Drug bioactivation predicting enzyme based biosensor

Drug discovery Clinical trials Computational model for CYP prediction. Phase I CYP Phase II Virtual screening Clinical trials Computational model for CYP prediction. Oxidation * Bioactivation * Uncoupling * Inhibition Conjugation

Where Do New Potential drug candidates Come From? Acquisition Compounds Natural Sources Endogenous Ligand Newly Synthesized Random Screening of Existing Compounds From other Pharmaceutical companies e.g. Natural peptide, hormone, etc. Combinatorial Chemistry Libraries

Hepatic microsomal enzymes (oxidation, conjugation) Adverse drug interactions are unpredictable from routine laboratory test in human. 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.

P450 metabolism GOOD or BAD????? In Smooth Endoplasmic Reticulum (microsome) - detoxification (cytochrome P-450s) - toxins, drugs and xenobiotics - O2 + RH R-OH and H2O - leakage of O2-. (Uncoupling) - metabolic activation - X. (Bioactivation)

Oxidative stress P450 Antioxidants Pro-oxidants Bioactivation Detoxification Oxidative stress Oxidative stress is caused by an imbalance between the production of reactive oxygen and a biological system's ability to readily detoxify the reactive intermediates or easily repair the resulting damage. Pro-oxidants Antioxidants

microsomal glutathione S-transferase 1 (mGST1) One of the enzyme known to protect the cell from reactive oxygen species consequently from oxidative stress. microsomal glutathione S-transferase 1 (mGST1) mGST1 is involved in cellular. defense against toxic, carcinogenic, and pharmacologically active electrophilic compounds e.g: Transferase Peroxidase halogenated alkenes and arenes, alpha, beta-unsaturated hydrocarbons and peroxides

SH mGST1 Activation of mGST1 via cystein 49 It is possible that activation is an alternative rapid response to toxic conditions in the cell. SX mGST1 Activity increased several fold. -mGST1 is activated by most alkylating agents, Cysteine 49, which is the target for electrophile activation.

How to measure reactive metabolites? Short lived Complicated methods ! Sensors detect modification of macromolecules Spin trapping Looking for easy methods for predicting drug bioactivation!

Modification of enzyme function e.g. mGST1 Oxidative Damage Short half life Difficult to detect Lipids (R-OO.) Proteins (-SH) Free Radicals and Reactive metabolites Modification of enzyme function e.g. mGST1 Scientific Question?? DNA/RNA (-OH.) Utilize it as a marker !!!

The relationship between drug metabolism and drug toxicity. mostly P450 P450 SH SX Activity increased several fold.

Drugs with reactive metabolites & never approved in US J. L.Walgren, M. D. Mitchell, & D. C. Thompson (2005) Crit. Rev. Toxicol. 35, 325-361 Drugs with reactive metabolites that have warnings of precautions for hepatoxicity Acetaminophen Carbamazepine Clozapine Diclofenac Disulfiram Halothane Leflunomide Methyldopa Rifampin Tacrin Tamoxifen Terbinafine Ticlopidine Zileuton Drugs with reactive metabolites & never approved in US Alpidem Amineptine Amodiaquine Cinchophen Dihydralazine Dilevaolo Ebrotidine Glafenine Ibufenac Isoxanine Niperotidien Perhexiline Pirprofen Tilbroquinol

Especially for the novel series of chemical entities under investigation. Sensing Scheme: Biosensor with immobilized P450 and mGST1 mGST1 modified Covalantaly Transferase Detection marker Enhanced activity of mGST1 Optical detection Peroxidase Biological monitor for reactive intermediates will be helpful in sorting out harmful compounds without prior knowledge of the intermediate structure of that particular compound.

Nopping bad drugs in the bud Support i-e Aluminium oxized Immobilization Fixation of Endoplasmic reticulum membranes Biomedical device Key benefit : high surface to volume mGST1 Detection marker In the case of reactive metabolites formation SH SX mGST1 P450-b5-reductase Enhanced activities of mGST1

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