The new drugs, called PCSK9 inhibitors, are monoclonal antibodies. They target and inactivate a specific protein in the liver. Knocking out this protein, called proprotein convertase subtilisin kexin 9, dramatically reduces the amount of harmful LDL cholesterol circulating in the bloodstream. Lower LDL translates into healthier arteries and fewer heart attacks, strokes, and other problems related to cholesterol-clogged arteries.

1. IDENTIFICATION OF POTENTIAL INHIBITORS
FOR PROPROTEIN CONVERTASE SUBTILISIN/KEXIN9
(PCSK9)
VIKAS REDDY .V – 1121210029
MOURYA .CH - 1121210038
PRASANTH .B - 1121210042
Under the guidance of Dr. P. Rathi
Suganya

2. INTRODUCTION
• Proprotein convertases are a family of proteins that activate other proteins.
• Many proteins are inactive when they are first synthesized, because they contain
chains of amino acids that block their activity.
• Proprotein convertases remove those chains and activate the protein.
• PCSK9, is an enzyme encoded by PCSK9 gene in humans.
• PCSK9 binds to the receptor for low density lipoprotein (LDL) cholesterol

3. • When PCSK9 binds to the LDL receptor, the receptor is broken down and
can no longer remove LDL cholesterol from the blood.
• Therefore, blocking PCSK9 can lower blood cholesterol levels. By reducing
the level of LDL cholesterol circulating in the bloodstream, it can decrease
of cardiovascular disease such as heart attacks.
• Evolocumab or Alirocumab are already known inhibitors for PCSK9 which
side effects like nasopharyngitis, common cold, upper respiratory tract
injection site reactions influenza and allergic reactions.

4. AIM
• To identify the potential inhibitors for PCSK9
OBJECTIVE
• XP docking of Binding database.
• E-pharmacophore generation for known inhibitor.
• Structure based screening of natural compounds.
• Virtual screening on best hits.
• Analysis of ADME properties.
• Binding free energy calculation studies for shortlisted compounds
• Molecular dynamics studies on potential lead compounds.

5. METHODOLOGY
step1
•3D structure of PCSK9 protein(2P4E) - PDB
•Protein preparation
step2
•Active Site Prediction using Site Map
•Grid Generation
step3
•Active Site Prediction using Site Map
•Grid Generation
step4
• E-pharmacophore generation
step5
• Structured based screening of natural compounds from ZINC database
step6
•Virtual screening on best hits
•ADME properties of short listed compounds
step7
• Molecular dynamics simulation using desmond

6. 3D STRUCTURE OF PCSK9 PROTEIN
Crystal Structure of PCSK9 (PDB ID: 2P4E) of Resolution 1.98Å

7. SITE SITE SCORE
2 1.032
1 1.028
3 0.953
4 0.861
5 0.711
ACTIVE SITE PREDICTION
FOR PCSK9
• Active sites of PCSK9 protein were predicted using sitemap
• Site 2 shows the best site score

8. XP DOCKING OF BINDING
DATABASE
• 4 known inhibitors were docked with protein PCSK9
• Ligand 3(BDBM50014066) shows best glide score among all and
it was taken for the generation of e pharmacophore
BINDING DB
GLIDE SCORE
(Kcal/mol )
GLIDE
ENERGY
(Kcal/mol )
XP
H-BOND
BDBM50280216 --4.96818 -27.1604 -1.14034
BDBM50067889 -5.53684 -31.8286 -0.55042
BDBM50014066 -7.62705 -45.5613 -1.27458
BDBM669996 -6.53684 -45.3158 -1.08332

9. H-bond interactions of ligand BDBM50014066 with VAL 664 and
ALA 637 of PCSK9 with H-bond 2.023 & 2.137 respectively

11. FEATURE SCORE
A1 -1.67
D6 -0.61
R8 -1.07
E-PHARMACOPHORE GENERATION
• The binding pose of ligand 3 was given as a input for e-pharmacaphore
generation
• 3 features were generated
 1 hydrogen bond acceptor
 1 hydrogen bond donor
 1 aromatic ring
E-pharmacophore generation for
BDBM50014066
E-pharmacophore features generated for
BDBM5001406

12. STRUCTURE BASED SCREENING OF NATURAL COMPOUNDS
• Around 20000 natural compounds from zinc database were screened
based on the pharmacophore hypothesis generated
• The compounds with fitness score above 1.5 were taken for further
studies
COMPOUND ID FITNESS SCORE
ZINC34237065 2.304714
ZINC01530713 2.262534
ZINC20464408 2.203128
ZINC71404899 2.193322
ZINC20464414 2.165204
ZINC20464417 2.164566
ZINC20464397 2.159519
ZINC20464404 2.154947
ZINC20464420 2.147802

13. Superimposition of BDBM50014066 with ZINC34237065

14. VIRTUAL SCREENING ON BEST HITS
• The best hits with good fitness score were taken for virtual screening (HTVS, SP, and XP)
ID XP Gscore glide energy glide emodel XP HBond
ZINC85625485 -13.03749 -57.629837 -91.670604 -3.264457
ZINC85625523 -12.925718 -79.264685 -119.027808 -3.629028
ZINC31167448 -12.348004 -72.306324 -89.342669 -5.058737
ZINC85625406 -12.583352 -77.525799 -111.040503 -2.591186
ZINC85625489 -12.428372 -72.79603 -124.659648 -5.78483

15. ADME properties of short listed compounds
Compound ID mol_MW donorHB AccptHB QPlogPo/w Human Oral
Absorption
(%)
ZINC85625485 563.689 5 7.45 4.528 42.635
ZINC85625523 463.527 6 9.15 1.437 31.978
ZINC31167448 504.935 6 12.3 0.81 5.219
ZINC85625406 659.775 7 9.9 4.611 35.467
ZINC85625489 497.544 6 8.2 1.985 25.394
Out of 5 shortlisted compounds two natural compounds ZINC85625485
(42.635%) and ZINC85625406 (35.465%) shows higher Human Oral
Absorption.

16. Molecular interactions (2D) of
ZINC85625485 with PCSK9
Molecular interactions (2D) of
ZINC85625406 with PCSK9

17. Molecular dynamics
• Molecular dynamics simulation predicts molecular interactions ,such as hydrogen bonds
between amino acids and substrate.
• There are two programmes that you will use for molecular dynamics :
Schrodinger
Desmond
• Molecular dynamics simulation (MD) further indicated that the conformation derived from
docking is basically consistent with the average structure extracted from MD simulation.

18. a) RMSD graph of 2P4E complex with ZINC85625485
(a) 2P4E complex with ZINC85625485 shows simulation range of backbone
atoms varied from 0.7 to 2.3 Å.

19. b) RMSD graph of 2P4E complex with ZINC85625406
(b) 2P4E complex with ZINC85625406 shows simulation range
of backbone atoms varied from 0.6 to 1.4 Å

20. CONCLUSION
• Known PCSK9 inhibitor has been screened and e-Pharmacophore has been generated.
• Based on hypothesis generated by e-Pharmacophore natural compounds from ZINC database has been
screened to investigate potential inhibitors of PCSK9.
• Binding orientation and protein-ligand interaction has been analyzed and found that VAL 644 and GLU
450 commonly known to interact with selected ligands.
• ADME properties of selected hits were found. Based on the docking results and ADME properties, we
report that two compounds (ZINC85625485 & ZINC85625406) that may act as the potential inhibitors
for PCSK9.

21. REFERENCES
• Gearing ME (2015-05-18). "A potential new weapon against heart disease: PCSK9
inhibitors“
• Joseph L, Robinson JG (2015). "Proprotein Convertase Subtilisin/Kexin Type 9
(PCSK9) Inhibition and the Future of Lipid Lowering Therapy". Progress in
Cardiovascular Diseases.
• http://www.medpagetoday.com/MeetingCoverage/ACC/50488. Sabatine MS, et al
"Efficacy and safety of evolocumab in reducing lipids and cardiovascular events"
N Engl J Med 2015; DOI: 10.1056/NEJMoa1500858.
• Tavori, H., Giunzioni , I., & Fazio, S. (2015). PCSK9 inhibition to reduce
cardiovascular disease risk: recent findings from the biology of PCSK9. Current
Opinion in Endocrinology, Diabetes and Obesity, 22(2), 126-132.

22. • Is there enough evidence with evolocumab and alirocumab (antibodies to proprotein convertase
substilisin-kexin type, PCSK9) on cardiovascular outcomes to use them widely? Evaluation of
Sabatine MS, Giugliano RP, Wiviott SD et al. Efficacy and safety of evolocumab in reducing
lipids and cardiovascular events. N Engl J Med 2015;372:1500-1509, and Robinson JG, Farnier
M, Krempf M et al. Efficacy and safety of alirocumab in reducing lipids and cardiovascular
events. N Engl J Med 2015; 372:1488-99.

23. THANK YOU