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2013/08/02

DISCOVERY OF RHO-KINASE INHIBITORS

BYDOCKING-BASED VIRTUAL SCREENING

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What is Rho-kinases?

Why ROCK is important Bio-target?

What is the current trend in ROCK inhibition?

What approach this Paper will use to do th e research ingeneral?

INTRODUCTION

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1.Serine/Threonine Protein Kinase(What is Kinase?)

2.There are two isoforms, Rock1&ROCK2

WHAT IS RHO-KINASES?

Crystal structure of human ROCK I

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ROCK kinase plays an essential role in several

process in cell, such as cell motility adhesion smoothcontractionetc.

In addition, abnormal activation of ROCK pathway

could lead to cardiovascular disease, neurologicaldisease, fibrotic disease, cancer.etc.

WHY ROCK IS AN IMPORTANT BIO-TARGET?

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The only official-proved ROCK inhibitors called Fasudil

There are still many ROCK inhibitors in clinical trial.

The current scaf folds for ROCK can be divided into 4 categories

(1)1H-imidazo[4,5-c]pyridine (2)isoquinoline

(3)pyridine (4)2-aminopyrimidine

WHAT IS THE CURRENT TREND IN ROCK INHIBITION ?

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The research can be divided in two major part:

WHAT APPROACH THIS PAPER WILL USE TO DO THE

RESEARCH IN GENERAL?

1Docking-based virtual screening

2Biochemistry experimental assays

3

Molecular dynamic interaction

Free energy decomposition

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Pt1.Validation of the performance of molecular docking

(DOCKING)

Pt2.Docking-based virtual screening

(DOCKINGVirtual Screening)

Pt3.ROCK1 kinase inhibition assay

Pt4.Cell culture and MTT assay

Pt5.Molecular dynamic simulation

Pt6.Binding free energy decomposition analysis

Pt7.Another Test for par ticular test compound

MATERIAL&METHOD

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1.De lete a chain of the homodimer

(What is the homodimer&dimer?)

2.Remove water

3.Add Hydrogen

4.Minimize the steric clash via OPLS2005 forcefield

(What is forcefield&OPLS2005?)

5.Let RMSD reach to 0.3A

(What is RMSD?)

PT1.(USING 2ESM&GLIDE)

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6.When Grid generation&Docking setting is deault by software?

(What is Grid generation for?)

7.Using dif ferent Scoring function mode to esclate the accuracy

(What is the Scoring fuction?)

(Which Scoring function We use?)

8.Choose 196 known inhibitors from Binding DB database

10000 compounds as non-inhibitors from ChemBridege via"Find Diverse Molecular" protocol in Discovery Studio2.5

PT1 CON.

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9.Preprocess by Ligprep

10.All structures are docked into ROCK1's active site and use 3

Scoring function mode (HTVS,SP and XP)

(1)High throughput virtual screening

(2)Standard precision

(3)Extra precision

P.S In the docking process, protein is rigid and the ligand is

flexible.(A.K.A Semi-flexible docking)

PT1 CON.

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1.Using database for screening (Specs & ChemBri dge)

2. Using Discovery studio for fur ther cleaned and fi ltered by

The clean protocol:

"duplicate removing"

"salt stripping"

"mixture splitt ing"

"functional group standard"

"charge neutralization

3. Using ~1.1 mill ion structures for docking and scoring in HTVS SPXP,Choose Top50% in HTVS Redocked and choose Top20% in

SPChemical similarity clustering Top2000 compounds identif ied by XP

mode was performed to maxmize the che mical diversity of the selected

compounds for biological assays in "select" module via SYBYL8.1

Top200 compounds from i ndividual clusters were chosen, only 174 were

available for purchase.

PT2

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Using Kit&Plate to Measure how good is those 174 potential

ROCK inhibitors are and finding who has capability of

inhibition above 50%.

PT3

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Culture the HeLa Cell & other Cancer cell line below

(1)NCI-358 NCI-H460 (Lung cancer)

(2)MCF-7 MDA-231 (Breast cancer)

(3)HepG-2 SMMC-7721 (hepatoma)

(4)LP1 OPM-2 (myeloma)

(5)K562 (leukemia)

(6)KB (Oral epidermoid carcinoma)

After cell are cultured, the MTT assay are used.

PT4

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RESULT OF PT1~4

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RESULT OF PT1~4

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RESULT OF PT1~4

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RESULT OF PT1~4

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After the Structural Analysis We found 2 novel scaf folds

compounds (13,24)

USE

Molecular Dynamic simulation

Free energy decomposition

to Observe the remain 2 potent lead compounds and figure

out how to do the optimization.

PT5

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Both Compound 13,24 have relatively good inhibition in

ROCK1 as new novel scaffolds

In addition, when experiment is taking in zebra fish,

Compound 24 was found to be a good candidate for the

therapy of cerebral hemorrhage.

RESULT OF PT5~6

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CONCLUSIONS

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END