IDENTIFICATION OF POTENTIAL INHIBITORS FOR PROPROTEIN CONVERTASE SUBTILISIN/KEXIN9 (PCSK9)

VIKAS REDDY .V – 1121210029MOURYA .CH - 1121210038PRASANTH .B - 1121210042Under the guidance of Dr. P. Rathi Suganya

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

•  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 the risk of cardiovascular disease such as heart attacks.

• Evolocumab or Alirocumab are already known inhibitors for PCSK9 which has side effects like nasopharyngitis, common cold, upper respiratory tract infections, injection site reactions influenza and allergic reactions.

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.

METHODOLOGYstep1 •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

compoundsstep7

• Molecular dynamics simulation using desmond

3D STRUCTURE OF PCSK9 PROTEIN

Crystal Structure of PCSK9 (PDB ID: 2P4E) of Resolution 1.98Å

SITE SITE SCORE2 1.0321 1.0283 0.9534 0.8615 0.711

ACTIVE SITE PREDICTION FOR PCSK9

• Active sites of PCSK9 protein were predicted using sitemap• Site 2 shows the best site score

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

ID

GLIDE SCORE(Kcal/mol )

GLIDE ENERGY

(Kcal/mol )

XP H-

BOND

BDBM50280216 --4.96818 -27.1604 -1.14034BDBM50067889 -5.53684 -31.8286 -0.55042BDBM50014066 -7.62705 -45.5613 -1.27458

BDBM669996 -6.53684 -45.3158 -1.08332

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

respectively

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

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 studiesCOMPOUND ID FITNESS SCORE

ZINC34237065 2.304714ZINC01530713 2.262534ZINC20464408 2.203128ZINC71404899 2.193322ZINC20464414 2.165204ZINC20464417 2.164566ZINC20464397 2.159519ZINC20464404 2.154947ZINC20464420 2.147802

Superimposition of BDBM50014066 with ZINC34237065

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

ADME properties of short listed compoundsCompound

IDmol_MW donorHB AccptHB QPlogPo/w Human

Oral Absorption

(%)ZINC856254

85563.689 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 25.394Out of 5 shortlisted compounds two natural compounds ZINC85625485 (42.635%) and ZINC85625406 (35.465%) shows

higher Human Oral Absorption.

Molecular interactions (2D) of ZINC85625485 with PCSK9

Molecular interactions (2D) of ZINC85625406 with PCSK9

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.

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.

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

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.

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.

• 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.

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