Advanced Epilepsy Research:

multidisciplinary approach - Sub project 4.

Progress report 6

M. V. Hosur & Tanusree Chaudhury

NIAS, Bangalore.

C. H. Janaki & Supriya Pal

CDAC-Bangalore
 AIMs of sub-project 4

1. Molecular Mechanism of action of AED’s: a) target

identifications and b) binding site based on molecular
modelling and molecular docking.

2. Use of data analysis tools to find new targets for drug

development.
Work done till now since last PRMC
1. Modeled the missense mutants in 17 genes. Submitting a
manuscript. Depositing 17 structures in PDB format to PMDB.
2. Docking study and identification of Potential active and natural
compounds.
3. Purification of Calpain1
4. SPR studies of miRNA binding at GABRA1 SNPs. (potential miRNA-
based drugs)
5. 3’-UTR SNPs as Potential biomarkers for MTLE ( Manuscript under
review)
Details will be presented later in the presentation
Work to be done (pandemic delay)

• 1. Synthesis of LEV-derivatives 1 and 2. IIT-BHU collaboration.

• 2. Purification of His-tag-free calpain1 core-domain
• 3. In-vitro measurements of Lev-der binding to calpain1.
• 4. Biological testing of lev-derivatives for their efficacies (Dr. Aparna Dixit).
• 5. Design and Testing of sRNA inhibitors of mR1275 and miR6769 as
• cure For drug-refractory MTLE patients (Dr. Aparna Dixit).
• 6. Crystallisation of calpain1 / lev-derivative complexes
• 7. X-ray crystal structure determination of calpain1-designed drug
complexes.
Levetiracetam Derivatives last review
• In our previous analysis, we have identified
two potential Levetiracetam derivatives that
bind to Calpain1 core domain.

BRV

Derivative 2 LEV
Derivative 1
LEV-DER1. DS = -9.9. Many stereo-
centres.
LEV-DER2 Fewer stereo-centres. DS =
-8.2

DS = -7.0
for SV2A

Organic synthesis
delayed by COVID-
19.
New contact
Dr. Gyan, IIT-BHU
 Other Active compounds as identified through
SwissSimilarity using Levetiracetam

DrugBank active 125

compounds
DrugBank Experimental 337
Compound
Zinc Database 400

• All compound’s smile notations were obtained SwissSimilarity list

• They were converted to SDF using https://cactus.nci.nih.gov/translate/
• These structures were subjected through HTVS pipeline in Schrodinger Maestro 2020 version.

Active compounds are either already approved drugs or easily available.

Docking Scores - binding site same
Sr. Compound Docking score
Number
1. LEV -3.19
2. L-Prolinamide -4.1
3. Piperidine-2- -5.95
carboxylic acid
tert-butylamide

4. Rufinamide -3.2
5. Oxcarbazepine -4.34
6. Enprofylline -3.5
7. BRV -4.15
8. LEV-DER1 -9.984
9. LEV-DER2 -8.228
Similar Natural compound
C22H35ClN6O6
Dscore -8.864
Quotation - very expensive
Purification of Calpain1 core domain
Calpain1 clone was obtained from Addgene (CAPN1:(Plasmid #25607))
Calpain1Purification: Protocol
• Plasmid extraction from bacterial clone( we used QIAPREP2.0)
• Plasmid transformation (Heat shock 90 s at 42, 1 min ice)
• Checking the level of Expression using 0.50 mM, 0.25 mM IPTG for
induction
• Purification
Ni-NTA
Size Exclusion chromatography
Calpain-1 expression check E.coli
BL21(DE3)

⁰C

⁰C
, 16

16

⁰C
rs

rs ,

, 37
6h

6h
G1

hrs
G1
IPT

G4
IPT
d

IPT
M
uce

uce

mM
5m

M
ind

ind

0.2

0.5

1m
un

un

120 0.5mM IPTG 16 hrs, 16⁰C

85 chosen for expression
50

35

25

20
 Calpain-1 Ni-NTA manual purification from
3l culture
250mM Imidazole

10

11
12

23
13
15

21
19
17
IN

FT

1
3

5
7
8
9
52
37
30

16

6.5

IN - Input

FT – Flow-through
Size Exclusion Chromatogram, SDS gel
HiLoad 16/600 Superdex 200pg column

52
37

30

MW is as expected. N-term sequencing

to be done after His-tag removal.
GABRA1 mutant in Drug-refractory
MTLE.
• Data from AIIMS deposition.
• Mapped on to hg-19 annotation.
• T > C mutation in 3’-UTR region of GABRA1 gene.
• Analysis of consequences of this SNV.
• miRDB search shows this mutation creates binding sites for
• Two miRNAS:
• miR1275 – found over-expressed in epilepsy-patients.
• miR6769 – found over expressed in brain injury seizures.
• miRNA binding inhibits protein translation. GABRA1 insufficiency
• Could contribute to the cause for epilepsy in these patients.
Analysis with SPR Assay
The below miRNAs were synthesized from GENSCRIPT.
•>con_5
UUUCAUCCACC
•>mut_5
UUUCACCCACC – SNP is in red
The above two were biotinylated at 5' end

•>hsa-miR-1275 MIMAT0005929
GUGGGGGAGAGGCUGUC

•>hsa-miR-6769b-5p MIMAT0027620
UGGUGGGUGGGGAGGAGAAGUGC
 SPR-Methodology
• Surface plasmon resonance experiments were conducted using a BiacoreT2000 instrument of Cytiva (formerly known as
GE healthCare).
• Oligonucleotides were synthesized through Genscript. Oligos representing target mRNAs were 11 nuc long and 5′
biotinylated for immobilization to the steptavidin chips.
• The miRNA sequences were synthesized as obtained through miRBase.
• Standard buffer HBS–EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 50 mM TRIS-HCl pH 7.4) was
used for the analyses carried out at 25 0C.
• Biotin–labeled nucleic acid solutions (~25 nM of mRNA is dissolved in HBS-EP buffer) and docked with a streptavidin–
coated chip to start a sensorgram with a 20 μl/min flow rate followed by injection of the activation buffer (1 M NaCl, 50
mM NaOH) for 1 min (20 μl) five to seven times to remove any unbound streptavidin from the sensor chip.
• We have chosen flow cell 1 as control and is left blank for subtraction.
• Flow cell 2 and 3 has been chosen for GABRA1 control and GABRA1 mutant subsequently.
• The number of RUs for channel 2 was 661.5 and for channel 3 was 679.5 indicating enough GABRA1 binding to the
chip.
• Hsa-miR-6769 and hsa-miR-1275 oligos were diluted in buffer (serial dilutions, 1X10-2, 2.5 X10-2, 5 X10-2, 7.5 X10-2,
1 X10-1 and 2 X10-1 µM) and injected over the flow cells for 100 s at 2 μl/min rate.
• The sensorgrams were double-referenced and were fit using a mathematical model of a simple 1:1 interaction. All
experiments were run in duplicate.
miR1275( Interaction using RNAcofold)
• >con(-9.20 kcal/mol)
UUUCAUCCACC&GUGGGGGAGAGGCUGUC
((((.(((((.&))))).)))).......

>mut(-11.70 kcal/mol)
UUUCACCCACC&GUGGGGGAGAGGCUGUC
((((.(((((.&))))).)))).......

miR1275 binds stronger to the mutant.

 Interaction with miR1275
(While modelling we have considered 71 residues and
the SPR was performed with 11 residues)
Control -9.20 kcal/mol

Mutant -11.70 kcal/mol

Ka=1.99E7 Kd= 1.00E-7

No interaction observed against GABRA1 control and miR1275
Residue interaction from mutant GABRA1
with miR1275 modeled using rosetta

C36
 Interaction with miR6769-5b and
GABRA1 Control

Control -14.60 kcal/mol

Ka=591455242.1 Kd=0.000280029
 Interaction with miR6769-5b and GABRA1
Mutant

Mutant -17.20 kcal/mol

Ka= 2007300000 Kd= 7.974E-09

 Mut Con Differences The difference between association and dissociation
energies from control to mutant
KA 2.01E+09 591455242 1415844758
In case of miR-6769-5b suggests that
KD 7.97E-09 0.00028003 -0.00028 1.miR-6769-5b can be a miRNA to bind to GABRA1
Table listing the KA and KD values for miR-6769-5b UTR even at normal conditions
2.This binding is being enhanced due to the T>C
mutation.

• In 2018 Shi et.al. identified Differential expression profiles of free microRNAs (miRNAs) in
cerebrospinal fluid (CSF) from patients with intracerebral haemorrhage (ICH) and
controls were analysed to identify miRNA markers associated with ICH-induced brain
injury.
• They have identified 77 miRNAs that were up-regulated (fold change > 2) and miR-6769-
5b is one of them.
• Seizures may be a presenting symptom for ICH as well.
• SoShi,
Qinghai in Dithat
Ge, Qicontext, even
Yang, Li Wang, miR-6769-5b
Jianfeng also should
Fu,MicroRNA profiling be considered
of cerebrospinal aswith
fluid from patients a biomarker.
intracerebral
haemorrhage, Frontiers in Laboratory Medicine, Volume 2, Issue 4,2018, Pages 141-145,ISSN 2542-3649,
Residue interaction from control GABRA1 and mutant
GABRA1 with miR6769-5b modeled using rosetta
Work to be done (pandemic delay)

• 1. Synthesis of LEV-derivatives 1 and 2

• 2. Purification of His-tag-free calpain1 core-domain
• 3. In-vitro measurements of Lev-der binding to calpain1.
• 4. Biological testing of lev-derivatives for their efficacies.
• 5. Design and Testing of sRNA inhibitors of mR1275 and miR6769 as
• cure For drug-refractory MTLE patients.
• 6. Crystallisation of calpain1 – levderivative complexes
• 7. X-ray crystal structure determination of calpain1-designed drug
complexes.
GANTT Chart
• Thank you.
 Levetiracetam
Docking Score
-3.191(S1),-3.644(C1N)

Active Site 1
L-Prolinamide
Docking Score -4.140
Piperidine-2-carboxylic acid tert-butylamide
Dscore -5.950
Rufinamide
Dscore -3.215
Oxcarbazepine
DSCore -4.341
Enprofylline
Dscore -3.534
Brivaracetam
Dscore -4.155
Docking Study for Identification of Natural
Compound • Natural compounds were downloaded
from Molprot Database
• Natural compounds were screened
based on similarity index using OCHEM
modelling environment
• We have used 1ZCM PDB id for calpain1.
• The ligand bound to this is
N-[(BENZYLOXY)CARBONYL]LEUCYL-N~1~-
[3-FLUORO-1-(4-HYDROXYBENZYL)-2-
OXOPROPYL]LEUCINAMIDE
C30 H40 F N3 O6
JCRSHQCFRMCMOC-GSDHBNRESA-N
• We searched natural compounds having
minimum 85% similarity to this
compound.
PDB Entries
PDBID Resolution Organism Ligand Present Expression system

2ARY 2.40 Homo sapiens NO Escherichia coli BL21(DE3)

1ZCM 2.00 Homo sapiens 11505008(Pubchem) Escherichia coli BL21(DE3)

DB04653(Drugbank
2R9F 1.60 Rattus norvegicus 49867185(Pubchem) Escherichia coli

2R9C 1.80 do 49867051 (Pubchem) do

2NQI 2.04 do 49867398 Structure Escherichia coli BL21

too flexible
2NQG 2.04 do 49867397 Escherichia coli BL21

2NQG 2.04 do 49867397

1TL9 1.80 Rattus norvegicus, 137348943 Escherichia coli BL21(DE3)

Streptomyces roseus
1QXP 2.80 Rattus norvegicus, Bos taurus No Escherichia coli

2G8J 1.61 Rattus norvegicus 6857712 Escherichia coli BL21

2G8E 2.25 do 11840936 do

1KXR 2.07 Rattus norvegicus No do

 Recent article “Identification of hsa-miR-1275 as a
Novel Biomarker Targeting MECP2 for Human
Epilepsy of Unknown Etiology” supports our SPR
result
• In recent work Zhao et al. conducted miRNA expression profiling of epilepsy of unknown etiology
(EUE) by microarray technology and identified 57 pathogenic changed miRNAs with significance.
• The data and bioinformatic analysis results indicated that among these miRNAs, hsa-microRNA
(miR)-1275 was highly associated with neurological disorders.
• Subsequently, they also collected samples of serum and cerebrospinal fluid were for validation
of hsa-miR-1275 expression by TaqMan assays.
• Results show that hsa-miR-1275 in serums of EUE were increased significantly, but in
cerebrospinal fluid, the miRNA was decreased.
• Moreover, through bioinformatics analysis they selected MECP2 gene as a hsa-miR-1275 target
based on target prediction tools and gene ontology analysis.
• Our study has also identified and validated through SPR assay GABRA1 T>C mutant as miR-1275
target.
• This suggest that both SNP and miR-1275 can as a biomarker in MTLE.
Ye Zhao, Congxia Lu, Huiling Wang, Qing Lin, Liangliang Cai, Fanrong Meng, Enque Biniam Tesfaye, Hsin-Chih Lai, Chi-Meng Tzeng,Identification of hsa-miR-1275 as a Novel Biomarker
Targeting MECP2 for Human Epilepsy of Unknown Etiology,Molecular Therapy - Methods & Clinical Development,Volume 19,2020,Pages 398-410,ISSN 2329-0501,