Journal of Biomolecular Research & Therapeutics

Establishing a Remedy for Phenylketonuria Disease from Indian Ayurvedic Herbs

Preenon Bagchi^1*, SophieVisvikis-Sieste¹ and Ajit Kar^{2 1}UMR INSERM U1122, IGE-PCV “Interactions Gene- Environnement en Physiopathologie Cardio-Vasculaire”, Faculté de Pharmacie, Université de Lorraine, Nancy, France
²Satsang Herbal Research Laboratory, Satsang, Deoghar, Jharkhand, India
^*Correspondence: Preenon Bagchi, UMR INSERM U1122, IGE-PCV “ Interactions Gene- Environnement en Physiopathologie Cardio-Vasculaire”, Faculté de Pharmacie, Université de Lorraine, Nancy, France, Tel: +33 751922837, Email:

Received: 27-Mar-2019 Published: 31-May-2019, DOI: 10.35248/2167-7956.19.8.177

Introduction

Phenylketonuria which is commonly known as PKU, is an inherited disorder that increases the levels of phenylalanine (which is a building block of proteins) in the blood that is obtained through the diet (it is found in all proteins and in some artificial sweeteners) [1-3]. If PKU is not timely treated, phenylalanine can build up to harmful levels of toxins in the body, causing brain damage. The U.S. Food and Drug Administration (FDA) has approved the drug sapropterin dihydrochloride (Kuvan^®) for the treatment of PKU. Kuvan^® is a form of BH4, which is a substance in the body that helps break down phenylalanine [4-6]. PKU is caused by mutations in the gene PAH, GCH1, MAOB, ALB, IGF1, ASCL1, among others Koch et al., Moyle et al., Naz and John. PKU`s symptoms include seizures, tremors or trembling and shaking, stunted growth, hyperactivity, skin conditions such as eczema, a musty odor (bad smell) in their breath, skin or urine. Infants with classic PKU appear normal until they are a few months old and without treatment, these children develop permanent intellectual disability. Children with PKU usually have lighter skin and hair than unaffected family members and more prone to have skin disorders like eczema [1-9].

Genes considered in this work

Classical PKU is an autosomal recessive disorder caused by mutations in both alleles of the gene coding for phenylalanine hydroxylase found on chromosome 12.

ASCL1 gene (achaete-scute family bHLH transcription factor 1)

It encodes a member of the basic helix- loop-helix (BHLH) family of transcription factors, a protein that activates transcription by binding to the E box (5'- CANNTG-3'). Dimerization with other BHLH proteins is required for efficient DNA binding and this protein plays a role in the neuronal commitment and differentiation and in the generation of olfactory and autonomic neurons. Mutations in this gene cause the congenital central hypoventilation syndrome (CCHS) phenotype in rare cases [10].

GCH1 gene (GTP cyclohydrolase 1)

It provides instructions for making an enzyme called GTP cyclohydrolase 1, which is involved in the first of three steps in the production of a molecule called tetrahydrobiopterin (BH4). Other enzymes help to carry out the second and third steps in this process. Tetrahydrobiopterin plays a significant role in processing several protein building blocks (amino acids) in the blood; specifically, tetrahydrobiopterin, which is involved in the production of two neurotransmitters called dopamine and serotonin. Among their many functions, dopamine spreads signals within the brain to produce smooth physical movements and serotonin regulates mood, emotion, sleep, and appetite. Since it helps enzymes carry out chemical reactions, tetrahydrobiopterin is known as a cofactor [11].

MAOB (Monoamine oxidase B)

The protein coded by this gene belongs to the flavin monoamine oxidase family and it is an enzyme located in the mitochondrial outer membrane. It catalyzes the oxidative deamination of biogenic and xenobiotic amines and plays an important role in the metabolism of neuroactive and vasoactive amines in the central nervous system and peripheral tissues. This protein preferentially degrades benzylamine and phenylethylamine [12].

Ayurvedic herbs used and their active components

• Wood betony: The principal chemical components present in this plant are Tannins, Betulinic acid, oleonilic acid, rosamarinic acid, rutin, urosolic acid, stachydrine and glycosides.

• Nettle: The principal chemical components present in this plant are histamine, formic acid, acetylcholine, serotonin and vitamins.

• Plantago ovate: The principal chemical components present in this plant are xylose, arabinose, alanine, valine, glutamic acid, glycine, cysteine, lysine, leucine, tyrosine and xylose.

• Turmeric: The principal chemical components present in this plant are curcumin and camphene.

• Dandelion: The principal chemical components present in this plant are taraxacin, laevulin, resin and inulin.

The main objective of this study is to establish a novel ligand as drug for the PKU from the phytocompounds of the above mentioned ayurvedic herbs.

Methodology

The proteins corresponding to the genes for the PKU were downloaded from Genbank database and their 3d structures were modeled using modeller [1,13]. Modeller is used for homology or comparative modeling of protein three-dimensional structures wherein the user provides an alignment of a sequence to be modeled with known related structures and modeller automatically calculates a model containing all non-hydrogen atoms [13]. The modeller generated models were verified using Ramachandran Plot [1,14]. Ramachandran Plot is a way to visualize energetically allowed regions for backbone dihedral angles ψ against φ of amino acid residues in protein structure [14]. The 3d structures of the phytocompounds mentioned above were downloaded from pubchem database. These compounds were docked with the PKU receptors using PATCHDOCK server which is a server for molecular docking [1,15]. ADME studies were done with the phytocompounds which showed best docking results with the PKU receptors [1,16,17]. ADME is “absorption, distribution, metabolism, and excretion” which is the disposition of a pharmaceutical compound within an organism. This is based on Lipinski's rule of five to evaluate drug-likeness or determine if a chemical compound with a certain pharmacological or biological activity has chemical properties and physical properties that would make it a likely orally active drug in humans [17].

Results and Discussion

Homology modelling

PKU (phenylketonuria) gene receptors were retrieved from GENBANK database Table 1. The homologous templates of the receptors in Table 1 were selected using BLAST search against Protein Data Bank (PDB) and the selected templates were downloaded from PDB Table 2A-C. Using Modeller, the 3d structures of the receptors in Table 1 were modelled [13]. The models were verified using Rampage Ramachandran Plot server Table 3A-C and Figure 1A-C [14].

Table 1: PKU genes with Genbank accession number.

Table 2(A): Homologous template of ASCL1.

Table 2(B): Homologous template of GCH 1.

Table 2(C): Homologous template of MAOB.

Table 3(A-C): Ramachandran Plot Analysis of modeler generated models.

Table 3(B)

Table 3 (C)

Figure 1A: Ramachandran plot analysis of the best model (5) of ASCL1 receptor.
Figure 1B: Ramachandran plot analysis of the best model (2) of GCH1 receptor.
Figure 1C: Ramachandran plot analysis of the best model (1) of MAOB receptor.

Docking

The selected models in Table 3 were docked with the phytocompounds from the Ayurvedic herbs using PATCHDOCK [15]. The docking scores were noted in Table 4A1-A5, B1- B5 and C1-C5. As per the virtual screening studies we find the phytocompounds betulinic acid, rutin, lecithin and curcumin docks with all the receptor genes. Hence we take these phycompounds into consideration (in comparison with the others used in this work) for further ADME studies to see which of these compounds show NO violations in the Lipinski rule of 5 (Figure 2A-C).

Table 4 (A1): Docking results of ASCL1 receptor with compounds from Wood Betony.

Table 4 (A2): Docking results of ASCL1 receptor with compounds from NETTLE.

Table 4 (A3): Docking results of ASCL1 receptor with compounds from PLANTAGO OVATO.

Table 4 (A4): Docking results of ASCL1 receptor with compounds from TURMERIC.

Table 4 (A5): Docking results of ASCL1 receptor with compounds from DANDELION.

Table 4 (B1): Docking results of GCH1 receptor with compounds from WOOD BETONY

Table 4(B2): Docking results of GCH1 receptor with compounds from NETTLE.

Table 4(B3): Docking results of GCH1 receptor with compounds from PLANTAGO OVATO.

Table 4(B4): Docking results of GCH1 receptor with compounds from TURMERIC.

Table 4(B5): Docking results of GCH1 receptor with compounds from DANDELION.

Table 4(C1): Docking results of MAOB receptor with compounds from WOOD BETONY.

Table 4(C2): Docking results of MAOB receptor with compounds from NETTLE.

Table 4(C3): Docking results of MAOB receptor with compounds from PLANTAGO OVATO.

Table 4(C4): Docking results of MAOB receptor with compounds from TURMERIC.

Table 4(C5): Docking results of MAOB receptor with compounds from DANDELION.

Figure 2A: ASCL1 docking images.
Figure 2B: GCH1 docking images.
Figure 2C: MAOB docking images.

ADME

The phytocompounds used in this work are subjected to ADME screening using molinspiration server [16-18]. The results are noted in Table 5 [1]. From the above table it is seen that the compounds betulinic acid, rutin and lecithin have 1, 3 and 1 violations respectively. Compound curcumin successfully clears ADME studies as it shows NO violations in the Lipinski rule of 5.

Table 5: ADME screening.

Conclusion

As per the virtual screening studies we find the phytocompounds betulinic acid, rutin, lecithin and curcumin docks with all the receptor genes. As per ADME studies compounds betulinic acid, rutin and lecithin cannot be considered as drug lead as they show 1, 3 and 1 violation respectively in ADME studies (as per Table 5). Compound curcumin successfully clears ADME studies (as per Table 5, there are no violations in the ADME properties of curcumin and hence the compound curcumin can be successfully considered as novel drug for phenylketonuria disease.

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