This document summarizes a Ph.D. thesis defense presentation by Hina Khalid from the Department of Bioinformatics and Biotechnology at GC University Faisalabad. The thesis involved an in silico and experimental investigation of synthetic compounds against Hepatitis C Virus. The presentation covered the need for the project, research objectives, methodology, and outcomes. The objectives were to develop assays to identify synthetic compounds with antiviral potential against HCV and minimal side effects, validate compounds' ability to inhibit the HCV polymerase NS5B, and characterize hit compounds' ability to restrict HCV replication in vitro. The methodology involved molecular virology experiments, compound preparation and screening, and computational modeling including molecular docking and dynamics simulations. Two potential hit

2. Ph.D. Thesis Defense
Insilico and experimental
investigation of the therapeutic
potential of synthetic compounds
against Hepatitis C Virus
Hina Khalid
Ph. D scholar
Department of Bioinformatics and
Biotechnology
GC University Faisalabad

3. Supervisor :
Dr. Usman Ali Ashfaq

4. Contents
• Introduction
• Need of the project
• Research Objectives
• Methodology
• Outcomes

6. Introduction
•
• .
.
Hepatitis C is a liver disease caused by the hepatitis C virus
leads to cirrhosis and HCC
Globally, an estimated 185 million people have chronic
hepatitis C infection.
Approximately 399,000 people die each year from hepatitis
C
Antiviral medicines can cure more than 95% of persons with
hepatitis C infection, but access to diagnosis and treatment
is low.
There is reduced antiviral efficacy in fibrotic and cirrhotic
subjects and no prophylactic approach available so far

7. HCV genome organization
Schematic illustration of the
9.6 kb positive-strand HCV
RNA genome.

8. HCV life cycle
• (a) receptor binding
and endocytosis;
• (b) fusion and
release of the single-
stranded viral RNA
genome into the cell
cytoplasm;
• (c) translation of
viral RNA and
processing of
polyprotein;
• (d) replication of
viral RNA ;
• (e) virion
morphogenesis;
• (f and g) viral
maturation and
release.

9. Structures of HCV non-nucleoside inhibitors
binding sites on NS5B.
(A) Binding sites of NNI-1
(cyan), NNI-2 (yellow), NNI-
3 (blue), and NNI-4 (green).

10. Synthetic compounds
• Antiviral activity was derived from the
compounds via
– binding to the viral envelope and inhibiting its
interaction with cells e-g tetrabutyl-calix
– interfere with the RNA synthesis initiation
phase e-g benzothiadiazines
– inactivate the enzyme from its active
conformation e-gThiopenes, Phenylalanines

11. Research Objectives
• Develop a cell-based assay enabling identification of the
synthetic compounds with antiviral potential along with minimal
side effects
• Validate the ability of hit compounds to inhibit HCV polymerase
(NS5B) functions
• Characterize hit compounds corresponding to their potential to
restrict/inhibit HCV replication in vitro

12. Research Methodology

13. Molecular Virology
Synthetic Compounds preparation
Cell culture
Evaluation of cytotoxicity
Antiviral potential of Compounds

14. Evaluation of Cytotoxicity

15. Antiviral assay

16. Western Blotting

17. Statistical analysis
1
• The statistical analysis was done using GraphPad Prism7
software
2
• The one-way analysis of variance (ANOVA) followed by Tukey
test was performed for determination of the difference between
control and treated groups.
3
• The p˂0.05 was considered as statistically significant

18. Computational Approach
Selection and refinement of protein
Compounds library preparation
Analysis of target active binding sites and Molecular docking
Molecular Dynamics Simulation
Pharmacokinetic profile prediction

19. Molecular Docking in MOE
1. Define and
prepare active
site
3. Best
conformation
3. Rank
compound
Virtual Synthetic
compounds
Most potent
inhibitor
Visual inspection
• Well buried in
active site ?
• H-bonds ?
• RMSD value ?
• Minimum score
?

20. Physiochemical property profile
and Toxicity Predictions
Drug-like properties are key
element to drug discovery
project
• MW< 500 Dalton
• H-bond donor < 5
• Sum of N and O
(H-bond acceptors)
< 10
• Mlogp<5

21. MD Simulation via Desmond

23. Section I
Results from
screening of
synthetic
compounds

24. Synthetic compounds
Benzothiazine Derivatives
Pyrazolobenzothiazine
Derivatives
Benzimidazole Derivatives
NORFLOXACIN Derivatives
Structural hybrids of
benzimidazole and
Benzothiazine
1
Selected ascribed
to their potent
antiviral activity
2

25. Cytotoxicity analysis of synthetic
compounds via MTT Assay
1.
The compounds evaluated with dose
ranging from 100 μM to 200 uM
exposed that administration of dose up
to 200 μM demonstrated >80% cell
viability for 11 of the compounds
including 2, 3, 4, 5a ,TB1S3, 5b, 5c, 6c
TBIS7,5d , TBIS9, TBIS11, TB1S14.
2.
However, 5d, 5e, TB1S13, TB1S15
exhibited cellular toxicity in HepG2
cells at the concentration of 200 μM
with 64%, 47%, 52%, and 79% of
cell viability respectively.

26. Cytotoxicity analysis of synthetic
compounds via MTT Assay
The graphical representation of MTT assay of
compounds (a:p ˂0.5, b:p˂0.1, c:p˂0.01 compared with DMSO)

27. Expression of NS5B HCV gene after 24 hours
treatment with compounds (2, 3, 4, TBIS3, 5a, 5b,
5c, and TBIS7)
Lane 1: Negative, 2: Vector, 3: compound 2 (100 µM), 4: compound 3 (100
µM), 5: compound 4 (100 µM), 6: compound TB1S3(100 µM), 7: compound
5a (100 µM), 8: compound 5a (200µM), 9: compound 5b (100 µM), 10:
compound 5b (200 µM), 11: compound 5c (100 µM), 12: compound 5c (200
µM), 13: compound TB1S7 (100 µM), 14: compound TB1S7 (200 µM), M:
100 bp DNA Ladder.

28. Expression of NS5B HCV gene after 24 hours
treatment with compounds (5d, TBIS9, and
TBIS11, 5e and TBIS13)
Lane 1: Negative, 2: compound TB1S9 (100 µM), 3: compound TB1S9 (200
µM), 4: compound TB1S11 (100 µM), 5: compound TB1S11 (200 µM), 6:
compound 5d (100 µM), 7: compound 5d (200 µM), 8: compound 5e (100
µM), 9: compound 5e (200 µM), 10: compound TB1S13 (100 µM), 11:
compound TB1S13 (200 µM), 12: Vector, M: 100 bp DNA Ladder.

29. First Potential hit
5b

30. 5b
Chemical Name • Chemical Structure
2-((1H-Benzo[d]imidazol-2-
yl)methyl)-N′-(4-
bromobenzylidene)-4-hydroxy-2H-
benzo[e][1,2]thiazine-3-
carbohydrazide 1,1-dioxide

31. Dose-dependent inhibition of HCV NS5B
gene by synthetic compound 5b
The concentration of 25, 50, 100
and 200 μM correlates with up to
0.4-fold, 0.45-fold, 0.7-fold, and
0.8-fold respectively as
compared to control.

32. Inhibition of NS5B expression at Protein level
The analysis of bands intensity using Image J
software depicted that the compound 5b at the
concentration of 50, 100, and 200 μM caused
inhibition of NS5B up to 20, 40, and 70 %
respectively
Effect of the compound 5b on the expression
level of HCV NS5B protein.

33. Molecular interactions of 5b with
allosteric sites

34. MD Simulation

35. Research Paper (IF 3.342)

36. Second Potential hit
6c

37. 6c
Chemical Name • Chemical Structure
2-(3,4-dimethyl-5,5-
dioxidobenzo[e]pyrazolo[4,3-
c][1,2]thiazin-2(4H)-yl)-N-(2-
fluorobenzyl)acetamide

38. Cytotoxicity analysis of synthetic
compounds via MTT Assay
The graphical
representation of
MTT assay of
compounds 6c

39. Anti-HCV NS5B Analysis in response to
6c
Expression of HCV NS5B gene
post 24 h treatment with
synthetic compound 6c.
Dose-dependent antiviral effect of 6c against HCV
NS5B. 24 h post-treatment with 6c, the RT-PCR method
was employed for the quantification of the expression
of HCV NS5B at mRNA level with GAPDH as an internal
control. **p˂0.01, **p˂0.001 versus DMSO.

40. The binding poses of compound 6c in the
respective binding pocket of NS5B polymerase

41. MD simulation
A B
C D

42. Research Papers (IF 2.557)

43. Section II
Results from
screening of
Phytochemicals

44. Phytochemicals
• Non-nutritive chemicals isolated from
plants
• cost-effective and have minimum side
effects
• Have protective or disease preventive
properties
anti-oxidants, anti-bacterial, anti-
viral and enzyme stimulators

45. The binding poses of the docked Betanin
and 3,5'-dihydroxythalifaboramine in
complex with NS5B

46. The binding poses of the docked Diarctigenin
and 6’-desmethylthalifaboramine in complex
with NS5B

47. The binding poses of the docked Cephalotaxine
and 5alpha-O-(3'-dimethylamino-3'-
phenylpropionyl) in complex with NS5B

48. The binding poses of the docked
IsoTetrandrine and Sofosbuvir in complex with
NS5B

49. Drug scan of Phytochemicals
Compound Molecular weight
(g/mol)
Number
of HBA
Number
of HBD
MLogP
Lipinski rule of five <500 <10 <5 <5
Betanin 550.47 13 8 -4.72
3,5'-dihydroxythalifaboramine 684.786 11 3 5.7
Diarctigenin 742.81 12 02 2.83
6’desmethylthalifaboramine 638.75 9 2 2.95
Cephalotaxine 545.62 10 2 1.40
5alpha-O-(3'-dimethylamino-3'-
phenylpropionyl) taxinine M
861.93 16 1 1.90
IsoTetrandrine 622.75 8 0 3.73
Sofosbuvir
529.46 12 3 0.64

50. The ADMET Profiling drug like parameters of
phytochemicals
A. ADMET Profiling
Compounds Betanin 3,5'-
dihydroxythal
ifaboramine
Diarctigenin 6’-
desmethylthalif
aboramine
Cephalotaxine 5alpha-O-(3'-
dimethylamino-3'-
phenylpropionyl)
taxinine M
IsoTetrandrine Sofosbuvir
A. Absorption
Blood-Brain Barrier + + + + + + + -
Gastro- Intestinal
Absorption
+ + + + + + + +
P-glycoprotein
substrate
No No Yes No No Yes No No
B. Metabolism
CYP450 1A2
Inhibitor
No No No No No No No No
CYP450 2C9
Inhibitor
No No No No No No No No
CYP450 2D6
Inhibitor
No No Yes No Yes No No No
Distribution
Subcellular
localization
Nucleu
s
Mitochondria Mitochondria Mitochondria Mitochondria Mitochondria Lysosomes Mitochondri
a
Toxicity

51. Toxicity assessment
Compounds
name
Predicted
toxicity
class
Prediction
accuracy
(%)
Hepatotoxicity
Probability
Cytotoxicity
Probability
Mutagenicity
Probability
1 Betanin IV 67.38 I 0.64 I 0.57 I 0.57
2 3,5'-
dihydroxythalifaborami
ne
IV 100 I 0.69 I 0.93 I 0.97
3 Diarctigenin IV 68.07 I 0.81 I 0.86 I 0.77
6’-
desmethylthalifaborami
ne
IV 97.58 I 0.95 I 0.72 A 0.53
5 Cephalotaxine II 100 I 0.88 A 0.63 I 0.66
6 5alpha-O-(3'-
dimethylamino-3'-
phenylpropionyl)
taxinine M
IV 67.38 I 0.82 I 0.55 I 0.54
7 IsoTetrandrine IV 100 I 0.95 I 0.69 A 0.84
8 Sofosbuvir VI 67.38 I 0.63 I 0.72 I 0.69

52. Research Paper (IF 1.339)

53. Conclusion
• It is revealed that novel discovered hits including 5b and 6c
compounds target simultaneously different sites of the NS5B
polymerase that would be a potential lead candidate for the
development of anti- HCV target-specific drugs targeting viral
polymerases
• This results and subsequent studies will kindle into the roles of
NS5B inhibition in the HCV treatment

54. Future Perspective
• It could be of significant importance to health professionals in
treating subjects particularly with HCV subjects with resistance-
associated substitutes and those with failure to DAA regimen
• The phytochemicals evaluated for drug-likeness and Toxicity
assessment suggest opportunities for the optimization of the
phytochemicals through experimental study
• Those patients diagnosed at the advanced stage could
significantly benefit from plant based bioactive compounds
discovered in the study ascribed to the minimal side effects and
pharmacological safety profile

55. Additional Articles

58. Publications (Impact Factor 24.926)
• Khalid, H., et al., Discovery of Novel HCV NS5B polymerase inhibitor, 2-(3, 4-dimethyl-5, 5-
dioxidobenzo [e] pyrazolo [4, 3-c][1, 2] thiazin-2 (4H)-yl)-N-(2-fluorobenzyl) acetamide via
molecular docking and experimental approach. Clinical and Experimental Pharmacology and
Physiology, 2021 (2.257)
• Ashfaq, U. A., Khalid, H. (2020). CRISPR/CAS9-Mediated Antiviral Activity: A Tool to Combat
Viral Infection. Critical Reviews™ in Eukaryotic Gene Expression, 30(1). (2.156)
• Shahid, F., Ashfaq, U. A., Javaid, A., Khalid, H. (2020). Immunoinformatics guided rational
design of a next generation multi epitope based peptide (MEBP) vaccine by exploring Zika
virus proteome. Infection, Genetics and Evolution, 80, 104199. (3.342)
• Javaid, A., Ashfaq, U., Zafar, Z., Akmal, A., Taj, S., Khalid, H. (2020). Phytochemical
Analysis and Antidiabetic Potential of Armoracia Rusticana: Pharmacological and
Computational Approach. Combinatorial Chemistry High Throughput Screening. (1.339)
• Khalid, H., Ashfaq, U. A. (2020). Exploring HCV genome to construct multi-epitope based
subunit vaccine to battle HCV infection: Immunoinformatics based approach. Journal of
Biomedical Informatics: 103498. (6.317)
• Khalid, H., et al. (2020). Discovery of novel Hepatitis C virus inhibitor targeting multiple
allosteric sites of NS5B polymerase. Infection, Genetics and Evolution: 104371. (3.342)

59. Cont.
• Khalid, H., Ashfaq, U. A. (2020). Molecular docking and Pharmacoinformatics studies reveal
potential phytochemicals against HCV NS5B Polymerase. Combinatorial Chemistry High
Throughput Screening. (1.339)
• Masoud, M. S., Ashfaq, U. A., Khalid, H. (2018). Interferon-Free Regimen for Hepatitis C:
Insight and Management. Critical Reviews™ in Eukaryotic Gene Expression, 28(4). (2.156)
• Ashfaq, U. A., Khalid, H. (2017). Mechanism of Hepatitis C Virus Induced Diabetes Mellitus.
Critical Reviews™ in Eukaryotic Gene Expression, 27(4) (2.156)
• Khalid, S., Idrees, S., Khalid, H., Hussain, B., Tiwari, S. (2015). Ab-InitioPrediction of Sequence
and Structural Biology of Fish Muscle Proteins Using Homology Modeling, Phylogeny and
Different Computational Approaches. MOJ Proteomics Bioinform 2 (3): 00047. DOI:
10.15406/mojpb. 2015.02. 00047 gi| 185132813, Q8UVF6 and gi| 49901349.
• Khalid, H., Masoud, M. S., Qasim, M., Ashfaq, U. A. (2014). Targeting cancer stem cells in
hepatocellular cancer: a review. European Journal of Oncology, 19(3), 130-143. (0.179)
• Hussain, B., Khalid, H., Nadeem, S., Sultana, T., Aslam, S. (2012). Phylogenetic and
Chronological Analysis of Proteins Causing Alzheimer’s, Parkinson’s and Huntington’s
Diseases. Int J Bioautomation, 16(3), 165-178. 2. (0.35)

60. Acknowledgement
• Supervisor I
– Dr. Usman Ali Ashfaq
• Co-Supervisor
– Dr. Matloob Ahmed
• Supervisory Committee
– Dr. Muhammad Shareef
Masoud
– Dr. Muhammad Qasim
Awan
• Collaborator
– Dr. Bushra Ijaz
• Faculty Members
– (Dept. of Bioinformatics &
Biotechnology)
• Funding Agency
– Higher Education
Commission
(HEC) Pakistan