Vivid SYP450 Screening Kits Vivid Substrates Cytochrome P450 BACULOSOMES Reagents RECO Reconstitution System...

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1 Products Vivid SYP450 Screening Kits Vivid Substrates Cytochrome P450 BACULOSOMES Reagents RECO Reconstitution System P450-Related Products Purified Cytochrome P450 Enzymes Cytochrome P450 Monoclonal Antibodies UDP-glycosyltransferases (UGTs) Glutathione Transferases (GSTs) Sulfotransferases (SULTs) Contract Services Trademark, Patent, and License Information

2 62 Drug Metabolizing Enzymes Products Vivid CYP450 Screening Kits Applications: High-throughput screening of enzyme-drug interactions Compound profiling for drug inhibition of cytochrome P450 isozymes Generate predictive SAR models to guide compound acquisition Features: Reliable Vivid Fluorogenic CYP450 Substrates offer superior fluorescent properties, kinetics, and specificity; improved aqueous solubilities Sensitive High signal to background ratio, broad dynamic range High enzyme turnover rate HTS Compatible with multiple assay formats from 96- well to 3,456-well, miniaturizable to 2 µl Superior specific activity The kits use recombinant Cytochrome P450 BACULOSOMES Reagents Room temperature protocol Easy and straightforward Vivid CYP450 Screening Kits represent a new generation of high-throughput fluorescence-based assays for detection of protein-drug interactions and CYP450 inhibition. They provide the high performance, throughput, and reliability you need to speed selection of novel compounds for drug development. Vivid CYP450 Fluorogenic Substrates are supplied with highly active and specific BACULOSOMES CYP450 Reagents in easy-to-use kits. CYP450 BACULOSOMES Reagents are microsomes prepared from insect cells expressing a human P450 isozyme and rabbit NADPH-P450 reductase. CYP450 BACULOSOMES Reagents offer a distinct advantage over human liver microsomes in that only one CYP450 enzyme is expressed, thereby preventing metabolism by other CYP450s. Vivid Fluorogenic Substrates offer significant benefits over conventional probes. They have superior fluorescence, solubility and kinetic properties compared to conventional fluorogenic probes. This results in higher sensitivity, greater signal to noise ratio, and better assay reproducibility. Together, Vivid Fluorogenic Substrates and BACULOSOMES Reagents provide a non-radioactive, optimized method for the study of isozyme-specific CYP450- drug interactions, metabolism, and inhibition. Product Quantity* Cat. no. Vivid CYP3A4 Red Screening Kit >300 Assays P2856 Vivid CYP3A4 Green Screening Kit >300 Assays P2857 Vivid CYP3A4 Blue Screening Kit >300 Assays P2858 Vivid CYP3A4 Cyan Screening Kit >300 Assays P2968 Vivid CYP2D6 Blue Screening Kit >300 Assays P2972 Vivid CYP2D6 Cyan Screening Kit >300 Assays P2862 Vivid CYP2C9 Red Screening Kit >300 Assays P2859 Vivid CYP2C9 Green Screening Kit >300 Assays P2860 Vivid CYP2C9 Blue Screening Kit >300 Assays P2861 Vivid CYP1A2 Blue Screening Kit >300 Assays P2863 Vivid CYP2C19 Blue Screening Kit >300 Assays P2864 Vivid CYP2B6 Blue Screening Kit >300 Assays P3019 Vivid CYP2B6 Cyan Screening Kit >300 Assays P3020 Vivid CYP2E1 Blue Screening Kit >300 Assays P3021 Vivid CYP3A5 Green Screening Kit >300 Assays P2969 Vivid CYP3A5 Blue Screening Kit >300 Assays P2970 Vivid CYP3A5 Cyan Screening Kit >300 Assays P2971 *Assay volume is 100 µl. Invitrogen Corporation 501 Charmany Drive Madison, WI USA

3 Products Technical Resource Guide 63 Vivid CYP450 Screening Kits, continued Vivid Substrates are blocked dyes that yield minimal fluorescence signal until cleaved. Cleavage at either of two potential cleavage sites (see arrows in Figure 16) releases the highly fluorescent product. Test compounds are analyzed for their capacity to inhibit the production of a fluorescence signal. The sensitivity of Vivid P450 Assays allows detection of weak inhibitors and miniaturization to as little as 2 µl per reaction. Vivid Assays are simple and homogeneous, with a threestep, mix and read format (Figure 17). No stop reagents are needed to conduct the kinetic reaction. The assays can also be done in endpoint mode to facilitate multi-plate screening. These reactions can be performed at room temperature or 37 C. Each kit provides all necessary reagents, including an NADPH-regeneration system, for at least three hundred 100 µl reactions. For more information on Vivid CYP450 Screening Kits, please visit Figure 16 Cleavage of Vivid Substrate Figure 17 Vivid Assay Step 1 Step 2 Step 3 Test Compound P450, NADPH Regeneration System NADP +, Vivid Substrate Read Fluorescence Signal No Inhibition (highly fluorescent) Blocked Dye (non-fluorescent) Free Dye (highly fluorescent) Inhibition (no fluorescence) Vivid Substrates Substrate No. of assays from 0.1 mg Cat. no. CYP3A4 Red 600 P2865 CYP3A4 Green 870 P2866 CYP3A4 Blue 320 P2867 CYP3A4 Cyan 560 P2973 CYP2D6 Blue 410 P2977 CYP2D6 Cyan 570 P2871 CYP2C9 Red 1410 P2868 CYP2C9 Green 440 P2869 CYP2C9 Blue 330 P2870 CYP1A2 Blue 410 P2872 CYP2C19 Blue 410 P2873 CYP2B6 Blue 640 P3022 CYP2B6 Cyan 1400 P3023 CYP2E1 Blue 410 P3027 CYP3A5 Green 340 P2974 CYP3A5 Blue 320 P2975 CYP3A5 Cyan 560 P2976 Phone: Fax: tech_service@invitrogen.com

4 64 Drug Metabolizing Enzymes Products Cytochrome P450 BACULOSOMES Reagents Recombinant Human P450 & NADPH-P450 Reductase Applications: Isozyme identification Kinetic analysis Isozyme specific metabolism and inhibition screening Cytochrome P450 BACULOSOMES Reagents are microsomes prepared from insect cells infected with recombinant baculovirus containing a cdna insert for a human P450 isozyme and a supplemental insert for rabbit NADPH-P450 reductase. P450 BACULOSOMES Reagents offer a distinct advantage over human liver microsomes (HLMs) in that only one P450 isozyme is expressed, thereby preventing metabolism by other P450s or other classes of drug metabolizing enzymes. The compound rankings based on metabolism and inhibition profiles observed with P450 BACULOSOMES Reagents are very similar to those seen with HLMs for most compounds tested. However, P450 BACULOSOMES Reagents activity and metabolic rates with most substrates is significantly higher than in preparations of human liver microsomes (HLMs). This results in a broad dynamic range and high sensitivity in assays utilizing P450 BACULOSOMES Reagents. The high level of activity and reproducibility of the enzyme component observed in the reaction with most probe substrates makes P450 BACULOSOMES Reagents well suited to high-throughput screening formats. Using P450 BACULOSOMES Reagents in assay development for ultra high-throughput screening is particularly successful. No background metabolism by endogenous insect P450s has been detected with any of the probe substrates tested so far. Four BACULOSOMES Reagents are now available: CYP2E1, CYP2B6, CYP2C9*2 (Cys-144), and CYP2C9*3 (Leu-359). Specifications: For CYP2E1, cells were infected with an additional cdna insert for human cytochrome b 5. For P450 content and specific activity, see Table 24. P450 Isozyme Quantity Cat. no. 1A2 0.5 nmol P2792 2B6 0.5 nmol P3028 2C9 0.5 nmol P2378 2C9*2 0.5 nmol P3052 2C9*3 0.5 nmol P3053 2C nmol P2570 2D6 0.5 nmol P2283 2E1 + Cytochrome b5 1.0 nmol P2948 3A4 0.5 nmol P2377 3A5 0.5 nmol P2512 P450 Control 5 mg P2315 Invitrogen Corporation 501 Charmany Drive Madison, WI USA

5 Products Technical Resource Guide 65 Table 24 Cytochrome P450 specifications Isozyme Format Amount of P450 Specific content Spectral P450/mg protein Cat. no. BACULOSOMES Reagents 0.5 nmol 0.01 P2792 1A2 RECO System 0.75 nmol 10 P2304 Purified 150 µg ( 1.5 nmol) 10 P2294 2B6 BACULOSOMES Reagents 0.5 nmol 0.05 P3028 BACULOSOMES Reagents 0.5 nmol 0.08 P2378 2C9 RECO System 0.75 nmol 10 P2362 Purified 100 µg ( 1 nmol) 10 P2355 2C9*2 BACULOSOMES Reagents 0.5 nmol 0.05 P3052 2C9*3 BACULOSOMES Reagents 0.5 nmol 0.08 P3053 BACULOSOMES Reagents 0.5 nmol 0.08 P2570 2C19 RECO System 0.75 nmol 10 P2787 Purified 150 µg 10 P2788 BACULOSOMES Reagents 0.5 nmol 0.08 P2283 2D6 RECO System 0.75 nmol 10 P2775 Purified 150 µg 10 P2774 2E1+cytochrome b5 BACULOSOMES Reagents 1.0 nmol 0.05 P2948 2E1 RECO System 3.75 nmol 10 P2306 Purified 150 µg ( 1.5 nmol) 10 P2223 BACULOSOMES Reagents 0.5 nmol 0.08 P2377 3A4 RECO System 3.75 nmol 10 P2305 Purified 150 µg ( 1.5 nmol) 10 P2194 3A5 BACULOSOMES Reagents 0.5 nmol 0.08 P2512 Control BACULOSOMES Reagents 5 mg Not detected P2315 Phone: Fax: tech_service@invitrogen.com

6 66 Drug Metabolizing Enzymes Products RECO Reconstitution System Purified, Recombinant Human With the RECO System, Invitrogen provides purified P450s in an easy-to-use, reconstituted format that includes a complete enzyme mix (consisting of the P450 isozyme, NADPH reductase, and cytochrome b 5) and a separate reaction buffer mix. This mix and metabolize configuration greatly simplifies the use of purified P450 isozymes with their associated proteins, making them as easy to use as microsomes while maintaining the inherent advantages associated with purified proteins. The ratio of the individual proteins contained in the RECO System is optimized for better metabolic rates. See the Certificate of Analysis for details. For example, the CYP3A4 RECO System contains a 1:2:1 mixture of P450 3A4, NADPH-P450 reductase, and cytochrome b 5. For specifications, please see Table 24 on page 65. P450 Isozyme Quantity Cat. no. 1A nmol P2304 2C nmol P2362 2E nmol P2306 3A nmol P2305 2D nmol P2775 2C nmol P2787 P450-Related Products NADPH-P450 reductase transfers electrons from NADPH to the various isoforms of cytochrome P450. It is typically used at a variety of ratios ranging from 0.5-5:1 (reductase: P450) for in vitro reconstitution assays. Invitrogen s human NADPH-P450 reductase is produced from insect cells infected with baculovirus containing the cdna for human P450 reductase. Cytochrome b 5 is a membrane-bound hemoprotein that enhances the catalytic efficiency of some P450 isoforms. Invitrogen s human cytochrome b 5 is overexpressed in E. coli and purified to homogeneity. P450 Isozyme Quantity Cat. no. NADPH-P450 Reductase, Purified, Recombinant Human 200 µg P2309 Cytochrome b5, Purified, Recombinant Human 100 µg P2252 Invitrogen Corporation 501 Charmany Drive Madison, WI USA

7 Products Technical Resource Guide 67 Purified Cytochrome P450 Enzymes Recombinant Human Invitrogen s purified recombinant human P450s are produced in E. coli. Although each P450 has been modified at its N-terminus to allow for optimal expression in E. coli, these modifications do not cause any significant differences in substrate specificity. Advantages of using purified cytochrome P450 enzymes include the lack of interfering activities commonly present in microsomal or tissue samples and the flexibility to optimize component ratios of P450, NADPH-P450 reductase, and cytochrome b 5 for specific applications. The physical purity for Purified Cytochrome P450 Enzymes is >85% for CYP1A2, CYP2E1, CYP3A4; >50% for CYP2C9 as assessed by visualization of 5 µg protein on a Coomassie Blue-stained SDS-polyacrylamide minigel. P450 Isozyme Quantity Cat. no. 1A2 150 µg P2294 2C9 100 µg P2355 2E1 150 µg P2223 3A4 150 µg P2194 2D6* 150 µg P2774 2C µg P2788 *Histidine-tagged Phone: Fax: tech_service@invitrogen.com

8 68 Drug Metabolizing Enzymes Products Cytochrome P450 Monoclonal Antibodies Human, for Inhibition Assays Inhibitory monoclonal antibodies are used to specifically inhibit cytochrome P450 isoforms in order to determine the specific P450 content in tissue and to examine an individual P450 isoform s contribution to the metabolism of xenobiotics and endobiotics. Cytochrome P450 Monoclonal Antibodies are supplied as ascites fluid derived from mouse hybridoma cells, and are specific to a unique antigen on each human cytochrome P450 isozyme. They do not cross-react with other isoforms, and have been shown to inhibit cytochrome activity by 90-95% using only 2-6 µl of antibody per inhibition assay. P450 Isozyme Immunogen Clone Quantity Cat. no. 1A1 Rat 1A µl P2686 2B6 Recombinant Human 2B µl P2687 2C8/9/18/19 Rat 2C µl P2688 2D6 Recombinant Human 2D µl P2689 2D6i Recombinant Human 2D µl P2690 2E1 Recombinant Human 2E µl P2691 3A4/5 Recombinant Human 3A µl P2692 2A6 Recombinant Human 2A µl P2732 1A2 Recombinant Human 1A µl P2733 Suitable for immunoblots Invitrogen Corporation 501 Charmany Drive Madison, WI USA

9 Products Technical Resource Guide 69 UDP-glycosyltransferases (UGTs) BACULOSOMES Reagents, Recombinant Human Examination of glucuronidation reactions catalyzed by the human UDP-glycosyltransferase (UGT) family of enzymes is essential when investigating the metabolic fate of any new drug, therapeutic agent, or potential environmental toxic agent. UGT activity is defined by the conjugation of glucuronic acid to a wide variety of xenobiotic and endogenous substrates containing sulfhydryl, hydroxyl, aromatic amino, or carboxylic acid moieties. The UGTs comprise a superfamily of integral membrane proteins of the endoplasmic reticulum, and have been subdivided into two families UGT1 and UGT2 based on the evolutionary divergence of their genes. Invitrogen s UGT BACULOSOMES Reagents are produced as a microsomal fraction of Sf-9 insect cells infected with a baculovirus strain containing cdna for a specific human UGT. Specifications: Source: Recombinant baculovirus-infected Sf-9 cells Concentration: 2-20 mg/ml Specific activity: UGT1A1 octyl gallate: 500 pmol/min/mg UGT1A6 naphthol: 500 pmol/min/mg UGT1A7 octyl gallate: 150 pmol/min/mg UGT1A10 naphthol: 30 pmol/min/mg UGT1A3 octyl gallate: 40 pmol/min/mg UGT2B7 hyodeoxycholic acid: 250 pmol/min/mg UGT Isozyme Quantity Cat. no. 1A1 5 mg P2327 1A6 5 mg P2358 1A3 5 mg P2470 1A7 5 mg P2447 1A10 5 mg P2472 2B7 5 mg P2636 UGT Control 5 mg P2448 Phone: Fax: tech_service@invitrogen.com

10 70 Drug Metabolizing Enzymes Products Glutathione Transferases (GSTs) Purified, Recombinant Human Glutathione transferases (GSTs) catalyze the formation of thioether conjugates between glutathione and xenobiotic compounds. Their major biological function is believed to be defense against electrophilic chemical species, many of which are formed by cellular oxidative reactions catalyzed by cytochrome P450 and other oxidases. GST A1-1 is found in only a few tissues of the body, including kidney, intestine, lung, and liver. GST M1-1 is also restricted to a few tissues (mainly liver); GST P1-1 is abundant in most types of tumor cells and is widely distributed throughout the body (except for its notable absence from the liver). GST M1-1 is the type b allelic variant, which corresponds to GST psi (Ψ) purified from human liver. Invitrogen s recombinant human GSTs are overexpressed in E. coli and purified by affinity chromatography. The physical and catalytic properties of all three recombinant enzymes are indistinguishable from the properties of enzymes purified from human tissue. Specifications: Source: Recombinant E. coli Concentration: 1-5 mg/ml Activity: Determined using spectrophotometric determination of 1-chloro-2,4-dinitrobenzene (CDNB) conjugation. Minimum acceptable specific activities, in µmol/min/mg are: 50 for GST A1-1, 100 for M1-1, and 75 for P1-1 Specificity: The different specificities of the three GST isozymes are demonstrated by determining the I 50 values obtained using the inhibitor S-hexylglutathione in the standard CDNB assay: I 50 (A1-1) < I 50 (M1-1) < I 50 (P1-1) Physical purity: >95% as assessed by visualization of 10 µg protein on a Coomassie Blue-stained SDS-polyacrylamide gel Storage buffer: 50% glycerol, 50 mm Tris (ph 7.5), 50 mm NaCl, 1 mm DTT, 1 mm EDTA GST Isozyme Quantity Cat. no. A µg P2175 M µg P2192 P µg P2177 Invitrogen Corporation 501 Charmany Drive Madison, WI USA

11 Products Technical Resource Guide 71 Sulfotransferases (SULTs) Cytosolic Extract, Recombinant Human The human cytosolic sulfotransferase isozymes are products of multiple genes that form a gene superfamily (SULT). Ten distinct SULT forms are known; two of these represent splice variants, whereas the others are encoded by separate genes. It is expected that other genes encoding sulfotransferases will continue to be identified. The nomenclature of the different genes and their mrna and protein products has recently been revised so that SULT is the accepted superfamily abbreviation. Allelic variants of sulfotransferase enzymes do exist, and the study of their expression levels and functional roles in drug disposition is an active area of research. Invitrogen s highly active cytosolic extracts are produced from insect cells infected with a baculovirus strain containing cdna encoding a specific human sulfotransferase. Specifications: Source: Recombinant baculovirus-infected Sf-9 cells Specific activity: SULT1A1*2 p-nitrophenol: 7,500 pmol/min/mg SULT1A1*1 p-nitrophenol: 3,000 pmol/min/mg SULT1A3 dopamine: 5,000 pmol/min/mg SULT1E estrone: 150 pmol/min/mg SULT2A1 DHEA: 300 pmol/min/mg SULT Isozyme Quantity Cat. no. 1A1*2 250 µg P2438 1A2*1 250 µg P2511 1A3 250 µg P2437 1E 250 µg P2436 2A1 250 µg P2435 SULT Control Cytosol 250 µg P2469 Phone: Fax: tech_service@invitrogen.com

12 72 Drug Metabolizing Enzymes Products Contract Services Invitrogen provides both a broad product line for drug metabolism experiments and a contract protein manufacturing facility staffed by a group of experienced scientists who work specifically in the area of drug metabolism. Our contract manufacturing capabilities include: Production of recombinant microsomes and purified enzymes Biosynthesis of metabolites for analytical and pharmacological assays High-throughput assay development Trademark, Patent, and License Information Trademarks Invitrogen, BACULOSOMES, RECO, and Vivid are registered trademarks of Invitrogen Corporation. Coomassie is a registered trademark of Imperial Chemical Industries, Ltd. Prism is a registered trademark of GraphPad, Inc. Ready Safe is a trademark of Beckman Coulter, Inc. TRITON is a registered trademark of Union Carbide Chemicals and Plastics Co., Inc. All other trademarks are the property of their respective owners. Patents The RECO System is covered by U.S. Patent No. 5,891,696 assigned to Invitrogen Corporation. Purified P450 isozymes from Invitrogen are covered under U.S. Patent No. 5,240,831 issued to the Board of Regents, University of Texas System on August 31, Vivid Probes are covered by one or more of the following U.S. Patents assigned to Invitrogen Corporation, as well as corresponding pending or issued foreign patents: 6,143,492; other U.S. patents pending. Licenses LULL no. 162: This product is the subject of one or more of US Patent 5,891,696, 6,143,492, and 6,420,130. The purchase of this product conveys to the buyer the non-transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer (whether the buyer is an academic or for-profit entity). The buyer cannot sell or otherwise transfer (a) this product (b) its components or (c) materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes. The buyer may transfer information or materials made through the use of this product to a scientific collaborator, provided that such transfer is not for any Commercial Purpose, and that such collaborator agrees in writing (a) to not transfer such materials to any third party, and (b) to use such transferred materials and/or information solely for research and not for Commercial Purposes. Commercial Purposes means any activity by a party for consideration and may include, but is not limited to: (1) use of the product or its components in manufacturing; (2) use of the product or its components to provide a service, information, or data; (3) use of the product or its components for therapeutic, diagnostic or prophylactic purposes; or (4) resale of the product or its components, whether or not such product or its components are resold for use in research. Invitrogen Corporation will not assert a claim against the buyer of infringement of the above patent claiming this product based upon the manufacture, use or sale of a therapeutic, clinical diagnostic, vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed, provided that neither this product nor any of its components was used in the manufacture of such product. If the purchaser is not willing to accept the limitations of this limited use statement, Invitrogen is willing to accept return of the product with a full refund. For information on purchasing a license to this product for purposes other than research, contact Licensing Department, Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, California Phone (760) Fax (760) Invitrogen Corporation 501 Charmany Drive Madison, WI USA