Screening cascade is biochemical

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Screening cascade is biochemical or cell based screen which is used to select the desired lead compound. During the screening cascade the compounds with the inferior properties are discarede and the compounds with good pharmacodynamic and pharmacokinetic properties pass through the screening cascade. Screening of large number of compounds againt enzyme or receptor is referred to as high through put screening. Compounds tested in HTS include existing compounds in the company and previously synthesised molecules.

Endothelins are family of vasoactive peptides that causes contraction of blood vessels and raise hypertension. It is widely distributed in the body. The isoforms of the endothelins are ET-1, ET-2 and ET-3 and the two important receptors are ET-A and ET-B. ET-A situated in the smooth muscle of blood vessels while ET-B found in endothelial cells. ET-A is responsible for increasing of vasoconstriction when the endothelin binds to the ET-A receptor that leads to the high blood pressure while ET-B receptors mediate vasodialation.

Endothelin receptor antagonists (ERA) are of three types. One is selective to ET-A while dual ERAs are selective towards both ET-A and ET-B and one selective to ET-B. In this work shop compounds are selected on the basis of anatagonist potency against the ET-A receptor.

These compounds are tested agaist different receptors such as endothelin converting enzyme(ECE), non catalytic Src homology (NCSH), Cholestereryl ester transfer protein(CEPT) and phosphodiesterase(PDE) by high throughput screening to check the inhibitory activity.

ACTIVITY IN ETA RECEPTOR BINDING ASSAY

COMPOUND

% INHIBITION

LN-208

100%

LN-015

100%

LN-142

90%

LN-292

95%

LN-127

60%

LN-216

95%

LN-047

90%

LN-209

60%

Test concentration 1x10-4M

All other compounds tested have inhibition < 10%


Sheet B1

ACTIVITY IN HIGH THROUGHPUT SCREENS

(A=Active I=Inactive)

Compound

% Inhib.

PDE

ECE

CETP

SH2

LN-208

100%

A

I

I

I

LN-015

100%

A

A

A

A

LN-142

90%

I

I

I

A

LN-292

95%

I

I

I

I

Sheet B2

LN-127

60%

I

I

I

I

LN-216

95%

I

I

I

I

LN-047

90%

I

I

A

I

LN-209

60%

I

I

I

I

LN-216 shows high percentage of inhibitory activity i.e, 95% but it is inactive towards endothelin converting enzyme(ECE), non catalytic Src homology (NCSH), Cholestereryl ester transfer protein(CEPT) and phosphodiesterase(PDE) receptors. This compound quite difficult to synthecise due to the complex amino acid structure and it is digested by the other enzymes to cause loss of activity. LN- 209 is unstable due to presense of sulphoxide bridges in its structure, therefore this compound discarded. LN-015active towards all the receptors and it shows 100% inhibition against ETA receptor. This compound has lack of specificity as it is active towards all the receptors it may elicit side effects. Out of the eight drugs ( LN-208, LN-105, LN-142, LN-292, LN-127, LN-216, LN-047 and LN-209), three drugs were not useful ( LN-216, LN-015 and LN-209). Remaining five drugs have good percentage of inhibition in addition to specificity. These five compounds are then carried to the further processes of screening.

Sheet C

IC50 DETERMINATION FOR ETA RECEPTOR BINDING

Compound

pIC50

LN-208

7

LN-142

8

LN-292

8

LN-127

<4

LN-047

7

Compounds from the previous process are then tested for IC50 against ETA receptor. IC50 can be defined as “The concentration of an inhibitor that is required for 50-percent inhibition”. Table 2 values are in the form of negative logarithm of IC50 (PIC50). All the compounds have higher IC50 values except for LN-127 (<4) indicates it has poor affinity towards the receptor. Therefore LN-127 discarded and other compounds are then carried into the next process of the screening.

Sheet D

pA2 DETERMINATION IN RAT AORTA vs ENDOTHELIN

Compound

pA2

LN-208

7

LN-142

8

Also contracts tissue

LN-292

8

LN-047

7

Compound from previous screening process are then tested for PA2 determination in rat aorta versus endothelin. PA2 can be defined as “ negative logarithm of antagonist that produces DR = 2”.

Sheet E

I.V. ACTIVITY IN ANAESTHETISED RAT vs ENDOTHELIN

Compound

ID50

LN-208

Inactive

LN-292

0.1 mg/kg

LN-047

0.5 mg/kg

ID50 can be defined as “concentration of the drug that is required for 50% of binding to the target receptor” Remaining three compounds ( LN-208, LN-292 AND LN-047) from previous screening process are then tested for ID50. It has been observed that LN-208 is inactive. Therefore this compound discarded for the next stage of screening process.

Sheet F

ORAL ACTIVITY IN CONSCIOUS RAT vs ENDOTHELIN

Compound

ID50

LN-292

100 mg/kg

LN-047

1 mg/kg

Compounds LN-292 and LN-047 were then tested for ID50­ in conscious rat vs endothelin (Oral activity). It has been observed that LN-292 has greater ID50 value (100 mg/kg) when compared to the LN-047 (1 mg/kg). This may be due to the first pass metabolism and decreased absorption of the drug.

Sheet G

ORAL DURATION OF ACTION IN CONSCIOUS RAT

% Inhibition with dose of 1 mg/kg

0.5 hr

1hr

3hr

5hr

LN-047