research note being presented on a research paper that showed some important aspects regarding phenylketonuria (PKU)

1. Therapeutic implication of
L-phenylalanine aggregation mechanism
and its modulation by D-phenylalanine in
phenylketonuria
Virender Singh1, Ratan Kumar Rai2, Ashish Arora3, Neeraj Sinha2 &
Ashwani Kumar Thakur1
1Department of Biological Sciences and Bioengineering, Indian Institute of Technology
Kanpur 208016, Uttar Pradesh, India,
2Centre of Biomedical Research, SGPGIMS Campus, Raibarelly Road, Lucknow 226014,
Uttar Pradesh, India, 3CSIR-Central Drug
Research Institute, 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226031,
Uttar Pradesh, India.
Research note by Roll no-11333
Riddhi Karnik
MES Garware PUNE
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2. Journal- SCIENTIFIC REPORTS
• Impact factor-5.6
• Online version- nature.com
• Article published on- 27th January 2014
• SUBJECT AREAS:SMALL MOLECULES,DRUG
DISCOVERY,BIOPHYSICAL CHEMISTRY
fig-phenylalaniner.r.karnik

3. Introduction to phenylketonuria
• Phenylketonuria (PKU) is an inborn metabolic disorder
linked with inability of infants to utilize
phenylalanine(Phe) due to lack of phenylalanine
hydroxylase (PAH) enzyme activity caused by a
genetic mutation.
• This drastically increases blood Phe concentration
from normal 50 mM/L to toxic 1200 mM/L level.
• It results in deficiency of tyrosine, dopamine,
epinephrine and norepinephrine molecules.
• This hampers proper brain development and results in
psychiatric disorder in patients.
• Currently, restricted Phe diet along with large
neutral amino acids (LNAA) and tetrahydrobiopterin
therapy are used to reduce toxic effects in PKU
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5. Difference
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6. Introduction
• Phe self-assembly results in high build-up of Phe in the
parietal cortex area of the brain, results in self-assembly
and fibril formation.
• Amyloid fibrils associated with several neurodegenerative
diseases like Alzheimer’s disease, Huntington’s disease and
Atrial amyloidosis.
• Presence of these amyloids and intermediate oligomers in
different regions of brain are associated with toxicity,
leading to neuronal
• Phe also form dimers in presence of its cationic and
zwitterionic state in aqueous solution
• Interactions responsible for Phe assembly are mainly
hydrogen bonding electrostatic groups
• However, it seems a reasonable assumption that Phe being
an aromatic amino acid can also form hydrophobic
interactions through 𝜋 − 𝜋 stacking.
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7. Abstract
• Self-assembly of phenylalanine is linked to amyloid
formation toxicity in phenylketonuria disease.
• We are demonstrating that L-phenylalanine self-assembles
to amyloid fibrils at varying experimental conditions and
transforms to a gel state at saturated concentration.
• Biophysical method nuclear magnetic resonance shows L
phenylalanine is governed mainly by hydrophobic
interactions.
• Interestingly, D-phenylalanine arrests the fibre formation
by L-phenylalanine and gives rise to flakes.
• These flakes do not propagate further and prevent fibre
formation by L-phenylalanine.
• This suggests the use of D-phenylalanine as modulator of
L-phenylalanine amyloid formation and may qualify as a
therapeutic molecule in phenylketonuria
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8. Hypothesis
• Non-natural amino acids have shown potential
as aggregation inhibitors by disrupting fibril
formation of proteins like tau in Alzheimer’s
disease.
• Also, D-amino acids have shown inhibitory
effects on biofilm development having amyloid
like characteristics.
• So, based on the available evidence of
inhibitory effects of D-enantiomers, test
was carried out to know the effect of D-Phe
on L-Phe fibril formationr.r.karnik

9. Material and methods
• L-Phe and D-Phe
• Self-assembly of phe-Different concentrations of
Phe ranging from 300 mM to 170 mM were
prepared by dissolving appropriate amount in double
distilled water at RT. Self assembly was checked at
different pH.
• RP-HPLC analysis.
• 90˚ LS, DLS and ThT binding fluorescence assay
• Scanning electron microscopy (SEM)
• Nuclear magnetic resonance (NMR).
• L-Phe titration with D-Phe.
• Homochiral andHeterochiral seeding
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10. Observations
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13. Results
• Kinetics of Phe self assembly showed with ThT
binding that enhaced flurosecence was obtained
abv conc. 3mM
• Aggregate structure and mechanism was seen
using SEM,NMR and titration and it was
confirmed that aggregation takes place at all
pH with different amount of time required
• L-phe aggegation modulation by D-phe
equimolar raecemic mixture showed no gel
formation rather fibril formation was inhibited
by flake formation.
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14. Disccusion
• It was proved that self assembly takes
place in phe leading to gel formation.
• Hydophobicity was responsible for
aggregation of phe
• Equimolar raecemic mixture of enantiomes
result no gel formation rather fibril
formation was inhibited by flake formation
which are non propogative thus this could
be used to immediately treat patients of
PKU suffering from amyloid formation
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15. • Studying toxicity of flakes formed
• Application of D-phe as competitive
inhibitor for L- phe in BBB to avoid
amyloid formation.
Future prospects
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