phe88 and four hydrophobic interactions with phe39, asp46, phe47, and ala84. Finally, the petunidin has three hydrogen bonds with glu71 (2) and arg81 and two hydrophobic interactions with glu71 and leu72. Overall, the interactions in Bcl2-xL show more hydrogen bonds than hydrophobic interactions.

Figure 2: Crystal docked structures of the anthocyanidins with Bcl2-xL. (a) Cyanidin, (b) delphinidin, (c) malvidin, (d) pelargonidin, (e) peonidin, and (f) petunidin

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Table 3: Noncovalent binding interactions of the anthocyanidin compounds to the inhibitory enzymes

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4E-BP

In 4E-BP, anthocyanidin forms hydrophobic interactions and hydrogen bonds, as shown in [Figure 3] and [Table 3]. Delphinidin has six hydrogen bonds with tyr59 (2), trp69, gln121, arg123, and lys128 and one hydrophobic interaction with trp69. In addition, delphinidin shows pi-stacking with trp23 (4) and trp69 (2). On the other hand, malvidin forms seven hydrogen bonds with tyr59 (2), trp69, glu70, gln121, arg123, and lys128 and one hydrophobic interaction with trp23. Malvidin also forms pi-stacking with trp23 (4) and trp69 (2). Meanwhile, peonidin exhibits six hydrogen bonds with tyr59, trp69, glu70, gln121, arg123, and lys128 and one hydrophobic interaction with trp69. It also has pi-stacking with trp23 (4) and trp69 (2). Cyanidin shows seven hydrogen bonds with tyr59, trp69, glu70, gln121, arg123 (2), and lys128 and four hydrophobic interactions with trp23 (2) and trp69 (2). In addition, cyanidin forms pi-stacking with trp23 (2). Pelargonidin shows six hydrogen bonds with tyr59 (2), trp69, gln121, arg123, and lys128. It also has pi-stacking with trp23 (4) and trp69 (2). Finally, petunidin has eight hydrogen bonds with tyr59 (2), trp69, glu70, gln121, arg123 (2), lys128, and two hydrophobic interactions with trp23 and trp69. Overall, the interactions in 4E-BP show more hydrogen bonds than hydrophobic interactions.

Figure 3: Crystal docked structures of the anthocyanidins with 4E-BP. (a) Cyanidin, (b) delphinidin, (c) malvidin, (d) pelargonidin, (e) peonidin, and (f) petunidin

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GSK3

In GSK3, anthocyanidin forms hydrophobic interactions and hydrogen bonds, as shown in [Figure 4] and [Table 3]. Peonidin has four hydrogen bonds with lys51, tyr94 (2), asp160 and four hydrophobic interactions with ile28, val36, leu92, and leu148. On the other hand, Delphinidin forms one hydrogen bond with asp160 and five hydrophobic interactions with ile28, ala49, leu92, thr98, and leu148. Pelargonidin has five hydrogen bonds with lys51, tyr94, val95, pro96, and asp160 and seven hydrophobic interactions with ile28, val36, lys51, val76, leu92, and leu148 (2). Cyanidin shows one hydrogen bond with val95 and six hydrophobic interactions with ile28, val36, ala49, leu92, thr98, and leu148. Malvidin has three hydrogen bonds with lys51, tyr94, and asp160 and four hydrophobic interactions with ile28, val36, thr98, and leu148. Finally, petunidin forms three hydrogen bonds with val95, glu97, and asp160 and four hydrophobic interactions with ala49, leu92, thr98, and leu148. Overall, the interactions in GSK3 show more hydrophobic interactions than hydrogen bonds.

Figure 4: Crystal docked structures of the anthocyanidins with GSK3. (a) Cyanidin, (b) delphinidin, (c) malvidin, (d) pelargonidin, (e) peonidin, and (f) petunidin

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Binding interaction of anthocyanidin with Nogo pathway-associated enzymes

RhoA

In RhoA, anthocyanidin forms hydrophobic interactions and hydrogen bonds, as shown in [Figure 5] and [Table 3]. Delphinidin forms nine hydrogen bonds with gly14, thr16, cys17, val32, arg40, arg40, lys115, ala158, and lys159 and three hydrophobic interactions in phe27, lys115, and lys159. In addition, delphinidin forms pi-cation interaction with lys115. Petunidin, on the other hand, has 11 hydrogen bonds with gly14, thr16 (2), cys17, val32 (2), arg40 (2), lys115, ala158, and lys159. Cyanidin shows seven hydrogen bonds with glu29, lys36, lys115 (3), ala158, and lys159 and three hydrophobic interactions with phe27 and lys115 (2). Malvidin has seven hydrogen bonds with thr16 (2), val30, val32, arg40 (2), and lys115 and one hydrophobic interaction with phe27. In addition, malvidin has pi-cation interactions with arg40 and lys115. Pelargonidin forms seven hydrogen bonds with val32, arg40 (2), lys115, asp117, ala158, and lys159 and one hydrophobic interaction with lys159. It also forms pi-stacking with phe27 (2) and pi-cation interaction with lys115. Finally, peonidin shows ten hydrogen bonds with gly14, val32 (2), arg40 (2), lys115, asp117, ser157, ala158, and lys159, and five hydrophobic interactions with phe27, lys115 (3), and lys159. Additionally, peonidin forms pi-cation interaction with lys115. Overall, the interactions in RhoA show more hydrogen bonds than hydrophobic interactions.

Figure 5: Crystal docked structures of the anthocyanidins with RhoA. (a) Cyanidin, (b) delphinidin, (c) malvidin, (d) pelargonidin, (e) peonidin, and (f) petunidin

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Rock2

In Rock2, anthocyanidin forms hydrophobic interactions and hydrogen bonds, as shown in [Figure 6] and [Table 3]. Mainly, delphinidin shows five hydrogen bonds with met146 (2), asp150 (2), and asp192 and four hydrophobic interactions in phe77, ala93 leu195, and phe358. On the other hand, cyanidin forms two hydrogen bonds on met146 and asp206 and three hydrophobic interactions with phe77, ala93, and phe358. Malvidin has two hydrogen bonds with asp150 and asp359 and four hydrophobic interactions with phe77, leu195, and phe358. Pelargonidin forms two hydrogen bonds with met146 (2) and three hydrophobic interactions with phe77, al93, and phe358. Peonidin shows one hydrogen bond with met146 and four hydrophobic interactions with phe77, ala93, and phe358. Finally, petunidin forms three hydrogen bonds with lys95, asp192, and asp206 and two hydrophobic interactions with phe77 (2). Overall, the interactions in Rock2 show more hydrophobic interactions than hydrogen bonds.

Figure 6: Crystal docked structures of the anthocyanidins with Rock2. (a) Cyanidin, (b) Delphinidin, (c) malvidin, (d) pelargonidin, (e) peonidin, and (f) petunidin

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Ret4

In Ret4, anthocyanidin forms hydrophobic interactions and hydrogen bonds, as shown in [Figure 7] and [Table 3]. Cyanidin has six hydrogen bonds with ile45, tyr142, gly175, val176, asn235, and phe302 and six hydrophobic interactions with ile45, tyr142, val143, thr146, val176, and leu259. On the other hand, delphinidin has six hydrogen bonds asn43, ile45, gly175, val176, asn235, and phe302 and six hydrophobic interactions with pro44, il45, tyr142, thr146, val176, and leu259. Malvidin has five hydrogen bonds with ile45, gly175, val176, asn235, and phe302 and five hydrophobic interactions with ile45, ty142, thr146, val176, and leu259. Pelargonidin has 6 hydrogen bonds with ile45, tyr142, gly175, val176, leu259, and phe302 and 6 hydrophobic interactions with ile45, tyr142, val143, thr146, val176, and leu259. Peonidin has 6 hydrogen bonds with ile45, tyr142, gly175, val176, asn235, and phe302 and 6 hydrophobic interactions with ile45, tyr142, val143, thr146, val176, and leu259. Finally, petunidin has five hydrogen bonds with ile45, gly175, val176, asn235, phe302 and six hydrophobic interactions with pro44, ile45, tyr142, thr146, val176, and leu259. Overall, the interactions in Ret4 show the same number of hydrophobic interactions and hydrogen bonds.

Figure 7: Crystal docked structures of the anthocyanidins with Ret4. (a) Cyanidin, (b) delphinidin, (c) malvidin, (d) pelargonidin, (e) peonidin, and (f) petunidin

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Binding interaction of anthocyanidin with transforming growth factor beta-associated enzymes

TGF-βR1

In TGF-βR1, anthocyanidin forms hydrophobic interactions and hydrogen bonds, as shown in [Figure 8] and [Table 3]. Particularly, delphinidin forms ten hydrogen bonds with arg45 (2), lys62 (2), glu75, tyr79, leu108, ser110, lys163, and asp177, and four hydrophobic interactions with val49, lys62 (2), and leu90. Cyanidin, on the other hand, has ten hydrogen bonds with arg45 (2), ala60, lys62 (2), leu108, ser110, lys163, and asp177 (2). Malvidin has six hydrogen bonds with lys62, glu75, ser117 (2), lys163, and asp177 and four hydrophobic interactions with val49, leu90, leu166, and ala176. Pelargonidin has five hydrogen bonds with lys62 (2), glu75, ser110, asp177, and six hydrophobic interactions with ile41, val49, ala60, lys62, leu90, and leu166. Peonidin has seven hydrogen bonds with lys62, ser110, tyr112, his113, gly116, asp120, and asp177 and four hydrophobic interactions with ala60, leu166 (2), and ala176. Finally, petunidin shows five hydrogen bonds with lys62, ser110, his113, asn164, and asp177 and four hydrophobic interactions with val49 (2), lys62, and leu166. Overall, the interactions in TGF-βR1 show more hydrogen bonds than hydrophobic interactions.

Figure 8: Crystal docked structures of the anthocyanidins with TGF-βR1. (a) Cyanidin, (b) delphinidin, (c) malvidin, (d) pelargonidin, (e) peonidin, and (f) petunidin

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TGF-βR2

In TGF-βR2, anthocyanidin forms hydrophobic interactions and hydrogen bonds, as shown in [Figure 9] and [Table 3]. Delphinidin has four hydrogen bonds with lys277, glu290, asn332, and asp379 and four hydrophobic interactions with val258, lys277, leu305, and leu386. Cyanidin, on the other hand, has six hydrogen bonds with lys277 (2), glu290 (2), asn332, and ser383 and six hydrophobic interactions with val258 (2), lys277 (2), leu305, and leu386. Malvidin has five hydrogen bonds with lys252 (2), lys381, ser383, and asp397 and three hydrophobic interactions with val258, leu305, and asp397. In contrast, pelargonidin has two hydrogen bonds with asn332 and asn384 and six hydrophobic interactions with val258, lys277 (2), leu305, thr325, and leu386. Peonidin has five hydrogen bonds with val250, lys277, glu290 (2), and asn332 and five hydrophobic interactions with val258 (2), lys277, leu305, and leu386. Finally, petunidin shows seven hydrogen bonds with lys252 (2), arg254, phe255, ala256, lys277, lys381, and three hydrophobic interactions, val258 (2) and leu386. Overall, the interactions in TGF-βR2 show more hydrogen bonds than hydrophobic interactions.

Figure 9: Crystal docked structures of the anthocyanidins with TGFβR2. (a) cyanidin, (b) delphinidin, (c) malvidin, (d) pelargonidin, (e) peonidin, and (f) petunidin

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Discussion

The Absorption, Distribution, Metabolism, Elimination, and Toxicity (ADMET) predict a compound's oral bioavailability.^[18] In Ro5, the physicochemical properties used to predict the ADMET property were molecular size, octanol/water partition coefficient (LogP), H bond acceptors, and the number of H bond donors.^[19] All the anthocyanidin compounds have ≤500 molecular mass, ≤5 octanol-water partition coefficients (LogP), H bond acceptor ≤10, and H bond donor ≤5. However, delphinidin had greater than five H bond donors. Studies suggest that more than five H bond donors may influence molecule diffusion on passive transport, contributing to protein folding that may affect the receptor–ligand configuration.^[20] More studies suggest that in drug discovery, the rule of 5 may predict the absorption or permeation of molecules and only holds for compounds that are not substrates for active transporters.^[21]

For compounds to cross the blood-brain barrier, the optimal Log D value at pH 7.4 is suggested to be between 1 and 3, a profile similar to that for drugs with optimal oral bioavailability.^[22] The Log D value of the anthocyanidins is ≤3, suggesting their high solubility across the blood-brain barrier.

To further analyze the potency of anthocyanidin aside from their oral bioavailability, they were docked with axonal regeneration-associated proteins. The negative docking score indicates higher binding affinity.^[23] Molecular docking uses algorithms to generate possible configurations for the interaction of energies.^[24] The docking process predicts the ligand conformation and posing within a target binding site.^[25] The researchers visualized their binding interactions with various inhibitory enzymes to better characterize the binding affinity of each anthocyanidin to each axon regeneration inhibitory enzyme.

In the mTOR pathway, most of the anthocyanidin binds with GSK3. The anthocyanidin's high binding affinity may be due to their prominent binding interaction with an abundance of hydrogen bonds and pi-cation interaction. Peonidin has the highest binding affinity in GSK3 and has more hydrogen bonds than peonidin in Bc12-XL; this may be due to the attachment of hydrogen directly to one of the most electronegative elements.^[26] On the other hand, their pi-cation interactions might have contributed to stabilizing and recognizing the binding affinity between the protein and anthocyanidin.^[27] The interaction of pi-cation may make it competitive with the H bond on drug-receptor and protein interactions.^[28]

In the Nogo pathway, most of the anthocyanidin binds with Rock2. The high binding affinity of the anthocyanidin may be due to the prominent binding interaction of having the highest number of hydrogen bonds and hydrophobic interactions. It also reveals the pi-cation interactions of delphinidin in the RhoA pathway. The pi-cation interactions have shown that neurotransmitters generally use a pi-cation interaction to bind to their receptors.^[28]

In the TGF-β pathway, most of the anthocyanidin binds with TGF-βR1. The high number of hydrogen bonds may be the reason for its high binding affinity to the enzyme. Due to a lot of hydrogen bonding in their structure, the intermolecular force of attraction was high, making the binding affinity stronger.^[29]

Among the different pathways, anthocyanidins had the strongest binding affinity with the TGF-β pathway, consisting of TGF-βR1 and TGFβR2, which could be attributed to the high hydrophobic interactions and hydrogen bonds. Delphinidin outranked all the ligand inhibitors in almost all the pathways except Bcl2-x1, GSK3, and Ret4. Among the anthocyanidin, peonidin, along with pelargonidin, showed a lower number of hydroxyl groups, but despite that, peonidin ranked highest in Bcl2-x1 and GSK2. Hydroxyl groups of anthocyanidin might have influenced the formation of H bonds with a specific enzyme. This instance suggests that the activity of anthocyanidin compounds depends on the enzyme and their binding affinity. The structures of the anthocyanidin compound and its non-covalent bonds might have affected its binding affinity with the protein of the different pathways.

Conclusion

Anthocyanidin follows Lipinski's rule of five, which indicates high oral bioavailability. Similarly, anthocyanidins appear to have favorable Log D at pH 7.4, which indicates their high solubility in the blood-brain barrier. The hydrogen bonding and hydrophobic interaction of the anthocyanidin compounds with the enzymes may have influenced its high binding affinity to the inhibitory enzymes. The anthocyanidins bind the highest with the GSK3 enzyme in the mTOR pathway, Ret4 enzyme in the Nogo pathway, and TGF-βR1 in the TGF-β pathway. Since anthocyanidin has high oral bioavailability and a high binding affinity to these inhibitory enzymes, anthocyanidin compounds may promote axonal regeneration via modulation of GSK3, RET4, and TGF-βR1. The inhibition of these enzymes may influence mTOR, Nogo, and TGF-β pathways, which may promote axonal regeneration. It is interesting to investigate whether the anthocyanidins can demonstrate the axonal regenerative effects in an actual neuron and other animal models; hence further studies are warranted.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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