Drug discovery

Virtual screening Phase I CYP

Phase II Clinical

trials

Computational model for CYP prediction.

Oxidation

* Bioactivation

* Uncoupling

* Inhibition

Conjugation

Where Do New Potential drug Where Do New Potential drug candidates Come From?candidates Come From?

DrugDrugDrugDrug

Acquisition Acquisition CompoundsCompoundsAcquisition Acquisition CompoundsCompounds

Natural Natural SourcesSourcesNatural Natural SourcesSources

Endogenous Endogenous LigandLigand

Endogenous Endogenous LigandLigand Newly Newly

SynthesizedSynthesizedNewly Newly

SynthesizedSynthesized

Random Screening Random Screening of Existing of Existing CompoundsCompounds

Random Screening Random Screening of Existing of Existing CompoundsCompounds

From other From other PharmaceuticalPharmaceuticalcompaniescompanies

e.g. Natural e.g. Natural peptide, hormone, peptide, hormone, etc.etc.

Combinatorial Combinatorial ChemistryChemistryLibrariesLibraries

Combinatorial Combinatorial ChemistryChemistryLibrariesLibraries

Hepatic microsomal enzymes (oxidation, conjugation)

Despite rigorous testing, approximately 75,000-135,000 deaths due to adverse drug reactions occur annually in the United States, making it the sixth leading cause of death.

Adverse drug interactions are unpredictable from routine laboratory test in human.

► In Smooth Endoplasmic Reticulum In Smooth Endoplasmic Reticulum (microsome)(microsome)

- detoxification (cytochrome P-450s)- detoxification (cytochrome P-450s)

- toxins, drugs and xenobiotics- toxins, drugs and xenobiotics

- O- O22 + RH + RH R-OH and H R-OH and H22OO

- leakage of O- leakage of O22-. -. (Uncoupling)(Uncoupling)

- metabolic activation - X- metabolic activation - X. .

(Bioactivation)(Bioactivation)

Oxidative stress is caused by an imbalance between the production of Oxidative stress is caused by an imbalance between the production of reactive oxygenreactive oxygen and a biological system's ability to readily detoxify and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. the reactive intermediates or easily repair the resulting damage.

Pro-Pro-

oxidantsoxidantsAntioxidantAntioxidant

ss

P450

Bioactivation Detoxification

Transferase Peroxidase

One of the enzyme known to protect the cell from reactive oxygen species consequently from oxidative stress.

mGST1 is involved in cellular. defense against toxic, carcinogenic, and pharmacologically active electrophilic compounds e.g:

SH

mGST1

SX mGST1

Activation of mGST1 via cystein 49

-mGST1 is activated by most alkylating agents, Cysteine 49, which is the target for electrophile activation.

Activity increased several fold.

It is possible that It is possible that activation is an activation is an alternative rapid alternative rapid response to toxic response to toxic conditions in the cell.conditions in the cell.

Spin trapping Sensors detect modification of macromolecules

Short lived

Looking for easy methods for predicting drug bioactivation!

Oxidative DamageOxidative Damage

Short half life

Difficult to detect

Free Radicals and Reactive metabolitesProteins

(-SH)

Lipids (R-OO.)

DNA/RNA (-OH.)

Modification of Modification of enzyme function enzyme function e.g. e.g.

mGST1mGST1Scientific Question??

Utilize it as a marker !!!

The relationship between drug metabolism and drug The relationship between drug metabolism and drug

toxicity.toxicity.

SH

SX

Activity increased several fold.

P450

J. L.Walgren, M. D. Mitchell, & D. C. Thompson J. L.Walgren, M. D. Mitchell, & D. C. Thompson (2005) (2005) Crit. Rev. Toxicol. 35Crit. Rev. Toxicol. 35, 325-361, 325-361

► Drugs with reactive Drugs with reactive metabolites that have metabolites that have warnings of precautions warnings of precautions for hepatoxicityfor hepatoxicity AcetaminophenAcetaminophen CarbamazepineCarbamazepine ClozapineClozapine DiclofenacDiclofenac DisulfiramDisulfiram HalothaneHalothane LeflunomideLeflunomide MethyldopaMethyldopa RifampinRifampin TacrinTacrin TamoxifenTamoxifen TerbinafineTerbinafine TiclopidineTiclopidine ZileutonZileuton

► Drugs with reactive Drugs with reactive metabolites & never metabolites & never approved in USapproved in US AlpidemAlpidem AmineptineAmineptine AmodiaquineAmodiaquine CinchophenCinchophen DihydralazineDihydralazine DilevaoloDilevaolo EbrotidineEbrotidine GlafenineGlafenine IbufenacIbufenac IsoxanineIsoxanine NiperotidienNiperotidien PerhexilinePerhexiline PirprofenPirprofen TilbroquinolTilbroquinol

Biosensor with immobilized P450 and mGST1

mGST1 modified Covalantaly

Enhanced activity of mGST1

Biological monitor for reactive intermediates will be helpful in sorting out harmful compounds without prior knowledge of the intermediate structure of that particular compound.

Transferase

Peroxidase

Especially for the novel series of chemical entities under investigation.

Sensing Scheme:

SH SX

mGST1

Support i-e Aluminium oxized

Key benefit : high surface to

volume

Fixation of Endoplasmic

reticulum membranes

Immobilization

mGST1 Detection marker

Biomedical device

P450-b5-reductase

In the case of reactive metabolites formation

Enhanced activities of

mGST1

ExpectedExpected outcomesoutcomes

Development of the biosensor using mGST1 activation Development of the biosensor using mGST1 activation as a surrogate detection marker which will be highly as a surrogate detection marker which will be highly valuable technology for the prediction and testing of valuable technology for the prediction and testing of new drug entries that form reactive intermediates. new drug entries that form reactive intermediates.